USO10941424B2

( 12 ) United States Patent ( 10 ) Patent No .: US 10,941,424 B2 Koch et al . (45 ) Date of Patent : * Mar . 9 , 2021

( 54 ) MICROORGANISMS AND METHODS FOR USPC 435/132 , 158 , 189 , 193 , 252.2 , 254.33 , THE CO - PRODUCTION OF ETHYLENE 435 / 320.1 GLYCOL AND THREE CARBON See application file for complete search history . COMPOUNDS ( 71 ) Applicant: Braskem S.A. , Camaçari ( BR ) (56 ) References Cited ( 72 ) Inventors: Daniel Johannes Koch , Camaçari U.S. PATENT DOCUMENTS ( BR ) ; Mateus Schreiner Lopes , 7,977,083 B1 7/2011 Sakakibara et al . Camaçari ( BR ) ; Ane Fernanda Beraldi 2007/0259411 A1 11/2007 Bramucci et al . Zeidler , Camaçari ( BR ) ; Lucas 2008/0293125 A1 11/2008 Subbian et al . Pedersen Parizzi , Camaçari ( BR ) 2010/0047878 A1 2/2010 Nagai et al . 2010/0311135 A1 12/2010 Takebayashi et al . (73 ) Assignee : BRASKEM S.A. , Camacari ( BR ) 2013/0280775 Al 10/2013 Grotkjaer et al. 2013/0316416 Al 11/2013 Stephanopoulos et al. ( * ) Notice: Subject to any disclaimer , the term of this 2014/0065686 A1 3/2014 Marliere patent is extended or adjusted under 35 2014/0141482 Al 5/2014 Pearlman et al . U.S.C. 154 ( b ) by 5 days . 2015/0147794 Al 5/2015 Chung et al . 2017/0260551 Al 9/2017 Koch et al . This patent is subject to a terminal dis 2018/0179558 A1 6/2018 Koch et al . claimer . ( 21 ) Appl . No .: 15 /726,978 FOREIGN PATENT DOCUMENTS ( 22 ) Filed : Oct. 6 , 2017 WO WO 2006/135075 Al 12/2006 WO WO 2009/008377 Al 1/2009 ( 65 ) Prior Publication Data WO WO 2011/012697 A2 2/2011 WO WO 2011/076691 A1 6/2011 US 2018/0179558 A1 Jun . 28 , 2018 WO WO 2011/130378 A1 10/2011 WO WO 2012/088467 A2 6/2012 Related U.S. Application Data WO WO 2013/126721 A1 8/2013 WO WO 2013/163230 A2 10/2013 ( 63 ) Continuation of application No. 15 / 453,094 , filed on WO WO 2014/004625 A1 1/2014 WO WO 2015/002977 Al 1/2015 Mar. 8 , 2017 WO WO 2015/032761 A1 3/2015 WO WO 2015/042588 A1 3/2015 ( 60 ) Provisional application No. 62 / 430,742, filed on Dec. WO WO 2016/079440 A1 5/2016 6 , 2016 , provisional application No. 62 / 305,814 , filed WO WO 2017/156166 A1 9/2017 on Mar. 9 , 2016 . ( 51 ) Int . Cl . OTHER PUBLICATIONS C12N 9/10 ( 2006.01 ) Devos et al . , ( Proteins: Structure, Function and Genetics, 2000 , vol . C12N 1/20 ( 2006.01 ) 41 : 98-107 . * C12P 7/18 ( 2006.01 ) C12P 7/28 ( 2006.01 ) (Continued ) C12N 9/02 ( 2006.01 ) Primary Examiner Robert B Mondesi C12N 9/12 ( 2006.01 ) CI2N 9/88 ( 2006.01 ) Assistant Examiner - Mohammad Y Meah C12N 9/90 ( 2006.01 ) ( 74 ) Attorney, Agent, or Firm - Morgan , Lewis & C12N 15/52 ( 2006.01 ) Bockius LLP C12P 5/02 ( 2006.01 ) C12P 7/04 ( 2006.01 ) ( 57 ) ABSTRACT C12P 7/26 ( 2006.01 ) The present application relates to recombinant microorgan ( 52) U.S. CI . isms useful in the biosynthesis of monoethylene glycol CPC C12P 7/28 ( 2013.01 ) ; C12N 9/0008 ( MEG ) and one or more three - carbon compounds such as ( 2013.01 ) ; C12N 9/1029 ( 2013.01 ) ; C12N acetone , isopropanol or propene. The MEG and one or more 9/1205 ( 2013.01 ) ; C12N 9/13 ( 2013.01 ) ; three - carbon compounds described herein are useful as C12N 9/88 ( 2013.01 ) ; C12N 9/90 ( 2013.01 ) ; starting material for production of other compounds or as C12N 15/52 ( 2013.01 ) ; C12P 5/026 ( 2013.01 ) ; end products for industrial and household use . The applica C12P 7/04 ( 2013.01 ) ; C12P 7/18 ( 2013.01 ) ; tion further relates to recombinant microorganisms co -ex C12P 7/26 ( 2013.01 ) ; C12Y 102/01021 pressing a C2 branch pathway and a C3 branch pathway for ( 2013.01 ) ; C12Y 203/01009 ( 2013.01 ) ; C12Y the production of MEG and one or more three - carbon 207701047 ( 2013.01 ) ; C12Y 208/03008 compounds. Also provided are methods of producing MEG ( 2013.01 ) ; C12Y 401/01004 ( 2013.01 ) ; C12Y and one or more three - carbon compounds using the recom 401/02013 ( 2013.01 ) ; C12Y 501/03 ( 2013.01 ) ; binant microorganisms, as well as compositions comprising C12P 2203/00 ( 2013.01 ) the recombinant microorganisms and / or optionally the prod ( 58 ) Field of Classification Search ucts MEG and one or more three - carbon compounds. CPC ...... C12N 9/90 ; C12N 9/1205 ; C12N 9/88 ; C12N 9/0008 ; C12N 15/52 ; C12Y 7 Claims , 15 Drawing Sheets 207/01047 ; C12P 7/28 , C12P 7/04 Specification includes a Sequence Listing . US 10,941,424 B2 Page 2

( 56 ) References Cited Hayward et al ., “ Structure and alternative splicing ofthe ketohexokinase gene ,” Eur . J. Biochem . ( 1998 ) , 257 : 85-91 . Itoh et al ., “ Purification and Characterization of D - Tagatose 3 - Epimerase OTHER PUBLICATIONS from Pseudomonas sp . ST- 24 ," Biosci . Biotech . Biochem ., 1994 , 58 ( 12 ) , 2168-2171 . Whisstock et al . , ( Quarterly Reviews of Biophysics 2003 , vol . 36 LeBlanc et al . , “ Metabolism of D - Arabinose: a New Pathway in ( 3 ) : 307-340 . * Escherichia coli , ” Journal of Bacteriology , Apr. 1971 , 106 ( 1 ) : 90-96 . Witkowski et al . , ( Biochemistry 38 : 11643-11650 , 1999. * Liu et al . , “ Biosynthesis of ethylene glycol in Escherichia coli, ” Kisselev L. , ( Structure, 2002 , vol . 10 : 8-9 . * Appl Microbiol Biotechnol ( 2013 ) 97 : 3409-3417 . Alkim et al . ( Microb cel fact 2015 , pp . 1-12 . * UniProtKB - B2TLNS (ADC_CLOBB ) Apr. 14 , 2009 , retrieved May et al . ( Met . Eng . 15 , 2013 , pp . 218-225 * from https://www.uniprot.org/uniprot/B2TLN8, 4 pages . Clomburg et al ( Appl micro boil biotec 2010 , 86 , 419-434 ) . * UniProtKBC1KKRI (DT3E_RHOSH ) Jan. 20 , 2016 , retrieved Alkim , Ceren , et al . “ Optimization of ethylene glycol production from https://www.uniprot.org/uniprot/CIKKR1, 6 pages. from ( D ) -xylose via a synthetic pathway implemented in Escherichia UniProtKB - E3PHWO ( E3PHWO_ECOH1 ) Jan. 11 , 2011 , retrieved coli .” Microbial Cell Factories ( 2015 ) ; 14.1 : 127 . from https: //www.uniprot.org.uniprot/E3PHWO , 4 pages. Boonstra, Birgitte , et al . “ The udhA gene of Escherichia coli UniProtKB - 050580 (DT3E_PSECI ) Dec. 1 , 2000 , retrieved from encodes a soluble pyridine nucleotide transhydrogenase .” Journal of https://www.uniprot.org/uniprot/050580 , 8 pages . Bacteriology ( 1999 ) ; 181.3 : 1030-1034 . UniProtKB — P00884 ( ALDOB_RAT ) Jul. 21 , 1986 , retrieved from Canonaco , Fabrizio , et al . “ Metabolic flux response to phosphoglucose https://www.uniprot.org/uniprot/P00884 , 9 pages. isomerase knock - out in Escherichia coli and impact of overexpres UniProtKB — P05062 ( ALDOB_HUMAN ) Aug. 13 , 1987 , retrieved sion of the soluble transhydrogenase UdhA .” FEMS Microbiology from https://www.uniprot.org/uniprot/P05062, 14 pages . Letters ( 2001 ) ; 204.2 : 247-252 . UniProtKB — P07097 ( THIL_ZOORA ) Apr. 1 , 1988 , retrieved from Charusanti, Pep , et al . " Genetic basis of growth adaptation of https://www.uniprot.org/uniprot/P07097 , 7 pages . Escherichia coli after deletion of pgi , a major metabolic gene. ” UniProtKB — P11553 ( FUCK_ECOLI) Oct. 1 , 1989 , retrieved from PLoS Genet ( 2010 ) ; 6.11 : e1001186 . https://www.uniprot.org/uniprot/P11553, 5 pages . Chen , Zhen , et al . “ Metabolic engineering of Corynebacterium UniProtKB — P14611 ( THIL_CUPNH ) Apr. 1 , 1990 , retrieved from glutamicum for the de novo production of ethylene glycol from https://www.uniprot.org/uniprot/P14611 , 7 pages . glucose. ” Metabolic Engineering ( 2016 ) ; 33 : 12-18 . UniProtKBP17764 ( THIL_RAT) Aug. 1 , 1990 , retrieved from Ehrensberger, Andreas H. , et al . “ Structure - guided engineering of https://www.uniprot.org/uniprot/P17764 , 9 pages. xylitol dehydrogenase cosubstrate specificity . ” Structure ( 2006 ) ; UniProtKBP23670 ( ADC_CLOAB ) Nov. 1 , 1991 , retrieved from 14.3 : 567-575 . https://www.uniprot.org/uniprot/P23670, 5 pages. Hao , Jijun, and Berry , Alan . " A thermostable variant of fructose UniProtKB - P23673 (CTFB_CLOAB ) Nov. 1 , 1991 , retrieved bisphosphate aldolase constructed by directed evolution also shows from https ://www.uniprot.org.uniprot/P23673 , 4 pages . increased stability in organic solvents. ” Protein Engineering Design UniProtKB - P24752 ( THIL_HUMAN ) Mar. 1 , 1992 , retrieved and Selection ( 2004 ) ; 17.9 : 689-697 . from https://www.uniprot.org/uniprot/P24752 , 14 pages . International Application No. PCT / US2017 / 021421 , International UniProtKBP33752 ( CTFA_CLOAB ) Feb. 1 , 1994 , retrieved from Search Report and Written Opinion dated Jul. 10 , 2017 , 15 pages . https://www.uniprot.org/uniprot/P33752 , 4 pages . International Application No. PCT / US2017 / 041732 , International UniProtKBP41338 ( THIL_YEAST) Feb. 1 , 1995 , retrieved from Search Report and Written Opinion dated Nov. 20 , 2017 , 19 pages . https://www.uniprot.org/uniprot/P41338 , 9 pages . Jarboe, Laura R. “ YqhD : a broad - substrate range aldehyde reductase UniProtKB — P50053 ( KHK_HUMAN ) Oct. 1 , 1996 , retrieved from with various applications in production of biorenewable fuels and https://www.uniprot.org/uniprot/P50053 , 9 pages. chemicals . ” Applied Microbiology and Biotechnology ( 2011 ) ; 89.2 : UniProtKBP76459 (ATOA_ECOLI ) Nov. 1 , 1997 , retrieved from 249-257 . https://www.uniprot.org/uniprot/P76459, 5 pages . Li , Hongmei , et al . “ Enhanced activity of yqhD oxidoreductase in UniProtKB_P76461 (ATOB_ECOLI ) Nov. 1 , 1997 , retrieved from synthesis of 1 , 3 -propanediol by error - prone PCR . ” Progress in https://www.uniprot.org/uniprot/P76461, 9 pages . Natural Science ( 2008 ) ; 18.12 : 1519-1524 . UniProtKBP79226 ( ALDOB_RABIT ) Nov. 1 , 1997 , retrieved Marmulla, R. , et al ., “ Linalool isomerase , a membrane - anchored from https://www.uniprot.org/uniprot/P79226 , 9 pages. enzyme in the anaerobic monoterpene degradation in Thauera UniProtKBP81336 ( ADC_CLOPA ) Jul. 15 , 1998 , retrieved from linaloolentis 47Lol . ” BMC Biochemistry ( 2016 ) 17 : 6 , pp . 1-11 . https://www.uniprot.org/uniprot/P81336 , 2 pages . Patel, Darshan H. , et al . “ Engineering of the catalytic site of xylose UniProtKBP97328 (KHK_MOUSE ) Jul. 15 , 1999 , retrieved from isomerase to enhance bioconversion of a non -preferential sub https://www.uniprot.org/uniprot/P97328, 6 pages . strate .” Protein Engineering Design and Selection ( 2012 ) ; 25 ( 7 ) : UniProtKB Q02974 ( KHK_RAT ) Jul. 1 , 1993 , retrieved from 331-336 . https://www.uniprot.org/uniprot/Q02974 , 6 pages . Sauer, Uwe, et al. “ The soluble and membrane - bound transhydrogenases UniProtKB - Q3A042 ( Q3A042_PELCD ) Nov. 22 , 2005 , retrieved UdhA and PntAB have divergent functions in NADPH metabolism from https://uniprot.org/uniprot/Q3A042, 4 pages . of Escherichia coli. ” Journal of Biological Chemistry ( 2004 ) ; 279.8 : UniProtKB Q5R171 (KHK_PONAB ) Jan. 24 , 2006 , retrieved 6613-6619 . from https://www.uniprot.org/uniprot/Q5RD71, 4 pages . Sulzenbacher , Gerlind , et al . “ Crystal structure of E. coli alcohol UniProtKB - Q7NSA6 ( ADC_CHRVO ) Mar. 1 , 2004 , retrieved dehydrogenase YqhD : evidence of a covalently modified NADP from https://www.uniprot.org/uniprot/Q7NSA6, 4 pages . coenzyme. ” Journal of Molecular Biology ( 2004 ) ; 342.2 : 489-502 . UniProtKB - Q89EP4 ( ADC2_BRADU ) Mar. 1 , 2004 , retrieved UniProtKB POAB87 ( Nov. 8 , 2005 ) [ retrieved on Jun . 18 , 2017 from https://www.uniprot.org/uniprot/Q89EP4 , 4 pages . from http://www.uniprot.org/uniprot/POAB87, 8 pages . UniProtKB - Q8QZTI ( THIL_MOUSE ) Sep. 13 , 2004 , retrieved Bermejo et al . , “ Expression of Clostridium acetobutylicum ATCC from https://www.uniprot.org/uniprot/Q8QZT1 , 9 pages . 824 Genes in Escherichia coli for Acetone Production and Acetate UniProtKB - Q8S4Y1 ( THIC1_ARATH ) Aug. 30 , 2005 , retrieved Detoxification , ” Applied and Enviromental Microbiology, Mar. from https://www.uniprot.org/uniprot/Q8S4Y1, 8 pages . 1998 , 64 ( 3 ) : 1079-1085 . UniProtKB - Q91797 ( ALDOB_MOUSE ) Apr. 23 , 2003 , retrieved Elsinghorst et al . , “ D - Arabinose Metabolism in Escherichia coli B : from https://www.uniprot.org/uniprot/Q8S4Y1, 9 pages . Induction and Cotransductional Mapping of the L - Fucose - D UniProtKBQ98FWO ( LR3E_RHILO ) Jan. 20 , 2016 , retrieved Arabinose Pathway Enzymes , ” Journal of Bacteriology, Dec. 1988 , from https://www.uniprot.org/uniprot/Q98FW0, 6 pages. 170 ( 12 ) : 5423-5432 . UniProtKBQ9BWD1 ( THIC_HUMAN ) Sep. 13 , 2004 , retrieved Hanai et al. , “ Engineered Synthetic Pathway for Isogropanol Pro fromhttps : //www.uniprot.org/uniprot/Q9BWD1, 11 pages . duction in Escherichia coli, ” Applied and Enviromental Microbi UniProtKBQ9RPK1 ( ADC_CLOBE ) Nov. 15 , 2002 , retrieved ology, Dec. 2007 , 73 ( 24 ) : 7814-7818 . from https://www.uniprot.org/uniprot/Q9RPK1, 3 pages . US 10,941,424 B2 Page 3

( 56 ) References Cited OTHER PUBLICATIONS Yoshida et al . , “ X - ray structures of the Pseudomonas cichorii D - tagatose 3 - epimerase mutant form C66S recognizing deoxy sug ars as substrates ,” Applied Microbiology and Biotechnology, Dec. 2016 , vol . 100 , Issue 24 , pp . 10403-10415 . Cabulong et al . , “ Enhanced yield of ethylene glycol production from d -xylose by pathway optimization in Escherichia coli, ” Enzyme and Microbial Technology 97 ( 2017 ) 11-20 . Cao et al . , " Metabolic Engineering of Escherichia coli for the Production of Xylonate ,” Plos One , Jul. 2013 , vol . 8 , Issue 7 , 7 pages . Extended European Search Report for European Application No. 17764027.3 dated Dec. 10 , 2019 , 9 pages . Haapalainen et al . , “ The thiolase superfamily : condensing enzymes with diverse reaction specificities, ” Trends In Biochemical Sci ences , Jan. 2006 , vol . 31 , No. 1 , pp . 64-71 . Partial Supplementary European Search Report for European Appli cation No. 17764027.3 dated Aug. 23 , 2019 , 11 pages . Wiesenborn et al . , “ Thiolase from Clostridium acetobutylicum ATCC 824 and Its Role in the Synthesis of Acids and Solvents , " Applied and Environmental Microbiology, Nov. 1988 , vol . 54 , No. 11 pp . 2717-2722 . * cited by examiner U.S. Patent Mar. 9 , 2021 Sheet 1 of 15 US 10,941,424 B2

Legends: Pentose Phosphate Pathway C2-stream

Pyruvate(0)Lactate

?. ?D-XyluloseXylulose-5P ???

l??iA FIG.1 D-Xylose

dte

fuck D-Ribulose12 ATP. FUCA RibuloseD- ATP VAD* NADH Xylose->MEG+0.5IPA1.5CO20.5NAD(P)H1ATP ADP Glycolic Acid MEG,20wt%IPAYmax(xylose)-0,618/8;41wt Glycolaldehyde aidafuco Stoichiometry: NADH? NAD* Ethylene Glycol U.S. Patent Mar. 9 , 2021 Sheet 2 of 15 US 10,941,424 B2

Legends: Pentose Phosphite Pathway ANN C2-stream

X-lactate

D-XyluloseXylulose5p-> ? xylB HAM1839 FIG.2 xylaV XyloseD- ATP ADP 12Xylulose-D aldos AD+ NADH Xylose->MEG+0.51PA1.5CO,0.5NAD(P)H+1ATP alda Glycolic Acid Ymax(xylose)=0,61g/;41wi%MEG,20wtPA Glycolaldehyde fuco 7 Stoichiometry: NADH NAD+ Ethylene Glycol U.S. Patent Mar. 9 , 2021 Sheet 3 of 15 US 10,941,424 B2

Legends: C2-stream ??LA XyloseD- KIVIA D-Xylulose lactatetal-PyruvateX XylB XYIB NADH ber K XylC xylD 2-ketodeoxy3xylonate yjhH, FIG.3 A yage XylonolactoneD- it D-xylonate Xalda Glycolic Acid Glycolaldehyde fuco 7 NADH NAQ+ Ethylene Glycol

Xylose->MEG+0.5IPA1.5CO20.5NAD(P)H AutoPropanol-2 41wt%MEG,20wtIPAYmax(xylase)-0,61g/s; Stoichiometry: U.S. Patent Mar. 9 , 2021 Sheet 4 of 15 US| 10,941,424 B2

zjouedoid-

FIG.4 ADRE U.S. Patent Mar. 9 , 2021 Sheet 5 of 15 US 10,941,424 B2

DHAPGlycerol

ADH1-5. ALDO Frutose-6P Frutose-1,6P TPII Ethanol Acetaldehyde Acetate NADPWADAS (atoAlfatoo) ACS),AC52 Ribose-SP Glyceraldehyde-3P PDC3.,PDCS,PDC6 Glucose-5Psensusesse th{A] Ribulose-SP IPDHC CoAAcetyl- Acetoacetyl-COA FIG.5 Glucose Pyruvate App Xylulose-5P Acetoacetate 2VADA murphT7] (adc) NADPZ.COM2 ATE,CQ COA,CO ]Xyl3[S1/ Malonyl-CoA Acetyl-coa GRE3(Xyli]/ Xyl2(] ATP DHAP Acetone D-Xylose 2 Xylitol ?D-Xylulose (adh) NADP (dte] GRE2fucol/( fuck)[ fucA][ Propanol2- D-Ribulose1P Ethyleneglycol D-Ribulosedifuck optoe Glycolaldehyde U.S. Patent Mar. 9 , 2021 Sheet 6 of 15 US 10,941,424 B2

) BIL ( MEG

goXylose

pupsinoo(y) FIG.6 cerevisiaeMEGinS.IPAandproductionofCo-

)EL ( PA , Xylose , Glucose U.S. Patent Mar. 9 , 2021 Sheet 7 of 15 US 10,941,424 B2

) l / g ( MEG

2. 0

timecourse(h) FIG7. E.coliproductioninMEG

15 6 m3 ) l / g ( Xylose , ) OD600 ( Biomass U.S. Patent Mar. 9 , 2021 Sheet 8 of 15 US 10,941,424 B2

Man

FIG.8 E.coliproductioninMEG

3 U.S. Patent Mar. 9 , 2021 Sheet 9 of 15 US 10,941,424 B2

FIG.9 '11 U.S. Patent Mar. 9 , 2021 Sheet 10 of 15 US 10,941,424 B2

/ g( Acetone , PA , MEG , acid Acetic

FIG.10 AcetoneMEG,IPAandproductionofCo- U.S. Patent Mar. 9 , 2021 Sheet 11 of 15 US 10,941,424 B2

FIG.11 YieldproductionCo- ...... :

...... '. U.S. Patent Mar. 9 , 2021 Sheet 12 of 15 US 10,941,424 B2

FIG.12

( 13 ) esix 00900 U.S. Patent Mar. 9 , 2021 Sheet 13 of 15 US 10,941,424 B2

YieldproductionCo- .'

.' U.S. Patent Mar. 9 , 2021 Sheet 14 of 15 US 10,941,424 B2

FIG.14 1 manenoha U.S. Patent Mar. 9 , 2021 Sheet 15 of 15 US 10,941,424 B2

..'

'.

FIG.15

.' US 10,941,424 B2 1 2 MICROORGANISMS AND METHODS FOR branch pathway for production of one or more three -carbon THE CO - PRODUCTION OF ETHYLENE compounds from DHAP or pyruvate. GLYCOL AND THREE CARBON The presently disclosed process of co - producing MEG COMPOUNDS and one or more three - carbon compounds is synergistic by 5 utilizing the excess NADH produced in the C3 branch CROSS REFERENCE TO RELATED pathway to feed the NADH requirement of the C2 branch APPLICATIONS pathway. In one aspect , the present application provides a recom This application is a Continuation Application of U.S. binant microorganism co - producing monoethylene glycol application Ser. No. 15 / 453,094 , filed on Mar. 8 , 2017 , 10 ( MEG ) and one or more three - carbon compounds. In one which claims benefit of priority to U.S. Provisional Appli- embodiment, the MEG and one or more three -carbon com cation No. 62 / 305,814 , filed Mar. 9 , 2016 , and to U.S. pounds are co - produced from xylose . In another embodi Provisional Application No. 62 / 430,742 , filed Dec. 6 , 2016 , ment, the recombinant microorganism comprises a deletion , the contents of each of which are incorporated herein by 15 insertion , or loss of function mutation in a gene encoding a reference in its entirety . D - xylulose - 5 - kinase and / or in a gene encoding a glycoal dehyde dehydrogenase. In some embodiments, the gene DESCRIPTION OF THE TEXT FILE encoding the D - xylulose - 5 -kinase is xylB . In some embodi SUBMITTED ELECTRONICALLY ments , the gene encoding the glycoaldehyde dehydrogenase The contents of the text file submitted electronically 20 is aldA . In some embodiments, MEG is produced through herewith are incorporated herein by reference in their the conversion of glycolaldehyde in a C2 branch pathway entirety : A computer readable format copy of the Sequence and one or more three - carbon compounds is produced Listing ( filename: BRAS_001_02 US_ST25.txt, date through the conversion of DHAP or pyruvate in a C3 branch recorded : Mar. 7 , 2017 , file size about 231 kilobytes ) . pathway. In other embodiments, at least a portion of the 25 excess NADH produced in the C3 branch pathway is used as TECHNICAL FIELD a source of reducing equivalents in the C2 branch pathway. In further embodiments , at least a portion of the excess This application relates to recombinant microorganisms NADH produced in the C3 branch pathway is used to useful in the biosynthesis of monoethylene glycol and one or produce ATP . In yet further embodiments, excess biomass more three - carbon compounds. The application further 30 formation is minimized and production of MEG and one or relates to methods of producing monoethylene glycol and more three - carbon compounds is maximized . one or more three - carbon compound using the recombinant In one embodiment, MEG is produced from xylose via microorganisms, as well as compositions comprising one or ribulose - 1 - phosphate . In another embodiment, MEG is pro more of these compounds and /or the recombinant microor- duced from xylose via xylulose - 1 - phosphate. In a further ganisms. 35 embodiment, MEG is produced from xylose via xylonate . In one embodiment, the one or more three - carbon com BACKGROUND pounds is acetone . In another embodiment, the one or more three - carbon compounds is isopropanol. In a further Organic compounds such as monoethylene glycol ( MEG ) , embodiment, the one or more three - carbon compounds is acetone , isopropanol (IPA ) and propene are valuable as raw 40 propene. material in the production of products like polyethylene In one preferred embodiment, MEG and acetone are terephthalate ( PET ) resins ( from MEG ) and the plastic co - produced from xylose using a ribulose - 1 - phosphate path polypropylene ( from propene ). These compounds also find way for the conversion of xylose to MEG and a C3 branch use directly for industrial or household purposes . pathway for the conversion of dihydroxyacetone - phosphate However, the compounds are currently produced from 45 ( DHAP ) to acetone . precursors that originate from fossil fuels, which contribute In one aspect , the present application relates to a recom to climate change. To develop more environmentally binant microorganism capable of co - producing monoethyl friendly processes for the production of MEG and three- ene glycol ( MEG ) and acetone from exogenous D - xylose , carbon compounds such as isopropanol, researchers have wherein the recombinant microorganism expresses one or engineered microorganisms with biosynthetic pathways to 50 more of the following : produce MEG or IPA separately . However, these pathways ( a ) at least one endogenous or exogenous nucleic acid are challenging to implement, with loss of product yield , molecule encoding an enzyme that catalyzes the con redox balance and excess biomass formation being some version of D - xylulose to D - ribulose ; major obstacles to overcome . ( b ) at least one endogenous or exogenous nucleic acid Thus there exists a need for improved biosynthesis path- 55 molecule encoding an enzyme that catalyzes the con ways for the production of MEG and three - carbon com version of D - ribulose from ( a ) to D - ribulose- 1 -phos pounds such as IPA . phate ; ( c ) at least one endogenous or exogenous nucleic acid SUMMARY OF THE DISCLOSURE molecule encoding an enzyme that catalyzes the con 60 version of D - ribulose - 1 -phosphate from ( b ) to glyco The present application relates to recombinant microor- laldehyde and dihydroxyacetonephosphate ( DHAP ) ; ganisms having one or more biosynthesis pathways for the ( d ) at least one endogenous or exogenous nucleic acid production of monoethylene glycol and one or more three- molecule encoding an enzyme that catalyzes the con carbon compounds . version of glycolaldehyde from ( c ) to MEG ; The present disclosure provides a combination of an easy 65 ( e ) at least one endogenous or exogenous nucleic acid to implement, high yield C2 branch pathway for MEG molecule encoding an enzyme that catalyzes the con production from xylose with an easy to implement C3 version of acetyl -CoA to acetoacetyl -CoA ; US 10,941,424 B2 3 4 ( f) at least one endogenous or exogenous nucleic acid aldolase is fucA , or homolog thereof. In a further embodi molecule encoding an enzyme that catalyzes the con- ment, the D - ribulose - 1 - phosphate aldolase comprises an version of acetoacetyl- CoA from ( e ) to acetoacetate ; amino acid sequence set forth in SEQ ID NO : 11. In yet a and / or further embodiment, the D - ribulose - 1 - phosphate aldolase is ( g ) at least one endogenous or exogenous nucleic acid 5 encoded by a nucleic acid sequence selected from the group molecule encoding an enzyme that catalyzes the con- consisting of SEQ ID NOs : 9 and 10 . version of acetoacetate from ( f) to acetone ; In one embodiment, the enzyme that catalyzes the con wherein the produced intermediate DHAP is converted to version of glycolaldehyde to MEG is a glycolaldehyde acetyl -CoA through the endogenous glycolysis pathway in reductase or aldehyde reductase . In a further embodiment, the microorganism , and wherein MEG and acetone are 10 the enzyme that catalyzes the conversion of glycolaldehyde co - produced . to MEG is encoded by one or more endogenous nucleic acid In one embodiment, the enzyme that catalyzes the con- molecules . In an alternative embodiment, the enzyme that version of D - xylulose to D - ribulose is a D - tagatose 3 - epi- catalyzes the conversion of glycolaldehyde to MEG is merase . In a further embodiment, the enzyme that catalyzes encoded by one or more exogenous nucleic acid molecules . the conversion of D -xylulose to D - ribulose is encoded by 15 In another embodiment, the enzyme is a glycolaldehyde one or more endogenous nucleic acid molecules . In an reductase or aldehyde reductase that is encoded by a nucleic alternative embodiment, the enzyme that catalyzes the con- acid molecule obtained from a microorganism selected from version of D - xylulose to D - ribulose is encoded by one or E. coli or S. cerevisiae . In some embodiments, the nucleic more exogenous nucleic acid molecules . In another embodi- acid molecule encoding glycolaldehyde reductase or alde ment, the enzyme is a D - tagatose 3 -epimerase that is 20 hyde reductase is selected from fuco , yqhD , dkgA ( yqhE ), encoded by a nucleic acid molecule obtained from a micro- dkgB ( yafB ), yeaE, yghZ , gldA , GRE2 , or homolog thereof. organism selected from Pseudomonas sp . , Mesorhizobium In another embodiment, the one or more nucleic acid mol sp . and Rhodobacter sp . In some embodiments, the nucleic ecules is yqhD . In some embodiments, the yqhD comprises acid molecule encoding D - tagatose 3 - epimerase is obtained a G149E mutation . In a further embodiment , the glycolal from a microorganism selected from Pseudomonas cichorii , 25 dehyde reductase comprises an amino acid sequence Pseudomonas sp . ST - 24 , Mesorhizobium loti and Rhodo- selected from the group consisting of SEQ ID NOs : 13 , 15 , bacter sphaeroides. In some embodiments, the nucleic acid 17 , 20 , 23 , 25 , 28 , 30 and 32. In yet a further embodiment, molecule encoding D -tagatose 3 - epimerase is dte, CIKKR1 , the glycolaldehyde reductase is encoded by a nucleic acid or homolog thereof. In some embodiments, the one or more sequence selected from the group consisting of SEQ ID nucleic acid molecules is FJ851309.1 or homolog thereof. In 30 NOs : 12 , 14 , 16 , 18 , 19 , 21 , 22 , 24 , 26 , 27 , 29 and 31 . a further embodiment, the D - tagatose 3 -epimerase com- In one embodiment, the enzyme that catalyzes the con prises an amino acid sequence selected from the group version of acetyl -CoA to acetoacetyl -CoA is a thiolase or consisting of SEQ ID NOs : 3 and 5. In yet a further acetyl coenzyme A acetyltransferase . In a further embodi embodiment, the D - tagatose 3 -epimerase is encoded by a ment, the enzyme that catalyzes the conversion of acetyl nucleic acid sequence selected from the group consisting of 35 CoA to acetoacetyl - CoA is encoded by one or more endog SEQ ID NOs : 1 , 2 and 4 . enous nucleic acid molecules . In an alternative embodiment, In one embodiment, the enzyme that catalyzes the con- the enzyme that catalyzes the conversion of acetyl- CoA to version of D - ribulose to D - ribulose - 1 - phosphate is a D - ribu- acetoacetyl- CoA is encoded by one or more exogenous lokinase . In a further embodiment, the enzyme that catalyzes nucleic acid molecules . In another embodiment, the enzyme the conversion of D - ribulose to D -ribulose - 1 - phosphate is 40 is a thiolase or acetyl coenzyme A acetyltransferase that is encoded by one or more endogenous nucleic acid molecules . encoded by a nucleic acid molecule obtained from a micro In an alternative embodiment, the enzyme that catalyzes the organism selected from Clostridium sp . , Bacillus sp . , E. coli , conversion of D - ribulose to D - ribulose - 1 - phosphate is Saccharomyces sp . and Marinobacter sp . In some embodi encoded by one or more exogenous nucleic acid molecules . ments, the nucleic acid molecule encoding thiolase or acetyl In another embodiment, the enzyme is a D - ribulokinase that 45 coenzyme A acetyltransferase is obtained from a microor is encoded by a nucleic acid molecule obtained from E. coli . ganism selected from Clostridium acetobutylicum , In some embodiments , the nucleic acid molecule encoding Clostridium thermosaccharolyticum , Bacillus cereus, E. D - ribulokinase is fuck , or homolog thereof. In a further coli, Saccharomyces cerevisiae and Marinobacter hydrocar embodiment, the D - ribulokinase comprises an amino acid bonoclasticus . In some embodiments, the nucleic acid mol sequence set forth in SEQ ID NO : 8. In yet a further 50 ecule encoding thiolase or acetyl coenzyme A acetyltrans embodiment, the D - ribulokinase is encoded by a nucleic ferase is th1A , atoB and / or ERG10 , or homolog thereof. In acid sequence selected from the group consisting of SEQ ID a further embodiment, the thiolase or acetyl coenzyme A NOs : 6 and 7 . acetyltransferase comprises an amino acid sequence selected In one embodiment, the enzyme that catalyzes the con- from the group consisting of SEQ ID NOs : 35 , 37 and 40 . version of D - ribulose - 1 -phosphate to glycolaldehyde and 55 In yet a further embodiment , thethiolase or acetyl coenzyme dihydroxyacetonephosphate (DHAP ) is a D -ribulose - 1- A acetyltransferase is encoded by a nucleic acid sequence phosphate aldolase . In a further embodiment, the enzyme selected from the group consisting of SEQ ID NOs : 33 , 34 , that catalyzes the conversion of D -ribulose - 1 - phosphate to 36 , 38 and 39 . glycolaldehyde and DHAP is encoded by one or more In one embodiment, the enzyme that catalyzes the con endogenoutous nucleic acid molecules . In an alternative 60 version of acetoacetyl - CoA to acetoacetate is an acetate : embodiment, the enzyme that catalyzes the conversion of acetoacetyl- CoA transferase or hydrolase . In some embodi D - ribulose - 1 - phosphate to glycolaldehyde and DHAP is ments, the transferase is an acetyl - CoA :acetoacetate - COA encoded by one or more exogenous nucleic acid molecules . transferase . In a further embodiment, the enzyme that cata In another embodiment, the enzyme is a D -ribulose - 1- lyzes the conversion of acetoacetyl -CoA to acetoacetate is phosphate aldolase that is encoded by a nucleic acid mol- 65 encoded by one or more endogenous nucleic acid molecules . ecule obtained from E. coli . In some embodiments, the In an alternative embodiment , the enzyme that catalyzes the nucleic acid molecule encoding D - ribulose - 1 - phosphate conversion of acetoacetyl - CoA to acetoacetate is encoded by US 10,941,424 B2 5 6 one or more exogenous nucleic acid molecules . In another deletion, insertion , or loss of function mutation in a gene embodiment, the enzyme is an acetate : acetoacetyl -CoA encoding a D - xylulose - 5 - kinase to prevent the conversion of transferase or hydrolase that is encoded by a nucleic acid D - xylulose to D -xylulose - 5 - phosphate and instead shunt the molecule obtained from a microorganism selected from reaction toward conversion of D - xylulose to D -xylulose - 1 Clostridium sp . and E. coli . In some embodiments, the 5 phosphate . nucleic acid molecule encoding acetate : acetoacetyl - CoA In one embodiment, the enzyme that catalyzes the con hydrolase is obtained from Clostridium acetobutylicum . In version of glycolaldehyde to glycolic acid is a glycolalde some embodiments, the nucleic acid molecules encoding hyde dehydrogenase. In some embodiments, the glycolal acetate : acetoacetyl - CoA transferase is obtained from E. coli . dehyde dehydrogenase is from Escherichia coli . In some acetateIn some :acetoacetyl embodiments -CoA , the transferase nucleic acid subunits molecules are encodingato A and 10 embodiments, the glycolaldehyde dehydrogenase is encoded atoD , or homologs thereof. In some embodiments, the by the aldA gene, or homolog thereof. nucleic acid molecules encoding acetate : acetoacetyl - CoA In some embodiments, a recombinant microorganism hydrolase subunits are ctfA and ctfB , or homologs thereof. producing MEG and a three - carbon compound comprises a In a further embodiment, the acetyl - CoA :acetoacetate - CoA 15 deletion , insertion , or loss of function mutation in a gene transferase or acetate :acetoacetyl -CoA hydrolase comprises encoding a glycolaldehyde dehydrogenase to prevent the an amino acid sequence selected from the group consisting production of glycolic acid from glycolaldehyde and instead of SEQ ID NOs : 43 , 46 , 97 , 99 , 101 and 103. In yet a further shunt the reaction toward conversion of glycolaldehyde to embodiment, the acetyl -CoA : acetoacetate - CoA transferase MEG . or acetate : acetoacetyl -CoA hydrolase is encoded by a 20 In one embodiment, the enzyme that catalyzes the con nucleic acid sequence selected from the group consisting of version of pyruvate to lactate is a lactate dehydrogenase . In SEQ ID NOs : 41 , 42 , 44 , 45 , 96 , 98 , 100 and 102 . particular embodiments, the enzyme converts pyruvate to In one embodiment, the enzyme that catalyzes the con- lactate . In some embodiments, the lactate dehydrogenase is version of acetoacetate to acetone is an acetoacetate decar- from Escherichia coli . In some embodiments, the lactate boxylase . In a further embodiment, the enzyme that cata- 25 dehydrogenase is encoded by the IdhA gene , or homolog lyzes the conversion of acetoacetate to acetone is encoded thereof . by one or more endogenous nucleic acid molecules . In an In some embodiments, a recombinant microorganism alternative embodiment, the enzyme that catalyzes the con producing MEG and a three - carbon compound comprises a version of acetoacetate to acetone is encoded by one or more exogenous nucleic acid molecules . In another embodiment, 30 deletion , insertion , or loss of function mutation in a gene the enzyme is an acetoacetate decarboxylase that is encoded encoding a lactate dehydrogenase to prevent the production by a nucleic acid molecule obtained from a microorganism of lactate from pyruvate and instead shunt the reaction selected from Clostridium sp . , Bacillus sp . , Chromobacte toward production of a three - carbon compound. rium sp . and Pseudomonas sp . In some embodiments , the In one embodiment, the recombinant microorganism fur nucleic acid molecule encoding acetoacetate decarboxylase 35 ther comprises an endogenous enzyme that catalyzes the is obtained from a microorganism selected from Clostridium conversion of D - xylose to D - xylulose . acetobutylicum , Clostridium beijerinckii , Clostridium cel In one preferred embodiment, MEG and acetone are lulolyticum , Bacillus polymyxa, Chromobacterium viola co - produced from xylose using a xylulose - 1 - phosphate pathway for the conversion of xylose to MEG and a C3 nucleicceum and acid Pseudomonas molecule encoding putida. acetoacetateIn some embodiments decarboxylase , the 40 branch pathway for the conversion of dihydroxyacetone is adc , or homolog thereof. In a further embodiment, the phosphate ( DHAP ) to acetone . acetoacetate decarboxylase comprises an amino acid In another aspect , the present application relates to a sequence selected from the group consisting of SEQ ID recombinant microorganism capable of co - producing mono NOs : 49 and 52. In yet another embodiment, the acetoac- ethylene glycol ( MEG ) and acetone from exogenous D -xy etate decarboxylase is encoded by a nucleic acid sequence 45 lose , wherein the recombinant microorganism expresses one selected from the group consisting of SEQ ID NOs : 47 , 48 , or more of the following: 50 and 51 . ( a ) at least one endogenous or exogenous nucleic acid In one embodiment, the recombinant microorganism fur molecule encoding an enzyme that catalyzes the con ther comprises one or more modifications selected from the version of D -xylulose to D -xylulose - 1 - phosphate ; group consisting of: 50 ( a ) a deletion, insertion, or loss of function mutation in a ( b ) at least one endogenous or exogenous nucleic acid gene encoding an enzyme that catalyzes the conversion molecule encoding an enzyme that catalyzes the con of D - xylulose to D -xylulose - 5 -phosphate ; version of D - xylulose - 1 - phosphate from ( a ) to glyco ( b ) a deletion, insertion , or loss of function mutation in a laldehyde and dihydroxyacetonephosphate ( DHAP ) ; gene encoding an enzyme that catalyzes the conversion 55 ( c ) at least one endogenous or exogenous nucleic acid of glycolaldehyde to glycolic acid ; and molecule encoding an enzyme that catalyzes the con ( c ) a deletion , insertion , or loss of function mutation in a version of glycolaldehyde from ( b ) to MEG ; gene encoding an enzyme that catalyzes the conversion ( d ) at least one endogenous or exogenous nucleic acid of pyruvate to lactate. molecule encoding an enzyme that catalyzes the con In one embodiment, the enzyme that catalyzes the con- 60 version of acetyl - CoA to acetoacetyl -CoA ; version of D - xylulose to D - xylulose - 5 -phosphate is a D -xy- ( e ) at least one endogenous or exogenous nucleic acid lulose - 5 - kinase . In some embodiments, the D -xylulose -5- molecule encoding an enzyme that catalyzes the con kinase is from Escherichia coli . In some embodiments , the version of acetoacetyl - CoA from ( d ) to acetoacetate ; D - xylulose - 5 - kinase is encoded by the xylB gene, or and / or homolog thereof. 65 ( f) at least one endogenous or exogenous nucleic acid In some embodiments, a recombinant microorganism molecule encoding an enzyme that catalyzes the con producing MEG and a three - carbon compound comprises a version of acetoacetate from ( e ) to acetone ; US 10,941,424 B2 7 8 wherein the produced intermediate DHAP is converted to selected from the group consisting of SEQ ID NOs : 13 , 15 , acetyl- CoA through the endogenous glycolysis pathway in 17 , 20 , 23 , 25 , 28 , 30 and 32. In yet a further embodiment, the microorganism , and wherein MEG and acetone are the glycolaldehyde reductase is encoded by a nucleic acid co - produced sequence selected from the group consisting of SEQ ID In one embodiment, the enzyme that catalyzes the con- 5 NOs : 12 , 14 , 16 , 18 , 19 , 21 , 22 , 24 , 26 , 27 , 29 and 31 . version of D -xylulose to D -xylulose - 1 -phosphate is a D -xy- In one embodiment, the enzyme that catalyzes the con lulose 1 - kinase . In a further embodiment, the enzyme that version of acetyl - CoA to acetoacetyl -CoA is a thiolase or catalyzes the conversion of D - xylulose to D -xylulose - 1- acetyl coenzyme A acetyltransferase . In a further embodi phosphate is encoded by one or more endogenous nucleic ment, the enzyme that catalyzes the conversion of acetyl acid molecules . In an alternative embodiment, the enzyme 10 CoA to acetoacetyl -CoA is encoded by one or more endog that catalyzes the conversion of D - xylulose to D - xylulose- enous nucleic acid molecules . In an alternative embodiment, 1 -phosphate is encoded by one or more exogenous nucleic the enzyme that catalyzes the conversion of acetyl- CoA to acid molecules . In another embodiment, the enzyme is a acetoacetyl -CoA is encoded by one or more exogenous D - xylulose 1 -kinase that is encoded by a nucleic acid nucleic acid molecules . In another embodiment, the enzyme molecule obtained from Homo sapiens. In one embodiment, 15 is a thiolase or acetyl coenzyme A acetyltransferase that is the Homo sapiens D -xylulose 1 -kinase is a ketohexokinase encoded by a nucleic acid molecule obtained from a micro C. In some embodiments , the nucleic acid molecule encod- organism selected from Clostridium sp . , Bacillus sp . , E. coli , ing human ketohexokinase C is khk - C , or homolog thereof. Saccharomyces sp . and Marinobacter sp . In some embodi In another embodiment, the one or more nucleic acid mol- ments , the nucleic acid molecule encoding thiolase or acetyl ecules encoding the D - xylulose 1 - kinase comprises an 20 coenzyme A acetyltransferase is obtained from a microor amino acid sequence set forth in SEQ ID NO : 55. In a further ganism selected from Clostridium acetobutylicum , embodiment, the one or more nucleic acid molecules encod- Clostridium thermosaccharolyticum , Bacillus cereus, E. ing the D - xylulose 1 - kinase is encoded by a nucleic acid coli, Saccharomyces cerevisiae and Marinobacter hydrocar sequence selected from the group consisting of SEQ ID bonoclasticus . In some embodiments, the nucleic acid mol NOs : 53 and 54 . 25 ecule encoding thiolase or acetyl coenzyme A acetyltrans In one embodiment, the enzyme that catalyzes the con- ferase is th1A , atoB and / or ERG10 , or homolog thereof. In version of D -xylulose - 1 - phosphate to glycolaldehyde and a further embodiment, the thiolase or acetyl coenzyme A dihydroxyacetonephosphate (DHAP ) is a D - xylulose - 1- acetyltransferase comprises an amino acid sequence selected phosphate aldolase . In a further embodiment, the enzyme from the group consisting of SEQ ID NOs : 35 , 37 and 40 . that catalyzes the conversion of D - xylulose - 1 - phosphate to 30 In yet a further embodiment, the thiolase or acetyl coenzyme glycolaldehyde and DHAP is encoded by one or more A acetyltransferase is encoded by a nucleic acid sequence endogenous nucleic acid molecules . In an alternative selected from the group consisting of SEQ ID NOs : 33 , 34 , embodiment, the enzyme that catalyzes the conversion of 38 and 39 . D - xylulose - 1 - phosphate to glycolaldehyde and DHAP is In one embodiment, the enzyme that catalyzes the con encoded by one or more exogenous nucleic acid molecules . 35 version of acetoacetyl - CoA to acetoacetate is a acetate : In another embodiment, the enzyme is a D -xylulose - 1- acetoacetyl- CoA transferase or hydrolase . In some embodi phosphate aldolase that is encoded by a nucleic acid mol- ments, the transferase is an acetyl -CoA :acetoacetate - CoA ecule obtained from Homo sapiens . In one embodiment, the transferase. In a further embodiment, the enzyme that cata Homo sapiens D -xylulose 1 - phosphate aldolase is an aldo- lyzes the conversion of acetoacetyl -CoA to acetoacetate is lase B. some embodiments , the nucleic acid molecule 40 encoded by one or more endogenous nucleic acid molecules . encoding human aldolase B is ALDOB , or homolog thereof. In an alternative embodiment, the enzyme that catalyzes the In some embodiments, the one or more nucleic acid mol- conversion of acetoacetyl - CoA to acetoacetate is encoded by ecules encoding the D -xylulose - 1 - phosphate aldolase com- one or more exogenous nucleic acid molecules . In another prises an amino acid sequence set forth in SEQ ID NO : 58 . embodiment, the enzyme is an acetate : acetoacetyl - CoA In some embodiments, the one or more nucleic acid mol- 45 transferase or hydrolase that is encoded by a nucleic acid ecules encoding the D - xylulose - 1 - phosphate aldolase is molecule obtained from a microorganism selected from encoded by a nucleic acid sequence selected from the group Clostridium sp . and E. coli . In some embodiments, the consisting of SEQ ID NOs : 56 and 57 . nucleic acid molecule encoding acetate : acetoacetyl- CoA In one embodiment, the enzyme that catalyzes the con- hydrolase is obtained from Clostridium acetobutylicum . In version of glycolaldehyde to MEG is a glycolaldehyde 50 some embodiments, the nucleic acid molecules encoding reductase or aldehyde reductase . In a further embodiment, acetate :acetoacetyl - CoA transferase is obtained from E. coli . the enzyme that catalyzes the conversion of glycolaldehyde In some embodiments, the nucleic acid molecules encoding to MEG is encoded by one or more endogenous nucleic acid acetate : acetoacetyl - CoA transferase subunits are ato A and molecules . In an alternative embodiment, the enzyme that atoD , or homologs thereof. In some embodiments, the catalyzes the conversion of glycolaldehyde to MEG is 55 nucleic acid molecules encoding acetate : acetoacetyl- CoA encoded by one or more exogenous nucleic acid molecules . hydrolase subunits are ctfA and ctfB , or homologs thereof. In another embodiment, the enzyme is a glycolaldehyde In a further embodiment, the acetyl -CoA : acetoacetate - COA reductase or aldehyde reductase that is encoded by a nucleic transferase or acetate :acetoacetyl - CoA hydrolase comprises acid molecule obtained from a microorganism selected from an amino acid sequence selected from the group consisting E. coli or S. cerevisiae. In some embodiments, the nucleic 60 of SEQ ID NOs : 43 , 46 , 97 , 99 , 101 and 103. In yet a further acid molecule encoding glycolaldehyde reductase or alde- embodiment, the acetyl- CoA : acetoacetate - CoA transferase hyde reductase is selected from fuco , yqhD , dkg A ( yqhE ), or acetate : acetoacetyl - CoA hydrolase is encoded by a dkgB ( yafB ), yeaE, yghZ , gldA , GRE2 , or homolog thereof. nucleic acid sequence selected from the group consisting of In another embodiment, the one or more nucleic acid mol- SEQ ID NOs : 41 , 42 , 44 , 45 , 96 , 98 , 100 and 102 . ecules is yqhD . In some embodiments, the yqhD comprises 65 In one embodiment, the enzyme that catalyzes the con a G149E mutation . In a further embodiment, the glycolal- version of acetoacetate to acetone is an acetoacetate decar dehyde reductase comprises an amino acid sequence boxylase . In a further embodiment, the enzyme that cata US 10,941,424 B2 9 10 lyzes the conversion of acetoacetate to acetone is encoded In some embodiments, a recombinant microorganism by one or more endogenous nucleic acid molecules . In an producing MEG and a three - carbon compound comprises a alternative embodiment, the enzyme that catalyzes the con- deletion , insertion , or loss of function mutation in a gene version of acetoacetate to acetone is encoded by one or more encoding a lactate dehydrogenase to prevent the production exogenous nucleic acid molecules . In another embodiment, 5 of lactate from pyruvate and instead shunt the reaction the enzyme is an acetoacetate decarboxylase that is encoded toward production of a three - carbon compound . by a nucleic acid molecule obtained from a microorganism In one embodiment, the recombinant microorganism fur selected from Clostridium sp . , Bacillus sp . , Chromobacte- ther comprises an endogenous enzyme that catalyzes the rium sp . and Pseudomonas sp . In some embodiments , the conversion of D - xylose to D - xylulose . nucleic acid molecule encoding acetoacetate decarboxylase 10 In another aspect , the present application relates to a is obtained from a microorganism selected from Clostridium recombinant microorganism capable of co -producing mono acetobutylicum , Clostridium beijerinckii, Clostridium cel- ethylene glycol ( MEG ) and acetone from exogenous D -xy lulolyticum , Bacillus polymyxa, Chromobacterium viola- lose and glucose , wherein the recombinant microorganism ceum and Pseudomonas putida . In some embodiments, the expresses one or more of the following: nucleic acid molecule encoding acetoacetate decarboxylase 15 ( a ) at least one exogenous nucleic acid molecule encoding is adc , or homolog thereof . In a further embodiment, the an enzyme that catalyzes the conversion of D - xylose to acetoacetate decarboxylase comprises an amino acid xylitol and at least one exogenous nucleic acid mol sequence selected from the group consisting of SEQ ID ecule encoding an enzyme that catalyzes the conversion NOs : 49 and 52. In yet another embodiment, the acetoac of xylitol to D - xylulose; etate decarboxylase is encoded by a nucleic acid sequence 20 ( b ) at least one exogenous nucleic acid molecule encoding selected from the group consisting of SEQ ID NOs : 47 , 48 , an enzyme that catalyzes the conversion of D - xylose to 50 and 51 . D - xylulose , and In one embodiment, the recombinant microorganism fur- wherein the microorganism further expresses one or more of ther comprises one or more modifications selected from the the following: group consisting of: 25 ( c ) at least one endogenous or exogenous nucleic acid ( a ) a deletion , insertion , or loss of function mutation in a molecule encoding a D - tagatose 3 - epimerase that cata gene encoding an enzyme that catalyzes the conversion lyzes the conversion of D - xylulose from ( a ) or ( b ) to of D -xylulose to D - xylulose - 5 - phosphate ; D - ribulose ; ( b ) a deletion , insertion , or loss of function mutation in a ( d ) at least one endogenous or exogenous nucleic acid gene encoding an enzyme that catalyzes the conversion 30 molecule encoding a D - ribulokinase that catalyzes the of glycolaldehyde to glycolic acid ; and conversion of D - ribulose from ( c ) to D - ribulose - 1 ( c ) a deletion , insertion, or loss of function mutation in a phosphate ; gene encoding an enzyme that catalyzes the conversion ( e ) at least one endogenous or exogenous nucleic acid of pyruvate to lactate . molecule encoding a D - ribulose - 1 - phosphate aldolase In one embodiment, the enzyme that catalyzes the con- 35 that catalyzes the conversion of D - ribulose - 1 -phos version of D - xylulose to D - xylulose - 5 - phosphate is a D -xy- phate from ( d ) to glycolaldehyde and dihydroxyaceto lulose - 5 - kinase . In some embodiments, the D -xylulose - 5 nephosphate ( DHAP ); kinase is from Escherichia coli . In some embodiments, the ( f) at least one endogenous or exogenous nucleic acid D - xylulose - 5 -kinase is encoded by the xylB gene , or molecule encoding a glycolaldehyde reductase or homolog thereof. 40 methylglyoxal reductase that catalyzes the conversion In some embodiments , a recombinant microorganism of glycolaldehyde from ( e ) to MEG ; producing MEG and a three -carbon compound comprises a ( g ) at least one endogenous or exogenous nucleic acid deletion , insertion , or loss of function mutation in a gene molecule encoding a thiolase that catalyzes the con encoding a D - xylulose - 5 - kinase to prevent the conversion of version of acetyl -CoA to acetoacetyl -CoA ; D - xylulose to D -xylulose - 5 - phosphate and instead shunt the 45 ( h ) at least one endogenous or exogenous nucleic acid reaction toward conversion of D - xylulose to D - xylulose - 1 molecule encoding an acetate : acetoacetyl - CoA trans phosphate . ferase or hydrolase that catalyzes the conversion of In one embodiment, the enzyme that catalyzes the con- acetoacetyl- CoA from ( g ) to acetoacetate; and / or version of glycolaldehyde to glycolic acid is a glycolalde- ( i ) at least one endogenous or exogenous nucleic acid hyde dehydrogenase. In some embodiments, the glycolal- 50 molecule encoding an acetoacetate decarboxylase that dehyde dehydrogenase is from Escherichia coli . In some catalyzes the conversion of acetoacetate from ( h ) to embodiments, the glycolaldehyde dehydrogenase is encoded acetone ; by the aldA gene , or homolog thereof. wherein the produced intermediate DHAP is converted to In some embodiments, a recombinant microorganism acetyl - CoA through the endogenous glycolysis pathway in producing MEG and a three - carbon compound comprises a 55 the microorganism , and wherein MEG and acetone are deletion , insertion , or loss of function mutation in a gene co - produced . encoding a glycolaldehyde dehydrogenase to prevent the In one embodiment, the enzyme that catalyzes the con production of glycolic acid from glycolaldehyde and instead version of D - xylose to xylitol is a xylose reductase or aldose shunt the reaction toward conversion of glycolaldehyde to reductase . In a further embodiment, the enzyme that cata MEG . 60 lyzes the conversion of D - xylose to xylitol is encoded by one In one embodiment, the enzyme that catalyzes the con- or more endogenous nucleic acid molecules . In an alterna version of pyruvate to lactate is a lactate dehydrogenase. In tive embodiment, the enzyme that catalyzes the conversion particular embodiments , the enzyme converts pyruvate to of D - xylose to xylitol is encoded by one or more exogenous lactate . In some embodiments, the lactate dehydrogenase is nucleic acid molecules . In another embodiment , the enzyme from Escherichia coli . In some embodiments , the lactate 65 is a xylose reductase or aldose reductase that is encoded by dehydrogenase is encoded by the IdhA gene , or homolog a nucleic acid molecule obtained from a microorganism thereof. selected from Hypocrea sp . , Scheffersomyces sp . , Saccha US 10,941,424 B2 11 12 romyces sp . , Pachysolen sp . , Pichia sp . , Candida sp . , Asper- In one embodiment, the enzyme that catalyzes the con gillus sp . , Neurospora sp . , and Cryptococcus sp . In some version of D - xylulose to D - ribulose is a D - tagatose 3 -epi embodiments, the nucleic acid molecule encoding the xylose merase . In a further embodiment, the enzyme that catalyzes reductase or aldose reductase is obtained from a microor- the conversion of D - xylulose to D - ribulose is encoded by ganism selected from Hypocrea jecorina , Scheffersomyces 5 one or more endogenous nucleic acid molecules . In an stipitis, S. cerevisiae, Pachysolen tannophilus, Pichia stipi- alternative embodiment, the enzyme that catalyzes the con tis, Pichia quercuum , Candida shehatae , Candida tenuis, version of D - xylulose to D - ribulose is encoded by one or Candida tropicalis, Aspergillus niger, Neurospora crassa more exogenous nucleic acid molecules . In another embodi and Cryptococcus lactativorus. In some embodiments, the ment, the enzyme is a D - tagatose 3 -epimerase that is nucleic acid molecule encoding xylose reductase or aldose 10 encoded by a nucleic acid molecule obtained from a micro reductase is xyli , GRE3, or homolog thereof. In some organism selected from Pseudomonas sp . , Mesorhizobium embodiments , the one or more nucleic acid molecules sp . and Rhodobacter sp . In some embodiments , the nucleic encoding the xylose reductase or aldose reductase comprises acid molecule encoding D - tagatose 3 - epimerase is obtained an amino acid sequence selected from the group consisting from a microorganism selected from Pseudomonas cichorii , of SEQ ID NOs : 84 and 87. In some embodiments , the one 15 Pseudomonas sp . ST - 24 , Mesorhizobium loti and Rhodo or more nucleic acid molecules encoding the xylose bacter sphaeroides. In some embodiments, the nucleic acid reductase or aldose reductase is encoded by a nucleic acid molecule encoding D - tagatose 3 - epimerase is dte, C1KKR1 , sequence selected from the group consisting of SEQ ID or homolog thereof. In some embodiments, the one or more NOs : 82 , 83 , 85 and 86 . nucleic acid molecules is FJ851309.1 or homolog thereof . In In one embodiment, the enzyme that catalyzes the con- 20 a further embodiment, the D - tagatose 3 - epimerase com version of xylitol to D - xylulose is a xylitol dehydrogenase. prises an amino acid sequence selected from the group In a further embodiment, the enzyme that catalyzes the consisting of SEQ ID NOs : 3 and 5. In yet a further conversion of xylitol to D - xylulose is encoded by one or embodiment, the D - tagatose 3 -epimerase is encoded by a more endogenous nucleic acid molecules . In an alternative nucleic acid sequence selected from the group consisting of embodiment, the enzyme that catalyzes the conversion of 25 SEQ ID NOs : 1 , 2 and 4 . xylitol to D -xylulose is encoded by one or more exogenous In one embodiment, the enzyme that catalyzes the con nucleic acid molecules . In another embodiment, the enzyme version of D -ribulose to D - ribulose - 1 -phosphate is a D -ribu is a xylitol dehydrogenase that is encoded by a nucleic acid lokinase . In a further embodiment, the enzyme that catalyzes molecule obtained from a microorganism selected from the conversion of D - ribulose to D - ribulose - 1 -phosphate is Scheffersomyces sp . , Trichoderma sp . , Pichia sp . , Saccha- 30 encoded by one or more endogenous nucleic acid molecules . romyces sp . , Gluconobacter sp . , Galactocandida sp . , Neu- In an alternative embodiment, the enzyme that catalyzes the rospora sp . , and Serratia sp . In some embodiments, the conversion of D - ribulose to D - ribulose - 1 -phosphate is nucleic acid molecule encoding the xylitol dehydrogenase is encoded by one or more exogenous nucleic acid molecules . obtained from a microorganism selected from Scheffersomy- In another embodiment, the enzyme is a D - ribulokinase that ces stipitis, Trichoderma reesei, Pichia stipitis, S. cerevisiae, 35 is encoded by a nucleic acid molecule obtained from E. coli . Gluconobacter oxydans, Galactocandida mastotermitis , In some embodiments, the nucleic acid molecule encoding Neurospora crassa and Serratia marcescens . In some D - ribulokinase is fuck , or homolog thereof. In a further embodiments , the one or more nucleic acid molecule encod- embodiment, the D - ribulokinase comprises an amino acid ing xylitol dehydrogenase is xy12 , xdh1, or homolog thereof. sequence set forth in SEQ ID NO : 8. In yet a further In some embodiments, the one or more nucleic acid mol- 40 embodiment, the D - ribulokinase is encoded by a nucleic ecules encoding the xylitol dehydrogenase comprises an acid sequence selected from the group consisting of SEQ ID amino acid sequence selected from the group consisting of NOs : 6 and 7 . SEQ ID NOs : 90 and 92. In some embodiments , the one or In one embodiment, the enzyme that catalyzes the con more nucleic acid molecules encoding the xylitol dehydro- version of D - ribulose - 1 - phosphate to glycolaldehyde and genase is encoded by a nucleic acid sequence selected from 45 dihydroxyacetonephosphate ( DHAP ) is a D - ribulose- 1 the group consisting of SEQ ID NOs : 88 , 89 and 91 . phosphate aldolase . In a further embodiment, the enzyme In one embodiment, the enzyme that catalyzes the con- that catalyzes the conversion of D - ribulose - 1 - phosphate to version of D -xylose to D - xylulose is a D - xylose isomerase . glycolaldehyde and DHAP is encoded by one or more In a further embodiment, the enzyme that catalyzes the endogenous nucleic acid molecules . In an alternative conversion of D - xylose to D -xylulose is encoded by one or 50 embodiment, the enzyme that catalyzes the conversion of more endogenous nucleic acid molecules . In an alternative D -ribulose - 1 -phosphate to glycolaldehyde and DHAP is embodiment, the enzyme that catalyzes the conversion of encoded by one or more exogenous nucleic acid molecules . D - xylose to D - xylulose is encoded by one or more exog- In another embodiment, the enzyme is a D - ribulose - 1 enous nucleic acid molecules . In another embodiment, the phosphate aldolase that is encoded by a nucleic acid mol enzyme is a D - xylose isomerase that is encoded by a nucleic 55 ecule obtained from E. coli . In some embodiments, the acid molecule obtained from E. coli . In another embodi- nucleic acid molecule encoding D - ribulose - 1 -phosphate ment, the xylose isomerase is encoded by one or more aldolase is fucA , or homolog thereof. In a further embodi nucleic acid molecules obtained from Pyromyces sp . In ment, the D - ribulose - 1 - phosphate aldolase comprises an some embodiments, the nucleic acid molecule encoding amino acid sequence set forth in SEQ ID NO : 11. In yet a D - xylose isomerase is xyla , or homolog thereof. In yet 60 further embodiment, the D -ribulose - 1 - phosphate aldolase is another embodiment, the one or more nucleic acid mol- encoded by a nucleic acid sequence selected from the group ecules encoding the xylose isomerase comprises an amino consisting of SEQ ID NOs : 9 and 10 . acid sequence set forth in SEQ ID NO : 95. In a further In one embodiment, the enzyme that catalyzes the con embodiment, the one or more nucleic acid molecules encod- version of glycolaldehyde to MEG is a glycolaldehyde ing the xylose isomerase is encoded by a nucleic acid 65 reductase or aldehyde reductase . In a further embodiment, sequence selected from the group consisting of SEQ ID the enzyme that catalyzes the conversion of glycolaldehyde NOs : 93 and 94 . to MEG is encoded by one or more endogenous nucleic acid US 10,941,424 B2 13 14 molecules . In an alternative embodiment, the enzyme that ase subunits are atoA and atoD , or homologs thereof. In catalyzes the conversion of glycolaldehyde to MEG is some embodiments, the nucleic acid molecules encoding encoded by one or more exogenous nucleic acid molecules . acetate :acetoacetyl -CoA hydrolase subunits are ctfA and In another embodiment, the enzyme is a glycolaldehyde ctfB , or homologs thereof. In a further embodiment, the reductase or aldehyde reductase that is encoded by a nucleic 5 acetyl- CoA : acetoacetate - CoA transferase or acetate : ac acid molecule obtained from a microorganism selected from etoacetyl -CoA hydrolase comprises an amino acid sequence E. coli or S. cerevisiae . In some embodiments , the nucleic acid molecule encoding glycolaldehyde reductase or alde selected from the group consisting of SEQ ID NOs : 43 , 46 , hyde reductase is selected from fuco , yqhD , dkgA ( yqhE ), 97 , 99 , 101 and 103. In yet a further embodiment, the dkgB ( yafB ), yeaE , yghZ , gldA , GRE2 , or homolog thereof. 10 acetyl - CoA :acetoacetate - CoA transferase or acetate :ac In another embodiment, the one or more nucleic acid mol etoacetyl - CoA hydrolase is encoded by a nucleic acid ecules is yqhD . In some embodiments, the yqhD comprises sequence selected from the group consisting of SEQ ID a G149E mutation . In a further embodiment, the glycolal NOs : 41 , 42 , 44 , 45 , 96 , 98 , 100 and 102 . dehyde reductase comprises an amino acid sequence In one embodiment, the enzyme that catalyzes the con selected from the group consisting of SEQ ID NOs : 13 , 15 , 15 version of acetoacetate to acetone is an acetoacetate decar 17 , 20 , 23 , 25 , 28 , 30 and 32. In yet a further embodiment, boxylase . In a further embodiment, the enzyme that cata the glycolaldehyde reductase is encoded by a nucleic acid lyzes the conversion of acetoacetate to acetone is encoded sequence selected from the group consisting of SEQ ID by one or more endogenous nucleic acid molecules . In an NOs : 12 , 14 , 16 , 18 , 19 , 21 , 22 , 24 , 26 , 27 , 29 and 31 . alternative embodiment, the enzyme that catalyzes the con In one embodiment, the enzyme that catalyzes the con- 20 version of acetoacetate to acetone is encoded by one or more version of acetyl -CoA to acetoacetyl - CoA is a thiolase or exogenous nucleic acid molecules . In another embodiment, acetyl coenzyme A acetyltransferase. In a further embodi- the enzyme is an acetoacetate decarboxylase that is encoded ment, the enzyme that catalyzes the conversion of acetyl- by a nucleic acid molecule obtained from a microorganism COA to acetoacetyl -CoA is encoded by one or more endog- selected from Clostridium sp . , Bacillus sp . , Chromobacte enous nucleic acid molecules . In an alternative embodiment, 25 rium sp . and Pseudomonas sp . In some embodiments , the the enzyme that catalyzes the conversion of acetyl- CoA to nucleic acid molecule encoding acetoacetate decarboxylase acetoacetyl- CoA is encoded by one or more exogenous is obtained from a microorganism selected from Clostridium nucleic acid molecules . In another embodiment, the enzyme acetobutylicum , Clostridium beijerinckii, Clostridium cel is a thiolase or acetyl coenzyme A acetyltransferase that is lulolyticum , Bacillus polymyxa, Chromobacterium viola encoded by a nucleic acid molecule obtained from a micro- 30 ceum and Pseudomonas putida . In some embodiments , the organism selected from Clostridium sp . , Bacillus sp . , E. coli, nucleic acid molecule encoding acetoacetate decarboxylase Saccharomyces sp . and Marinobacter sp . In some embodi is adc , or homolog thereof. In a further embodiment, the ments , the nucleic acid molecule encoding thiolase or acetyl acetoacetate decarboxylase comprises an amino acid ganismcoenzyme selectedA acetyltransferase from Clostridium is obtained fromacetobutylicum a microor , 35 sequence selected from the group consisting of SEQ ID Clostridium thermosaccharolyticum , Bacillus cereus, E. NOs : 49 and 52. In yet another embodiment, the acetoac coli , Saccharomyces cerevisiae and Marinobacter hydrocar etate decarboxylase is encoded by a nucleic acid sequence bonoclasticus. In some embodiments, the nucleic acid mol selected from the group consisting of SEQ ID NOs : 47 , 48 , ecule encoding thiolase or acetyl coenzyme A acetyltrans 50 and 51 . ferase is th1A , atoB and / or ERG10 , or homolog thereof. In 40 In one embodiment, the recombinant microorganism fur a further embodiment, the thiolase or acetyl coenzyme A ther comprises one or more modifications selected from the acetyltransferase comprises an amino acid sequence selected group consisting of : from the group consisting of SEQ ID NOs : 35 , 37 and 40 . ( a ) a deletion , insertion , or loss of function mutation in a In yet a further embodiment, the thiolase or acetyl coenzyme gene encoding an enzyme that catalyzes the conversion A acetyltransferase is encoded by a nucleic acid sequence 45 of D - xylulose to D - xylulose - 5 - phosphate; and selected from the group consisting of SEQ ID NOs: 33 , 34 , ( b ) a deletion, insertion , or loss of function mutation in a 36 , 38 and 39 . gene encoding an enzyme that catalyzes the conversion In one embodiment, the enzyme that catalyzes the con- of D -xylulose - 5 -phosphate to D -xylulose . version of acetoacetyl -CoA to acetoacetate is a acetate : In one embodiment, the enzyme that catalyzes the con acetoacetyl - CoA transferase or hydrolase. In some embodi- 50 version of D - xylulose to D -xylulose - 5 - phosphate is a D -xy ments , the transferase is an acetyl -CoA :acetoacetate - CoA lulose - 5 - kinase . In some embodiments , the D - xylulose - 5 transferase. In a further embodiment, the enzyme that cata- kinase is from Saccharomyces cerevisiae . In some lyzes the conversion of acetoacetyl -CoA to acetoacetate is embodiments the D -xylulose - 5 -kinase is encoded by the encoded by one or more endogenous nucleic acid molecules . XKS1 gene , or homolog thereof. In some embodiments, the In an alternative embodiment, the enzyme that catalyzes the 55 D - xylulose - 5 - kinase is from Pichia stipitis. In some embodi conversion of acetoacetyl - CoA to acetoacetate is encoded by ments the D - xylulose - 5 - kinase is encoded by the XYL3 one or more exogenous nucleic acid molecules . In another gene , or homolog thereof. embodiment, the enzyme is an acetate :acetoacetyl -CoA In some embodiments, a recombinant microorganism transferase or hydrolase that is encoded by one or more producing MEG and a three - carbon compound comprises a nucleic acid molecule obtained from a microorganism 60 deletion, insertion, or loss of function mutation in a gene selected from Clostridium sp . and E. coli . In some embodi- encoding a D -xylulose - 5 - kinase to prevent the conversion of ments , the nucleic acid molecules encoding acetate :ac- D - xylulose to D -xylulose - 5 - phosphate and instead shunt the etoacetyl- CoA hydrolase is obtained from Clostridium reaction toward conversion of D - xylulose to D - xylulose -1 acetobutylicum . In some embodiments, the nucleic acid phosphate . molecules encoding acetate : acetoacetyl -CoA transferase is 65 In one embodiment, the enzyme that catalyzes the con obtained from E. coli . In some embodiments, the nucleic version of D - xylulose - 5 -phosphate to D - xylulose is an alka acid molecules encoding acetate : acetoacetyl -CoA transfer- line phosphatase . In some embodiments , the alkaline phos US 10,941,424 B2 15 16 phatase is from S. cerevisiae. In some embodiments, the Haloferax volcanii, Halorubrum lacusprofundi and Tricho alkaline phosphatase is encoded by the PHO13 gene , or derma reesei. In some embodiments, the nucleic acid mol homolog thereof. ecule encoding xylose dehydrogenase is selected from xylB , In some embodiments , a recombinant microorganism xdh ( HVO_B0028 ) , xydl, or homolog thereof. In a further producing MEG and a three -carbon compound comprises a 5 embodiment, the one or more nucleic acid molecules encod deletion , insertion, or loss of function mutation in a gene ing the xylose dehydrogenase comprises an amino acid encoding an alkaline phosphatase to prevent the production sequence selected from the group consisting of SEQ ID of D - xylulose from D - xylulose - 5 - phosphate . NOs : 61 , 63 and 65. In yet another embodiment, the one or In one embodiment, the recombinant microorganism more nucleic acid molecules encoding the xylose dehydro capable of co - producing monoethylene glycol ( MEG ) and 10 genase is encoded by a nucleic acid sequence selected from acetone from exogenous D - xylose and glucose is a fungus. the group consisting of SEQ ID NOs : 59 , 60 , 62 and 64 . In one preferred embodiment, MEG and acetone are In one embodiment, the enzyme that catalyzes the con co -produced from xylose using a xylonate pathway for the version of D - xylonolactone to D - xylonate is a xylonolacto conversion of xylose to MEG and a C3 branch pathway for nase . In a further embodiment, the enzyme that catalyzes the the conversion of dihydroxyacetone - phosphate ( DHAP ) to 15 conversion of D - xylonolactone to D - xylonate is encoded by acetone . one or more endogenous nucleic acid molecules . In an In another aspect , the present application relates to a alternative embodiment, the enzyme that catalyzes the con recombinant microorganism capable of co - producing mono- version of D - xylonolactone to D -xylonate is encoded by one ethylene glycol ( MEG ) and acetone from exogenous D -xy- or more exogenous nucleic acid molecules . In another lose , wherein the recombinant microorganism expresses one 20 embodiment, the enzyme is a xylonolactonase that is or more of the following: encoded by a nucleic acid molecule obtained from a micro ( a ) at least one endogenous or exogenous nucleic acid organism selected from Caulobacter sp . and Haloferax sp . molecule encoding an enzyme that catalyzes the con- In some embodiments, the nucleic acid molecule encoding version of D - xylose to D - xylonolactone ; the xylonolactonase is obtained from a microorganism ( b ) at least one endogenous or exogenous nucleic acid 25 selected from Caulobacter crescentus, Haloferax volcanii molecule encoding an enzyme that catalyzes the con- and Haloferax gibbonsii . In some embodiments, the nucleic version of D - xylonolactone from ( a ) to D - xylonate ; acid molecule encoding xylonolactonase is xylc , or ( c ) at least one endogenous or exogenous nucleic acid homolog thereof. In a further embodiment, the one or more molecule encoding an enzyme that catalyzes the con- nucleic acid molecules encoding the xylonolactonase com version of D - xylonate from ( b ) to 2 - keto - 3 - deoxy- 30 prises an amino acid sequence set forth in SEQ ID NO : 67 . xylonate ; In yet another embodiment, the one or more nucleic acid ( d ) at least one endogenous or exogenous nucleic acid molecules encoding the xylonolactonase is encoded by a molecule encoding an enzyme that catalyzes the con- nucleic acid sequence set forth in SEQ ID NO : 66 . version of 2 -keto - 3 - deoxy - xylonate from ( c ) to glyco- In one embodiment, the enzyme that catalyzes the con laldehyde and pyruvate; 35 version of D -xylonate to 2 - keto - 3 - deoxy -xylonate is a ( e ) at least one endogenous or exogenous nucleic acid xylonate dehydratase . In a further embodiment, the enzyme molecule encoding an enzyme that catalyzes the con- that catalyzes the conversion of D - xylonate to 2 -keto - 3 version of glycolaldehyde from ( d ) to MEG ; deoxy - xylonate is encoded by one or more endogenous ( f) at least one exogenous nucleic acid molecule encoding nucleic acid molecules . In an alternative embodiment, the an enzyme that catalyzes the conversion of acetyl - CoA 40 enzyme that catalyzes the conversion of D -xylonate to to acetoacetyl -CoA ; 2 -keto - 3 -deoxy -xylonate is encoded by one or more exog ( g ) at least one endogenous or exogenous nucleic acid enous nucleic acid molecules . In another embodiment, the molecule encoding an enzyme that catalyzes the con- enzyme is a xylonate dehydratase that is encoded by a version of acetoacetyl- CoA from ( f) to acetoacetate ; nucleic acid molecule obtained from a microorganism and /or 45 selected from Caulobacter sp . , Haloferax sp . , Sulfolobus sp . ( h ) at least one exogenous nucleic acid molecule encoding and E. coli . In some embodiments , the nucleic acid molecule an enzyme that catalyzes the conversion of acetoacetate encoding the xylonate dehydratase is obtained from a micro from ( g ) to acetone ; organism selected from Caulobacter crescentus, Haloferax wherein the produced intermediate pyruvate is converted to volcanii , E. coli and Suffolobus soffataricus. In some acetyl - CoA through the endogenous glycolysis pathway in 50 embodiments , the nucleic acid molecule encoding xylonate the microorganism , and wherein MEG and acetone are dehydratase is selected from xylD , yjhG , yagF , xad , or co - produced . homolog thereof. In a further embodiment, the one or more In one embodiment, the enzyme that catalyzes the con- nucleic acid molecules encoding the xylonate dehydratase version of D - xylose to D - xylonolactone is a xylose dehy- comprises an amino acid sequence selected from the group drogenase. In a further embodiment, the enzyme that cata- 55 consisting of SEQ ID NOs : 69 , 72 and 75. In yet another lyzes the conversion of D -xylose to D -xylonolactone is embodiment, the one or more nucleic acid molecules encod encoded by one or more endogenous nucleic acid molecules . ing the xylonate dehydratase is encoded by a nucleic acid In an alternative embodiment, the enzyme that catalyzes the sequence selected from the group consisting of SEQ ID conversion of D - xylose to D -xylonolactone is encoded by NOs : 68 , 70 , 71 , 73 and 74 . one or more exogenous nucleic acid molecules . In another 60 In one embodiment, the enzyme that catalyzes the con embodiment, the enzyme is a xylose dehydrogenase that is version of 2 -keto - 3 -deoxy -xylonate to glycolaldehyde and encoded by a nucleic acid molecule obtained from a micro- pyruvate is a 2 -keto - 3 -deoxy - D -pentonate aldolase . In a organism selected from Caulobacter sp . , Haloarcula sp . , further embodiment, the enzyme that catalyzes the conver Haloferax sp . , Halorubrum sp . and Trichoderma sp . In some sion of 2 - keto - 3 - deoxy -xylonate to glycolaldehyde and embodiments, the nucleic acid molecule encoding the xylose 65 pyruvate is encoded by one or more endogenous nucleic acid dehydrogenase is obtained from a microorganism selected molecules . In an alternative embodiment, the enzyme that from Caulobacter crescentus, Haloarcula marismortui, catalyzes the conversion of 2 -keto - 3 -deoxy - xylonate to gly US 10,941,424 B2 17 18 colaldehyde and pyruvate is encoded by one or more exog- A acetyltransferase is encoded by a nucleic acid sequence enous nucleic acid molecules . In another embodiment, the selected from the group consisting of SEQ ID NOs : 33 , 34 , enzyme is a 2 -keto - 3 -deoxy - D -pentonate aldolase that is 36 , 38 and 39 . encoded by a nucleic acid molecule obtained from a micro- In one embodiment, the enzyme that catalyzes the con organism selected from Pseudomonas sp . and E. coli . In 5 version of acetoacetyl - CoA to acetoacetate is a acetate : some embodiments , the nucleic acid molecule encoding acetoacetyl - CoA transferase or hydrolase. In some embodi 2 -keto - 3 -deoxy - D -pentonate aldolase is selected from yjhH , ments, the transferase is an acetyl - CoA :acetoacetate - COA yagE , or homolog thereof. In a further embodiment, the one transferase . In a further embodiment, the enzyme that cata or more nucleic acid molecules encoding the 2 -keto - 3- lyzes the conversion of acetoacetyl- CoA to acetoacetate is deoxy - D -pentonate aldolase comprises an amino acid 10 encoded by one or more endogenous nucleic acid molecules . sequence selected from the group consisting of SEQ ID In an alternative embodiment, the enzyme that catalyzes the NOs : 78 and 81. In yet another embodiment, the one or more conversion of acetoacetyl - CoA to acetoacetate is encoded by nucleic acid molecules encoding the 2 -keto - 3 - deoxy - D- one or more exogenous nucleic acid molecules . In another pentonate aldolase is encoded by a nucleic acid sequence embodiment, the enzyme is an acetate : acetoacetyl- CoA selected from the group consisting of SEQ ID NOs : 76 , 77 , 15 transferase or hydrolase that is encoded by one or more 79 and 80 . nucleic acid molecules obtained from a microorganism In one embodiment, the enzyme that catalyzes the con- selected from Clostridium sp . and E. coli . In some embodi version of glycolaldehyde to MEG is a glycolaldehyde ments, the nucleic acid molecules encoding acetate :ac reductase or aldehyde reductase. In a further embodiment, 20 etoacetyl -CoA hydrolase are obtained from Clostridium the enzyme that catalyzes the conversion of glycolaldehyde acetobutylicum . In some embodiments, the nucleic acid to MEG is encoded by one or more endogenous nucleic acid molecules encoding acetate : acetoacetyl -CoA transferase are molecules . In an alternative embodiment, the enzyme that obtained from E. coli . In some embodiments , the nucleic catalyzes the conversion of glycolaldehyde to MEG is acid molecules encoding acetate : acetoacetyl -CoA transfer encoded by one or more exogenous nucleic acid molecules . 25 ase subunits are ato A and atoD , or homologs thereof. In In another embodiment, the enzyme is a glycolaldehyde some embodiments, the one or more nucleic acid molecules reductase or aldehyde reductase that is encoded by a nucleic encoding acetate : acetoacetyl - CoA hydrolase subunits are ctfA and ctfB , or homologs thereof. In a further embodi acid molecule obtained from a microorganism selected from ment, the acetyl - CoA : acetoacetate - CoA transferase or E.acid coli molecule or S. cerevisiae encoding . Inglycolaldehyde some embodiments reductase, the ornucleic alde- 30 acetate :acetoacetyl - CoA hydrolase comprises an amino acid hyde reductase is selected from fuco , yqhD , dkgA (yqhE ), sequence selected from the group consisting of SEQ ID dkgB ( yafB ), yeaE , yghZ , gldA , GRE2 , or homolog thereof. NOs : 43 , 46 , 97 , 99 , 101 and 103. In yet a further embodi ment, the acetyl - CoA : acetoacetate - CoA transferase or In another embodiment, the one or more nucleic acid mol acetate :acetoacetyl - CoA hydrolase is encoded by a nucleic ecules is yqhD . In some embodiments, the yqhD comprises 35 acid sequence selected from the group consisting of SEQ ID a G149E mutation . In a further embodiment, the glycolal NOs : 41 , 42 , 44 , 45 , 96 , 98 , 100 and 102 . dehyde reductase comprises an amino acid sequence In one embodiment, the enzyme that catalyzes the con selected from the group consisting of SEQ ID NOs : 13 , 15 , version of acetoacetate to acetone is an acetoacetate decar 17 , 20 , 23 , 25 , 28 , 30 and 32. In yet a further embodiment, boxylase . In a further embodiment, the enzyme that cata the glycolaldehyde reductase is encoded by a nucleic acid 40 lyzes the conversion of acetoacetate to acetone is encoded sequence selected from the group consisting of SEQ ID by one or more endogenous nucleic acid molecules . In an NOs : 12 , 14 , 16 , 18 , 19 , 21 , 22 , 24 , 26 , 27 , 29 and 31 . alternative embodiment, the enzyme that catalyzes the con In one embodiment, the enzyme that catalyzes the con- version of acetoacetate to acetone is encoded by one or more version of acetyl- CoA to acetoacetyl - CoA is a thiolase or exogenous nucleic acid molecules . In another embodiment, acetyl coenzyme A acetyltransferase. In a further embodi- 45 the enzyme is an acetoacetate decarboxylase that is encoded ment, the enzyme that catalyzes the conversion of acetyl- by one or more nucleic acid molecule obtained from a CoA to acetoacetyl -CoA is encoded by one or more endog- microorganism selected from Clostridium sp . , Bacillus sp . , enous nucleic acid molecules . In an alternative embodiment, Chromobacterium sp . and Pseudomonas sp . In some the enzyme that catalyzes the conversion of acetyl- CoA to embodiments, the nucleic acid molecule encoding acetoac acetoacetyl- CoA is encoded by one or more exogenous 50 etate decarboxylase is obtained from a microorganism nucleic acid molecules . In another embodiment , the enzyme selected from Clostridium acetobutylicum , Clostridium is a thiolase or acetyl coenzyme A acetyltransferase that is beijerinckii , Clostridium cellulolyticum , Bacillus polymyxa, encoded by a nucleic acid molecule obtained from a micro- Chromobacterium violaceum and Pseudomonas putida. In organism selected from Clostridium sp . , Bacillus sp . , E. coli, some embodiments, the nucleic acid molecule encoding Saccharomyces sp . and Marinobacter sp . In some embodi- 55 acetoacetate decarboxylase is adc , or homolog thereof. In a ments , the nucleic acid molecule encoding thiolase or acetyl further embodiment, the acetoacetate decarboxylase com coenzyme A acetyltransferase is obtained from a microor- prises an amino acid sequence selected from the group ganism selected from Clostridium acetobutylicum , consisting of SEQ ID NOs : 49 and 52. In yet another Clostridium thermosaccharolyticum , Bacillus cereus , E. embodiment, the acetoacetate decarboxylase is encoded by coli, Saccharomyces cerevisiae and Marinobacter hydrocar- 60 a nucleic acid sequence selected from the group consisting bonoclasticus. In some embodiments, the nucleic acid mol- of SEQ ID NOs : 47 , 48 , 50 and 51 . ecule encoding thiolase or acetyl coenzyme A acetyltrans- In one embodiment, the recombinant microorganism fur ferase is th1A , atoB and / or ERG10 , or homolog thereof. In ther comprises one or more modifications selected from the a further embodiment, the thiolase or acetyl coenzyme A group consisting of: acetyltransferase comprises an amino acid sequence selected 65 ( a ) a deletion , insertion , or loss of function mutation in a from the group consisting of SEQ ID NOs : 35 , 37 and 40 . gene encoding an enzyme that catalyzes the conversion In yet a further embodiment, the thiolase or acetyl coenzyme of D - xylose to D - xylulose ; US 10,941,424 B2 19 20 ( b ) a deletion , insertion , or loss of function mutation in a ( f) at least one endogenous or exogenous nucleic acid gene encoding an enzyme that catalyzes the conversion molecule encoding an enzyme that catalyzes the con of glycolaldehyde to glycolic acid ; and version of acetoacetyl -CoA from ( e ) to acetoacetate ; ( c ) a deletion , insertion , or loss of function mutation in a and / or gene encoding an enzyme that catalyzes the conversion 5 ( g ) at least one exogenous nucleic acid molecule encoding of pyruvate to lactate . an enzyme that catalyzes the conversion of acetoacetate In one embodiment, the enzyme that catalyzes the con from ( f) to acetone ; wherein the produced intermediate pyruvate is converted to version of D -xylose to D - xylulose is a D -xylose isomerase . acetyl- CoA through the endogenous glycolysis pathway in In some embodiments , the D - xylose isomerase is from 10 the microorganism , and wherein MEG and acetone are Escherichia coli . In some embodiments, the D - xylose co -produced isomerase is encoded by the xylA gene , or homolog thereof. In one embodiment, the enzyme that catalyzes the con In some embodiments, a recombinant microorganism version of D - xylose to D -xylonate is a xylose dehydroge producing MEG and a three - carbon compound comprises a nase . In a further embodiment, the enzyme that catalyzes the deletion, insertion, or loss of function mutation in a gene 15 conversion of D - xylose to D - xylonate is encoded by one or encoding a D - xylose isomerase to prevent conversion of more endogenous nucleic acid molecules . In an alternative D - xylose to D - xylulose and instead shunt the reaction embodiment, the enzyme that catalyzes the conversion of toward the conversion of D -xylose to D - xylonate . D - xylose to D -xylonate is encoded by one or more exog In one embodiment, the enzyme that catalyzes the con- enous nucleic acid molecules . In another embodiment, the version of glycolaldehyde to glycolic acid is a glycolalde- 20 enzyme is a xylose dehydrogenase that is encoded by a hyde dehydrogenase. In some embodiments , the glycolal- nucleic acid molecule obtained from a microorganism dehyde dehydrogenase is from Escherichia coli . In some selected from Caulobacter sp . , Haloarcula sp . , Haloferax embodiments , the glycolaldehyde dehydrogenase is encoded sp . , Halorubrum sp . and Trichoderma sp . In some embodi by the aldA gene , or homolog thereof. ments, the nucleic acid molecule encoding the xylose dehy In some embodiments , a recombinant microorganism 25 drogenase is obtained from a microorganism selected from producing MEG and a three - carbon compound comprises a Caulobacter crescentus, Haloarcula marismortui, deletion , insertion , or loss of function mutation in a gene Haloferax volcanii, Halorubrum lacusprofundi and Tricho encoding a glycolaldehyde dehydrogenase to prevent the derma reesei. In some embodiments, the nucleic acid mol production of glycolic acid from glycolaldehyde and instead ecule encoding xylose dehydrogenase is selected from xylB , shunt the reaction toward conversion of glycolaldehyde to 30 xdhembodiment (HVO_B0028 , the one ), xyd1or more, or nucleichomolog acid thereof molecules. In a furtherencod MEG . ing the xylose dehydrogenase comprises an amino acid In one embodiment, the enzyme that catalyzes the con sequence selected from the group consisting of SEQ ID version of pyruvate to lactate is a lactate dehydrogenase . In NOs : 61 , 63 and 65. In yet another embodiment, the one or particular embodiments , the enzyme converts pyruvate to 35 more nucleic acid molecules encoding the xylose dehydro lactate . In some embodiments , the lactate dehydrogenase is genase is encoded by a nucleic acid sequence selected from from Escherichia coli . In some embodiments , the lactate the group consisting of SEQ ID NOs : 59 , 60 , 62 and 64 . dehydrogenase is encoded by the IdhA gene , or homolog In one embodiment, the enzyme that catalyzes the con thereof. version of D - xylonate to 2 -keto - 3 -deoxy -xylonate is a In some embodiments , a recombinant microorganism 40 xylonate dehydratase. In a further embodiment, the enzyme producing MEG and a three - carbon compound comprises a that catalyzes the conversion of D -xylonate to 2 -keto - 3 deletion , insertion , or loss of function mutation in a gene deoxy - xylonate is encoded by one or more endogenous encoding a lactate dehydrogenase to prevent the production nucleic acid molecules . In an alternative embodiment, the of lactate from pyruvate and instead shunt the reaction enzyme that catalyzes the conversion of D - xylonate to toward production of a three - carbon compound . 45 2 - keto - 3 - deoxy -xylonate is encoded by one or more exog In another aspect , the present application relates to a enous nucleic acid molecules . In another embodiment, the recombinant microorganism capable of co - producing mono- enzyme is a xylonate dehydratase that is encoded by a ethylene glycol ( MEG ) and acetone from exogenous D - xy- nucleic acid molecule obtained from a microorganism lose , wherein the recombinant microorganism expresses one selected from Caulobacter sp . , Haloferax sp . , Sulfolobus sp . or more of the following: 50 and E. coli . In some embodiments, the nucleic acid molecule ( a ) at least one endogenous or exogenous nucleic acid encoding the xylonate dehydratase is obtained from a micro molecule encoding an enzyme that catalyzes the con- organism selected from Caulobacter crescentus, Haloferax version of D -xylose to D - xylonate ; volcanii , E. coli and Suffolobus soffataricus. In some ( b ) at least one endogenous or exogenous nucleic acid embodiments, the nucleic acid molecule encoding xylonate molecule encoding an enzyme that catalyzes the con- 55 dehydratase is selected from xylD , yjhG , yagF , xad , or version of D - xylonate from ( a ) to 2 -keto - 3 - deoxy- homolog thereof. In a further embodiment, the one or more xylonate ; nucleic acid molecules encoding the xylonate dehydratase ( c ) at least one endogenous or exogenous nucleic acid comprises an amino acid sequence selected from the group molecule encoding an enzyme that catalyzes the con- consisting of SEQ ID NOs : 69 , 72 and 75. In yet another version of 2 -keto - 3 -deoxy - xylonate from ( b ) to glyco- 60 embodiment, the one or more nucleic acid molecules encod laldehyde and pyruvate ; ing the xylonate dehydratase is encoded by a nucleic acid ( d ) at least one exogenous nucleic acid molecule encoding sequence selected from the group consisting of SEQ ID an enzyme that catalyzes the conversion of glycolal- NOs : 68 , 70 , 71 , 73 and 74 . dehyde from ( c ) to MEG ; In one embodiment, the enzyme that catalyzes the con ( e ) at least one exogenous nucleic acid molecule encoding 65 version of 2 - keto - 3 - deoxy -xylonate to glycolaldehyde and an enzyme that catalyzes the conversion of acetyl - CoA pyruvate is a 2 -keto - 3 - deoxy - D - pentonate aldolase . In a to acetoacetyl -CoA ; further embodiment, the enzyme that catalyzes the conver US 10,941,424 B2 21 22 sion of 2 -keto - 3 - deoxy - xylonate to glycolaldehyde and a further embodiment, the thiolase or acetyl coenzyme A pyruvate is encoded by one or more endogenous nucleic acid acetyltransferase comprises an amino acid sequence selected molecules . In an alternative embodiment, the enzyme that from the group consisting of SEQ ID NOs : 35 , 37 and 40 . catalyzes the conversion of 2 -keto - 3 - deoxy -xylonate to gly- In yet a further embodiment, the thiolase or acetyl coenzyme colaldehyde and pyruvate is encoded by one or more exog- 5 A acetyltransferase is encoded by a nucleic acid sequence enous nucleic acid molecules . In another embodiment, the selected from the group consisting of SEQ ID NOs : 33 , 34 , enzyme is a 2 -keto - 3 -deoxy - D - pentonate aldolase that is 36 , 38 and 39 . encoded by a nucleic acid molecule obtained from a micro- In one embodiment, the enzyme that catalyzes the con organism selected from Pseudomonas sp . and E. coli . In version of acetoacetyl -CoA to acetoacetate is a acetate : some embodiments , the nucleic acid molecule encoding the 10 acetoacetyl - CoA transferase or hydrolase . In some embodi 2 - keto - 3 - deoxy - D - pentonate aldolase is obtained from a ments , the transferase is an acetyl - CoA : acetoacetate -COA microorganism selected from E. coli . In some embodiments , transferase. In a further embodiment, the enzyme that cata the nucleic acid molecule encoding 2 -keto -3 -deoxy -D -pen- lyzes the conversion of acetoacetyl - CoA to acetoacetate is tonate aldolase is selected from yjhH , yagE , or homolog encoded by one or more endogenous nucleic acid molecules . thereof. In a further embodiment, the one or more nucleic 15 In an alternative embodiment, the enzyme that catalyzes the acid molecules encoding the 2 -keto - 3 -deoxy - D -pentonate conversion of acetoacetyl -CoA to acetoacetate is encoded by aldolase comprises an amino acid sequence selected from one or more exogenous nucleic acid molecules . In another the group consisting of SEQ ID NOs : 78 and 81. In yet embodiment, the enzyme is an acetate : acetoacetyl- CoA another embodiment, the one or more nucleic acid mol- transferase or hydrolase that is encoded by one or more ecules encoding the 2 -keto - 3 - deoxy - D - pentonate aldolase is 20 nucleic acid molecules obtained from a microorganism encoded by a nucleic acid sequence selected from the group selected from Clostridium sp . and E. coli . In some embodi consisting of SEQ ID NOs : 76 , 77 , 79 and 80 . ments , the nucleic acids molecule encoding acetate :ac In one embodiment, the enzyme that catalyzes the con- etoacetyl- CoA hydrolase are obtained from Clostridium version of glycolaldehyde to MEG is a glycolaldehyde acetobutylicum . In some embodiments, the nucleic acid reductase or aldehyde reductase . In a further embodiment, 25 molecules encoding acetate : acetoacetyl - CoA transferase are the enzyme that catalyzes the conversion of glycolaldehyde obtained from E. coli . In some embodiments , the nucleic to MEG is encoded by one or more endogenous nucleic acid acid molecules encoding acetate :acetoacetyl -CoA transfer molecules . In an alternative embodiment, the enzyme that ase subunits are atoA and atoD , or homologs thereof. In catalyzes the conversion of glycolaldehyde to MEG is some embodiments, the nucleic acid molecules encoding encoded by one or more exogenous nucleic acid molecules . 30 acetate : acetoacetyl - CoA hydrolase subunits are ctfA and In another embodiment, the enzyme is a glycolaldehyde ctfB , or homologs thereof. In a further embodiment, the reductase or aldehyde reductase that is encoded by a nucleic acetyl -CoA :acetoacetate - CoA transferase or acetate :ac acid molecule obtained from a microorganism selected from etoacetyl - CoA hydrolase comprises an amino acid sequence E. coli or S. cerevisiae . In some embodiments , the nucleic selected from the group consisting of SEQ ID NOs : 43 , 46 , acid molecule encoding glycolaldehyde reductase or alde- 35 97 , 99 , 101 and 103. In yet a further embodiment, the hyde reductase is selected from fuco , yqhD , dkg A ( yqhE ) , acetyl -CoA : acetoacetate - CoA transferase or acetate : ac dkgB ( yafB ), yeaE, yghZ , gldA , GRE2 , or homolog thereof. etoacetyl - CoA hydrolase is encoded by a nucleic acid In another embodiment, the one or more nucleic acid mol- sequence selected from the group consisting of SEQ ID ecules is yqhD . In some embodiments, the yqhD comprises NOs : 41 , 42 , 44 , 45 , 96 , 98 , 100 and 102 . a G149E mutation . In a further embodiment, the glycolal- 40 In one embodiment, the enzyme that catalyzes the con dehyde reductase comprises an amino acid sequence version of acetoacetate to acetone is an acetoacetate decar selected from the group consisting of SEQ ID NOs : 13 , 15 , boxylase . In a further embodiment, the enzyme that cata 17 , 20 , 23 , 25 , 28 , 30 and 32. In yet a further embodiment, lyzes the conversion of acetoacetate to acetone is encoded the glycolaldehyde reductase is encoded by a nucleic acid by one or more endogenous nucleic acid molecules . In an sequence selected from the group consisting of SEQ ID 45 alternative embodiment, the enzyme that catalyzes the con NOs : 12 , 14 , 16 , 18 , 19 , 21 , 22 , 24 , 26 , 27 , 29 and 31 . version of acetoacetate to acetone is encoded by one or more In one embodiment, the enzyme that catalyzes the con- exogenous nucleic acid molecules . In another embodiment, version of acetyl -CoA to acetoacetyl - CoA is a thiolase or the enzyme is an acetoacetate decarboxylase that is encoded acetyl coenzyme A acetyltransferase. In a further embodi- by a nucleic acid molecule obtained from a microorganism ment, the enzyme that catalyzes the conversion of acetyl- 50 selected from Clostridium sp . , Bacillus sp . , Chromobacte COA to acetoacetyl -CoA is encoded by one or more endog- rium sp . and Pseudomonas sp . In some embodiments , the enous nucleic acid molecules . In an alternative embodiment, nucleic acid molecule encoding acetoacetate decarboxylase the enzyme that catalyzes the conversion of acetyl -CoA to is obtained from a microorganism selected from Clostridium acetoacetyl- CoA is encoded by one or more exogenous acetobutylicum , Clostridium beijerinckii, Clostridium cel nucleic acid molecules . In another embodiment, the enzyme 55 lulolyticum , Bacillus polymyxa, Chromobacterium viola is a thiolase or acetyl coenzyme A acetyltransferase that is ceum and Pseudomonas putida. In some embodiments, the encoded by a nucleic acid molecule obtained from a micro- nucleic acid molecule encoding acetoacetate decarboxylase organism selected from Clostridium sp . , Bacillus sp . , E. coli , is adc , or homolog thereof. In a further embodiment, the Saccharomyces sp . and Marinobacter sp . In some embodi- acetoacetate decarboxylase comprises an amino acid ments , the nucleic acid molecule encoding thiolase or acetyl 60 sequence selected from the group consisting of SEQ ID coenzyme A acetyltransferase is obtained from a microor- NOs : 49 and 52. In yet another embodiment, the acetoac ganism selected from Clostridium acetobutylicum , etate decarboxylase is encoded by a nucleic acid sequence Clostridium thermosaccharolyticum , Bacillus cereus , E. selected from the group consisting of SEQ ID NOs : 47 , 48 , coli, Saccharomyces cerevisiae and Marinobacter hydrocar- 50 and 51 . bonoclasticus. In some embodiments, the nucleic acid mol- 65 In one embodiment, the recombinant microorganism fur ecule encoding thiolase or acetyl coenzyme A acetyltrans- ther comprises one or more modifications selected from the ferase is th1A , atoB and / or ERG10 , or homolog thereof. In group consisting of: US 10,941,424 B2 23 24 ( a ) a deletion, insertion , or loss of function mutation in a 652 , Alcaligenes eutrophus, Clostridium ragsdalei, gene encoding an enzyme that catalyzes the conversion Clostridium beijerinckii, Clostridium carboxidivorans , of D - xylose to D - xylulose ; Thermoanaerobacter brockii, Thermoanaerobacter ethano ( b ) a deletion , insertion , or loss of function mutation in a licus ( Clostridium thermohydrosulfuricum ), Rhodococcus gene encoding an enzyme that catalyzes the conversion 5 ruber, Methanobacterium palustre, methanogenic archaea of glycolaldehyde to glycolic acid ; and Methanogenium liminatans, parasitic protist Entamoeba his ( c ) a deletion , insertion, or loss of function mutation in a tolytica , parasitic protozoan Tritrichomonas foetus and gene encoding an enzyme that catalyzes the conversion human parasite Trichomonas vaginalis. In some embodi of pyruvate to lactate . ments , the one or more nucleic acid molecule encoding In one embodiment, the enzyme that catalyzes the con- 10 secondary alcohol dehydrogenase is adh , adhB , EhAdhl, or version of D - xylose to D - xylulose is a D - xylose isomerase . homolog thereof. In some embodiments, the S - ADH is In some embodiments, the D - xylose isomerase is from predicted from homology and can be from Thermoanaero Escherichia coli. In some embodiments, the D -xylose bacter mathranii, Micrococcus luteus, Nocardiopsis alba , isomerase is encoded by the xylA gene, or homolog thereof. Mycobacterium hassiacum , Helicobacter suis , Candida In some embodiments, a recombinant microorganism 15 albicans, Candida parapsilosis , Candida orthopsilosis, producing MEG and a three - carbon compound comprises a Candida metapsilosis, Grosmannia clavigera and Scheffer deletion , insertion , or loss of function mutation in a gene somyces stipitis . In a further embodiment, the alcohol dehy encoding a D - xylose isomerase to prevent conversion of drogenase comprises an amino acid sequence selected from D - xylose to D - xylulose and instead shunt the reaction the group consisting of SEQ ID NOs : 106 and 108. In yet toward the conversion of D - xylose to D - xylonate. 20 another embodiment, the alcohol dehydrogenase is encoded In one embodiment, the enzyme that catalyzes the con- by a nucleic acid sequence selected from the group consist version of glycolaldehyde to glycolic acid is a glycolalde- ing of SEQ ID NOs : 104 , 105 and 107 . hyde dehydrogenase. In some embodiments, the glycolal- In any of the above - described aspects and embodiments, dehyde dehydrogenase is from Escherichia coli . In some the recombinant microorganism may further comprise at embodiments, the glycolaldehyde dehydrogenase is encoded 25 least one nucleic acid molecule encoding an enzyme that by the aldA gene , or homolog thereof. catalyzes the conversion of isopropanol to propene. In one In some embodiments , a recombinant microorganism embodiment, the enzyme that catalyzes the conversion of producing MEG and a three - carbon compound comprises a isopropanol to propene is encoded by one or more endog deletion , insertion , or loss of function mutation in a gene enous nucleic acid molecules . In an alternative embodiment, encoding a glycolaldehyde dehydrogenase to prevent the 30 the enzyme that catalyzes the conversion of isopropanol to production of glycolic acid from glycolaldehyde and instead propene is encoded by one or more exogenous nucleic acid shunt the reaction toward conversion of glycolaldehyde to molecules . In one embodiment, the enzyme that catalyzes MEG . the conversion of isopropanol to propene is a dehydratase. In one embodiment, the enzyme that catalyzes the con- In one embodiment, MEG is produced through the con version of pyruvate to lactate is a lactate dehydrogenase . In 35 version of glycolaldehyde in a C - 2 branch pathway and particular embodiments, the enzyme converts pyruvate to acetone is produced through the conversion of DHAP or lactate . In some embodiments , the lactate dehydrogenase is pyruvate in a C - 3 branch pathway. In another embodiment, from Escherichia coli . In some embodiments , the lactate MEG is produced through the conversion of glycolaldehyde dehydrogenase is encoded by the IdhA gene , or homolog in a C - 2 branch pathway and IPA is produced through the thereof. 40 conversion of DHAP or pyruvate in a C - 3 branch pathway. In some embodiments , a recombinant microorganism In a further embodiment, MEG is produced through the producing MEG and a three -carbon compound comprises a conversion of glycolaldehyde in a C - 2 branch pathway and deletion , insertion , or loss of function mutation in a gene propene is produced through the conversion of DHAP or encoding a lactate dehydrogenase to prevent the production pyruvate in a C - 3 branch pathway . of lactate from pyruvate and instead shunt the reaction 45 In one embodiment, at least a portion of the excess NADH toward production of a three - carbon compound . produced in the C - 3 branch is used as a source of reducing In any of the above - described aspects and embodiments, equivalents in the C - 2 branch . In another embodiment, at the recombinant microorganism may further comprise at least a portion of the excess NADH produced in the C - 3 least one nucleic acid molecule encoding an enzyme that branch is used to produce ATP . catalyzes the conversion of acetone to isopropanol. In one 50 In one embodiment, the co - produced MEG and acetone embodiment, the enzyme that catalyzes the conversion of comprise a yield potential greater than 90 % of the theoreti acetone to isopropanol is encoded by one or more endog- cal maximum yield potential without carbon fixation . In enous nucleic acid molecules . In an alternative embodiment, another embodiment, the co - produced MEG and IPA com the enzyme that catalyzes the conversion of acetone to prise a yield potential greater than 90 % of the theoretical isopropanol is encoded by one or more exogenous nucleic 55 maximum yield potential without carbon fixation . In a acid molecules . In one embodiment, the enzyme that cata- further embodiment , the co - produced MEG and propene lyzes the conversion of acetone to isopropanol is a second- comprise a yield potential greater than 90 % of the theoreti ary alcohol dehydrogenase ( S - ADH ) . In another embodi- cal maximum yield potential without carbon fixation . ment, the enzyme is a secondary alcohol dehydrogenase that In one embodiment, excess biomass formation is mini is encoded by a nucleic acid molecule obtained from a 60 mized and production of MEG and acetone is maximized . In microorganism selected from Burkholderia sp , Alcaligenes another embodiment, excess biomass formation is mini sp . , Clostridium sp . , Thermoanaerobacter sp . , Phytomonas mized and production of MEG and IPA is maximized . In a sp . , Rhodococcus sp . , Methanobacterium sp . , Methanoge- further embodiment, excess biomass formation is minimized nium sp . , Entamoeba sp . , Trichomonas sp . , and Tritrichomo- and production of MEG and propene is maximized . nas sp . In some embodiments , the nucleic acid molecule 65 In yet another aspect , the present application provides a encoding the secondary alcohol dehydrogenase is obtained method of producing MEG and a three carbon compound from a microorganism selected from Burkholderia sp . AIU using a recombinant microorganism as described above , US 10,941,424 B2 25 26 wherein the method comprises cultivating the recombinant SEQUENCES microorganism in a culture medium containing a feedstock providing a carbon source until the MEG and the three A sequence listing for SEQ ID NO : 1 - SEQ ID NO : 120 is carbon compound is produced. In some embodiments, the part of this application and is incorporated by reference three carbon compound is selected from acetone , isopropa- 5 herein . The sequence listing is provided at the end of this nol , and propene . document. In yet another aspect , the present application provides a method of producing a recombinant microorganism that DETAILED DESCRIPTION co -produces , produces or accumulates MEG and a three carbon compound. In some embodiments , the three carbon 10 Definitions compound is selected from acetone , isopropanol, and pro pene . The following definitions and abbreviations are to be used In yet another aspect , the present application provides a for the interpretation of the disclosure . recombinant microorganism co - producing monoethylene 15 formsAs used" a. " " hereinan , " and and " the in "the include appended plural claims referents , the unless singular the glycol ( MEG ) and a three carbon compound . In some context clearly dictates otherwise . Thus, for example , ref embodiments, the three carbon compound is selected from erence to " a three - carbon compound ” includes a plurality of acetone , isopropanol, and propene . such three - carbon compounds and reference to “ the micro organism ” includes reference to one or more microorgan BRIEF DESCRIPTION OF DRAWINGS 20 isms , and so forth . As used herein , the terms " comprises, " " comprising , " Illustrative embodiments of the disclosure are illustrated “ includes , ” “ including, " " has, ” “ having , " contains, " " con in the drawings, in which : taining ," or any other variation thereof, are intended to cover FIG . 1 illustrates MEG and isopropanol co - production a non - exclusive inclusion . A composition , mixture, process , pathway via ribulose - 1 - phosphate. 25 method , article , or apparatus that comprises a list of ele FIG . 2 illustrates MEG and isopropanol co -production ments is not necessarily limited to only those elements but pathway via xylulose - 1 - phosphate . may include other elements not expressly listed or inherent FIG . 3 illustrates MEG and isopropanol co -production to such composition , mixture , process , method , article, or pathway via xylonate . apparatus. Further, unless expressly stated to the contrary, FIG . 4 illustrates possible three carbon co - products for 30 “ or ” refers to an inclusive “ or” and not to an exclusive “ or. ” MEG . The terms “ about ” and “ around ," as used herein to modify FIG . 5 illustrates MEG and isopropanol co - production a numerical value , indicate a close range surrounding that pathway from xylose and glucose , via ribulose - 1 - phosphate , explicit value . If “ X ” were the alue , " about X ” or “ around in S. cerevisiae. X ” would indicate a value from 0.9x to 1.1x , or, in some FIG . 6 illustrates MEG and isopropanol co -production 35 embodiments, a value from 0.95x to 1.052 . Any reference to from xylose and glucose in S. cerevisiae . “ about X ” or “ around X ” specifically indicates at least the FIG . 7 illustrates MEG production from xylose in E. coli . values X , 0.95x , 0.96x , 0.97 % , 0.98 % , 0.99x , 1.01x , 1.02x , FIG . 8 illustrates improved MEG production from xylose 1.03x , 1.04x , and 1.05x . Thus, “ about X ” and “ around X ” in E. coli . are intended to teach and provide written description support FIG . 9 illustrates overall yield ( g products/ g xylose ) of 40 for a claim limitation of, e.g. , " 0.98x . ” ethylene glycol , isopropanol and / or acetone produced using ismAs ,” usedand “ microorganismherein , the terms ” include “ microbial any , organism” “ microbial that organexists a ribulose - 1 - phosphate pathway in six E. coli strains as a microscopic cell that is included within the domains of described in Example 3 and Table 2 . archaea , bacteria or eukarya, the latter including yeast and FIG . 10 illustrates co - production of MEG , isopropanol 45 filamentous fungi, protozoa, algae , or higher Protista. There and acetone using a xylulose - 1 - phosphate pathway in E. coli fore, the term is intended to encompass prokaryotic or as described in Example 4 . eukaryotic cells or organisms having a microscopic size and FIG . 11 illustrates overall yield ( g products / g xylose ) of includes bacteria, archaea, and eubacteria of all species as ethylene glycol , isopropanol and acetone produced using a well as eukaryotic microorganisms such as yeast and fungi. xylulose - 1 - phosphate pathway as described in Example 4 . 50 Also included are cell cultures of any species that can be FIG . 12 illustrates co - production of MEG , isopropanol cultured for the production of a chemical. As described herein , in some embodiments, the recom and acetone using a xylonate pathway in E. coli as described binant microorganisms are prokaryotic microorganism . In in Example 5 . some embodiments, the prokaryotic microorganisms are FIG . 13 illustrates overall yield ( g products / g xylose ) of 55 bacteria . “ Bacteria " , or " eubacteria ” , refers to a domain of ethylene glycol , isopropanol and acetone produced using a prokaryotic organisms. Bacteria include at least eleven dis xylonate pathway as described in Example 5 . tinct groups as follows: ( 1 ) Gram - positive ( gram + ) bacteria , FIG . 14 shows an SDS - PAGE of soluble fraction of of which there are two major subdivisions: ( 1 ) high G + C assays ( a ) to ( e ) as described in Example 6. The arrow group (Actinomycetes , Mycobacteria , Micrococcus, others ) indicates LinD expression in ( b ) , ( c ) , ( d ) and ( e ) . 60 ( 2 ) low G + C group (Bacillus , Clostridia , Lactobacillus, FIG . 15 illlustrates that assays ( d ) and ( e ) showed the Staphylococci, Streptococci, Mycoplasmas ); ( 2 ) Proteobac production of propylene and isopropanol in IPA + LinD can teria , e.g. , Purple photosynthetic + non - photosynthetic didates . Assay ( a ) showed isopropanol production of Gram -negative bacteria ( includes most “ common ” Gram pZs * 13_IPA and a small amount of propylene . Assays ( b ) negative bacteria ); ( 3 ) Cyanobacteria, e.g. , oxygenic photo and ( c ) showed propylene production in medium supple- 65 trophs; ( 4 ) Spirochetes and related species ; ( 5 ) Planctomy mented with 3.0 g / L isopropanol using glycerol and glucose ces ; ( 6 ) Bacteroides , Flavobacteria ; ( 7 ) Chlamydia ; ( 8 ) as carbon source , respectively . Green sulfur bacteria; ( 9 ) Green non - sulfur bacteria ( also US 10,941,424 B2 27 28 anaerobic phototrophs ); ( 10 ) Radioresistant micrococci and where appropriate, single stranded or double stranded , sense relatives ; ( 11 ) Thermotoga and Thermosipho thermophiles. or antisense ribonucleic acid ( RNA ) of any length , including “ Gram -negative bacteria ” include cocci , nonenteric rods, siRNA . The term “ nucleotide ” refers to any of several and enteric rods . The genera of Gram - negative bacteria compounds that consist of a ribose or deoxyribose sugar include, for example, Neisseria , Spirillum , Pasteurella , Bru- 5 joined to a purine or a pyrimidine base and to a phosphate cella , Yersinia, Francisella , Haemophilus, Bordetella , group , and that are the basic structural units ofnucleic acids . Escherichia , Salmonella , Shigella , Klebsiella , Proteus, The term “ nucleoside ” refers to a compound ( as guanosine Vibrio , Pseudomonas, Bacteroides, Acetobacter, Aerobacter, or adenosine ) that consists of a purine or pyrimidine base Agrobacterium , Azotobacter, Spirilla, Serratia , Vibrio, combined with deoxyribose or ribose and is found especially Rhizobium , Chlamydia , Rickettsia, Treponema, and Fuso- 10 in nucleic acids . The term “ nucleotide analog ” or “ nucleo bacterium . side analog ” refers, respectively, to a nucleotide or nucleo " Gram positive bacteria ” include cocci , nonsporulating side in which one or more individual atoms have been rods , and sporulating rods . The genera of gram positive replaced with a different atom or with a different functional bacteriaClostridium include , Corynebacterium , for example , Erysipelothrix, Actinomyces, ,Lactobacil- Bacillus, 15 group. Accordingly, the term polynucleotide includes lus, Listeria , Mycobacterium , Myxococcus, Nocardia, nucleic acids of any length , DNA , RNA , analogs and Staphylococcus, Streptococcus, and Streptomyces. fragments thereof. A polynucleotide of three or more nucleo The term “ recombinant microorganism ” and “ recombi tides is also called nucleotidic oligomer or oligonucleotide . nant host cell” are used interchangeably herein and refer to It is understood that the polynucleotides described herein microorganisms that have been genetically modified to 20 include " genes ” and that the nucleic acid molecules express or to overexpress endogenous enzymes , to express described herein include " vectors ” or “ plasmids. ” Accord heterologous enzymes , such as those included in a vector , in ingly , the term “ gene ” , also called a “ structural gene” refers an integration construct, or which have an alteration in to a polynucleotide that codes for a particular sequence of expression of an endogenous gene . By " alteration ” it is amino acids , which comprise all or part of one or more meant that the expression of the gene, or level of a RNA 25 proteins or enzymes , and may include regulatory (non molecule or equivalent RNA molecules encoding one or transcribed ) DNA sequences, such as promoter sequences, more polypeptides or polypeptide subunits, or activity of which determine for example the conditions under which the one or more polypeptides or polypeptide subunits is up gene is expressed . The transcribed region of the gene may regulated or down regulated , such that expression , level , or include untranslated regions , including introns, 5' - untrans activity is greater than or less than that observed in the 30 lated region ( UTR ) , and 3 ' - UTR , as well as the coding absence of the alteration . For example, the term “ alter ” can sequence . mean " inhibit , ” but the use of the word " alter ” is not limited The term " enzyme ” as used herein refers to any substance to this definition . It is understood that the terms " recombi- that catalyzes or promotes one or more chemical or bio nant microorganism ” and “ recombinant host cell ” refer not chemical reactions , which usually includes enzymes totally only to the particular recombinant microorganism but to the 35 or partially composed of a polypeptide or polypeptides , but progeny or potential progeny of such a microorganism . can include enzymes composed of a different molecule Because certain modifications may occur in succeeding including polynucleotides . generations due to either mutation or environmental influ- As used herein , the term “ non -naturally occurring, " when ences, such progeny may not , in fact, be identical to the used in reference to a microorganism organism or enzyme parent cell , but are still included within the scope of the term 40 activity of the disclosure , is intended to mean that the as used herein . microorganism organism or enzyme has at least one genetic The term " expression ” with respect to a gene sequence alteration not normally found in a naturally occurring strain refers to transcription of the gene and , as appropriate , of the referenced species , including wild - type strains of the translation of the resulting mRNA transcript to a protein . referenced species . Genetic alterations include , for example , Thus, as will be clear from the context, expression of a 45 modifications introducing expressible nucleic acids encod protein results from transcription and translation of the open ing metabolic polypeptides, other nucleic acid additions , reading frame sequence . The level of expression of a desired nucleic acid deletions and / or other functional disruption of product in a host cell may be determined on the basis of the microorganism's genetic material. Such modifications either the amount of corresponding mRNA that is present in include, for example, coding regions and functional frag the cell , or the amount of the desired product encoded by the 50 ments thereof, for heterologous, homologous , or both het selected sequence . For example, mRNA transcribed from a erologous and homologous polypeptides for the referenced selected sequence can be quantitated by qRT- PCR or by species . Additional modifications include, for example , non Northern hybridization ( see Sambrook et al . , Molecular coding regulatory regions in which the modifications alter Cloning: A Laboratory Manual, Cold Spring Harbor Labo- expression of a gene or operon . Exemplary non -naturally ratory Press ( 1989 ) ) . Protein encoded by a selected sequence 55 occurring microorganism or enzyme activity includes the can be quantitated by various methods, e.g. , by ELISA , by hydroxylation activity described above . assaying for the biological activity of the protein , or by The term “ exogenous ” as used herein with reference to employing assays that are independent of such activity, such various molecules , e.g. , polynucleotides , polypeptides, as western blotting or radioimmunoassay, using antibodies enzymes , etc., refers to molecules that are not normally or that recognize and bind the protein . See Sambrook et al . , 60 naturally found in and / or produced by a given yeast , bacte 1989 , supra . rium , organism , microorganism , or cell in nature . The term “ polynucleotide ” is used herein interchangeably On the other hand, the term “ endogenous” or “ native ” as with the term “ nucleic acid ” and refers to an organic used herein with reference to various molecules , e.g. , poly polymer composed of two or more monomers including nucleotides, polypeptides, enzymes, etc. , refers to molecules nucleotides , nucleosides or analogs thereof, including but 65 that are normally or naturally found in and / or produced by not limited to single stranded or double stranded , sense or a given yeast, bacterium , organism , microorganism , or cell antisense deoxyribonucleic acid (DNA ) of any length and, in nature . US 10,941,424 B2 29 30 The term “ heterologous” as used herein in the context of ity in biological function may vary , but in one embodiment, a modified host cell refers to various molecules , e.g. , poly- is at least 1 % , at least 2 % , at least 3 % , at least 4 % , at least nucleotides, polypeptides, enzymes , etc. , wherein at least 5 % , at least 6 % , at least 7 % , at least 8 % , at least 9 % , at least one of the following is true : ( a ) the molecule ( s ) is / are foreign 10 % , at least 20 % , at least 30 % , at least 40 % , at least 50 % , ( " exogenous " ) to ( i.e. , not naturally found in ) the host cell ; 5 at least 60 % , at least 65 % , at least 70 % , at least 75 % , at least ( b ) the molecule ( s ) is / are naturally found in ( e.g. , is “ endog- 80 % , at least 85 % , at least 90 % , at least about 91 % , at least enous to ” ) a given host microorganism or host cell but is about 92 % , at least about 93 % , at least about 94 % , at least either produced in an unnatural location or in an unnatural about 95 % , at least about 96 % , at least about 97 % , at least amount in the cell ; and / or ( c ) the molecule ( s ) differ ( s ) in about 98 % , or at least 98.5 % , or at least about 99 % , or at nucleotide or amino acid sequence from the endogenous 10 least 99.5 % , or at least 99.8 % , or at least 99.9 % , according nucleotide or amino acid sequence ( s) such that the molecule to one or more assays known to one skilled in the art to differing in nucleotide or amino acid sequence from the determine a given biological function . endogenous nucleotide or amino acid as found endog- The term “ variant ” refers to any polypeptide or enzyme enously is produced in an unnatural ( e.g. , greater than described herein . A variant also encompasses one or more naturally found ) amount in the cell . 15 components of a multimer , multimers comprising an indi The term “ homolog ," as used herein with respect to an vidual component, multimers comprising multiples of an original enzyme or gene of a first family or species , refers to individual component ( e.g. , multimers of a reference mol distinct enzymes or genes of a second family or species ecule ) , a chemical breakdown product, and a biological which are determined by functional, structural, or genomic breakdown product. In particular, non - limiting embodi analyses to be an enzyme or gene of the second family or 20 ments , a linalool dehydratase / isomerase enzyme may be a species which corresponds to the original enzyme or gene of " variant” relative to a reference linalool dehydratase / the first family or species . Homologs most often have isomerase enzyme by virtue of alteration ( s ) in any part of the functional, structural, or genomic similarities . Techniques polypeptide sequence encoding the reference linalool dehy are known by which homologs of an enzyme or gene can dratase / isomerase enzyme. A variant of a reference linalool readily be cloned using genetic probes and PCR . Identity of 25 dehydratase / isomerase enzyme can have enzyme activity of cloned sequences as homologs can be confirmed using at least 10 % , at least 30 % , at least 50 % , at least 80 % , at least functional assays and / or by genomic mapping of the genes . 90 % , at least 100 % , at least 105 % , at least 110 % , at least A protein has “ homology” or is “ homologous ” to a second 120 % , at least 130 % or more in a standard assay used to protein if the amino acid sequence encoded by a genehas a measure enzyme activity of a preparation of the reference similar amino acid sequence to that of the second gene . 30 linalool dehydratase / isomerase enzyme. In some embodi Alternatively, a protein has homology to a second protein if ments, a variant may also refer to polypeptides having at the two proteins have “ similar ” amino acid sequences. Thus, least 50 % , at least 60 % , at least 70 % , at least 80 % , at least the term “ homologous proteins ” is intended to mean that the 90 % , at least 91 % , at least 92 at least 93 % , at least 94 % , two proteins have similar amino acid sequences. In certain at least 95 % , at least 96 % , at least 97 % , at least 98 % , or at instances, the homology between two proteins is indicative 35 least 99 % sequence identity to the full - length , or unpro of its shared ancestry , related by evolution . The terms cessed linalool dehydratase / isomerase enzymes of the pres " homologous sequences ” or “ homologs” are thought, ent disclosure . In some embodiments, a variant may also believed , or known to be functionally related . A functional refer to polypeptides having at least 50 % , at least 60 % , at relationship may be indicated in any one of a number of least 70 % , at least 80 % , at least 90 % , at least 91 % , at least ways , including, but not limited to : ( a ) degree of sequence 40 92 % , at least 93 % , at least 94 % , at least 95 % , at least 96 % , identity and / or ( b ) the same or similar biological function . at least 97 % , at least 98 % , or at least 99 % sequence identity Preferably, both ( a ) and ( b ) are indicated . The degree of to the mature , or processed linalool dehydratase / isomerase sequence identity may vary , but in one embodiment, is at enzymes of the present disclosure. least 50 % (when using standard sequence alignment pro- The term “ signal sequence " as used herein refers to an grams known in the art ), at least 60 % , at least 65 % , at least 45 amino acid sequence that targets peptides and polypeptides 70 % , at least 75 % , at least 80 % , at least 85 % , at least 90 % , to cellular locations or to the extracellular environment. at least about 91 % , at least about 92 % , at least about 93 % , Signal sequences are typically at the N - terminal portion of at least about 94 % , at least about 95 % , at least about 96 % , a polypeptide and are typically removed enzymatically. at least about 97 % , at least about 98 % , or at least 98.5 % , or Polypeptides that have their signal sequences are referred to at least about 99 % , or at least 99.5 % , or at least 99.8 % , or 50 as being full -length and / or unprocessed . Polypeptides that at least 99.9 % . Homology can be determined using software have had their signal sequences removed are referred to as programs readily available in the art, such as those discussed being mature and / or processed. in Current Protocols in Molecular Biology ( F. M. Ausubel et The term “ yield potential ” as used herein refers to a yield al . , eds . , 1987 ) Supplement 30 , section 7.718 , Table 7.71 . of a product from a biosynthetic pathway. In one embodi Some alignment programs are MacVector ( Oxford Molecu- 55 ment, the yield potential may be expressed as a percent by lar Ltd , Oxford , U.K. ) and ALIGN Plus ( Scientific and weight of end product per weight of starting compound . Educational Software, Pennsylvania ). Other non - limiting The term “ thermodynamic maximum yield ” as used alignment programs include Sequencher (Gene Codes, Ann herein refers to the maximum yield of a product obtained Arbor, Mich . ) , AlignX , and Vector NTI ( Invitrogen , Carls- from fermentation of a given feedstock , such as glucose , bad , Calif. ) . A similar biological function may include , but 60 based on the energetic value of the product compared to the is not limited to : catalyzing the same or similar enzymatic feedstock . In a normal fermentation , without use of addi reaction ; having the same or similar selectivity for a sub- tional energy sources such as light, hydrogen gas or methane strate or co - factor ; having the same or similar stability; or electricity, for instance , the product cannot contain more having the same or similar tolerance to various fermentation energy than the feedstock . The thermodynamic maximum conditions ( temperature , pH , etc.) ; and / or having the same 65 yield signifies a product yield at which all energy and mass or similar tolerance to various metabolic substrates, prod- from the feedstock is converted to the product. This yield ucts , by - products, intermediates, etc. The degree of similar- can be calculated and is independent of a specific pathway . US 10,941,424 B2 31 32 If a specific pathway towards a product has a lower yield ( DHAP ) ) . The C2 stream is easy to implement at high than the thermodynamic maximum yield , then it loses mass efficiency, but the C3 stream is very difficult to implement at and can most likely be improved upon or substituted with a high efficiency via metabolic engineering. Several pathway more efficient pathway towards the product. options for DHAP- > MEG exist , all of which are difficult to The term “ redox balanced ” refers to a set of reactions, 5 implement. Furthermore , the overall process is ATP neutral. which taken together produce as much redox cofactors as Thus, some glucose and therefore yield will be lost in order they consume . Designing metabolic pathways and engineer- to obtain some surplus ATP required for cell growth and ing an organism such that the redox cofactors are balanced maintenance. or close to being balanced usually results in a more efficient, A further demonstrated fermentative production of MEG higher yield production of the desired compounds. Redox 10 from xylose ( Alkim et al . , Microb Cell Fact ( 2015 ) 14 : 127 ) , reactions always occur together as two half -reactions hap- via xylulose - 1 - phosphate, is very similar to the route pening simultaneously, one being an oxidation reaction and described by WO2013126721A1 . It has the same high yield the other a reduction reaction . In redox processes , the potential ( 82 wt % ) , but the C3 stream for MEG production reductant transfers electrons to the oxidant. Thus, in the via DHAP is difficult to implement and there is an ATP reaction , the reductant or reducing agent loses electrons and 15 shortage . is oxidized , and the oxidant or oxidizing agent gains elec- A further fermentative production of MEG was demon trons and is reduced . In one embodiment, the redox reactions strated from glucose ( Chen et al . , Met . Eng . ( 2016 ) 33: 12 take place in a biological system . Biological energy is 18 ) . It uses exclusively a pathway identical to one of the C3 frequently stored and released by means of redox reactions. stream solutions of WO2013126721A1 , going via DHAP Photosynthesis involves the reduction of carbon dioxide into 20 and then ethanolamine to glyceraldehyde to MEG . Only in sugars and the oxidation of water into molecular oxygen . this case , DHAP is derived from glucose , not from xylose . The reverse reaction , respiration , oxidizes sugars to produce Thus it suffers even more from the technical difficulty to carbon dioxide and water . As intermediate steps, the reduced implement a high productivity and high yield pathway from carbon compounds are used to reduce nicotinamide adenine DHAP to MEG . It furthermore has a reduced total yield dinucleotide (NAD + ), which then contributes to the creation 25 potential of 69 wt % versus the thermodynamic maximum of a proton gradient, which drives the synthesis of adenosine yield for the product MEG derived from glucose ( 82 wt % ) . triphosphate ( ATP ) and is maintained by the reduction of The pathway is furthermore ATP neutral, not generating any oxygen . The term redox state is often used to describe the ATP that the cells need for growth and maintenance . This balance of GSH / GSSG , NAD + /NADH and NADP + /NA- pathway is also not redox balanced and has a high excess of DPH in a biological system such as a cell or organ . The 30 2 mol NADH per mol of consumed glucose , all of which redox state is reflected in the balance of several sets of needs to be re -oxidized for the cell to be viable . In an aerobic metabolites ( e.g. , lactate and pyruvate, beta -hydroxybu fermentation , this NADH can be used to generate ATP, tyrate , and acetoacetate ), whose interconversion is depen- which however would be in high excess ( 2 NADH > 6 ATP ), dent on these ratios . An abnormal redox state can develop in leading to excess biomass formation during the production a variety of deleterious situations, such as hypoxia , shock , 35 phase and therefore reduced product formation and yield . and sepsis . The only described solution for the loss of yield potential for The terms “ C2 pathway ”, “ C2 branch pathway ” or “ C2 MEG production from glucose is the production of MEG stream ” as used herein refers to a biochemical pathway from xylose with a high yield potential. The only described wherein MEG can be produced via glycolaldehyde . solution for the excess NADH production in the MEG from The terms “ C3 pathway ”, “ C3 branch pathway ” or “ C3 40 glucose process is the production of MEG from xylose stream ” as used herein refers to a biochemical pathway which can be redox neutral. wherein MEG or one or more three - carbon compounds can A demonstrated fermentative production of IPA via be produced via pyruvate or dihydroxyacetonephosphate acetoacetyl- CoA (US 2010/0311135 , which is herein refer ( DHAP ) . enced in its entirety ) has excess NADH ( 2 mol per mol of 45 consumed glucose ) and low yield potential ( 34 wt % ) . This INTRODUCTION pathway has excess ATP ( 2 mol per mol of consumed glucose ) , more than is required for cell maintenance during The present disclosure combines the production of mono- the production phase , thereby favoring biomass formation ethylene glycol ( MEG ) and one or more three carbon over production . If the NADH is not utilized via carbon compounds in different hosts . In some embodiments , the 50 fixation, it needs to be re - oxidized for the cell to stay viable, three carbon compound is isopropanol ( IPA ). The present further losing glucose in this process . Alternatively, NADH disclosure thereby avoids some of the biggest pathway can be oxidized through ATP production, which would lead engineering challenges for known MEG and IPA pathways to even more unwanted excess ATP. demonstrated so far. Surprisingly, the combination of a Other potential solutions exist for reducing NADH excess pathway for MEG production and a pathway for production 55 and increasing IPA yield potential ( thermodynamic max of a three carbon compound complements each other and is yield = 47 wt % ) : re - capturing CO2 produced in excess during highly synergistic , avoiding or overcoming the biggest chal- the fermentation and in doing so also re - oxidizing excess lenges and shortcomings of each pathway alone , establish- NADH ( CO , fixation ). Or avoid excess CO2 and NADH ing a good redox balance but also delivering required ATP, release altogether by diverting some flux from glycolysis to without production of excess ATP. 60 a phosphoketolase ( PK ) / phosphotransacetylase (PTA ) path A demonstrated fermentative production of MEG from way to generate more acetyl -CoA and less CO2 and NADH . xylose ( W02013126721A1 , which is herein referenced in However, so far none of these options have been technically its entirety ), via ribulose - 1 -phosphate , has a high yield demonstrated in the context of IPA production and are potential ( 82 wt % = 0.82 g MEG / g xylose ) . MEG is pro- generally known to be very challenging. duced via two different pathways which are active in par- 65 The present disclosure combines one of three easy to allel , a 2 - carbon ( C2 ) stream ( via glycolaldehyde ) and a implement, high yield C2 - streams for MEG production from 3 - carbon ( C3 ) stream ( via dihydroxyacetonephosphate xylose with an easy to implement IPA production stream via US 10,941,424 B2 33 34 the DHAP pathway. Surprisingly, the problem of the IPA and pyruvate ) is part of natural E. coli metabolism . One pathway, excess NADH production , complements the molecule of acetyl - CoA is condensed to another molecule of NADH requiring C2 part of MEG production . The combi- acetyl -CoA by the enzyme thiolase to produce acetoacetyl nation of these pathways leads to a high total yield potential COA . The CoA from acetoacetyl- CoA is recycled to a of 61 wt % , which is close to the maximum energetic yield 5 molecule of acetate by acetate : acetoacetyl -CoA transferase of 65 wt % for degradation of xylose into MEG and IPA , or hydrolase , generating acetyl - CoA and acetoacetate . assuming these products are produced in a 2 : 1 ratio . This Acetoacetate is decarboxylated by acetoacetate decarboxy high yield potential stems from the synergies of coupling the lase to acetone which is further reduced to IPA by a IPA pathway with the C2 - branch of MEG production from secondary alcohol dehydrogenase enzyme. IPA can further xylose . 10 be converted to propene by a dehydratase ( FIG . 4 ) . The proposed pathway in its basic form is not redox In another embodiment, the pathway for MEG + IPA co neutral, but has a small excess of 0.5 mol NADH per mol of production in E. coli comprises the following enzymes for consumed xylose. In an aerobic fermentation, oxidation of IPA production : thiolase , acetate : acetoacetyl- CoA transfer NADH can deliver just enough ATP to obtain sufficient, but ase or hydrolase, acetoacetate decarboxylase and secondary not excessive, ATP required for growth and maintenance 15 alcohol dehydrogenase . The MEG pathway via D -xylulose during the production phase without having a significantly 1 - phosphate comprises the following enzymes : D - xylulose negative impact on product formation . 1 - kinase , D -xylulose - 1 - phosphate aldolase and glycolalde The present disclosure solves a number of problems hyde reductase . In order to increase carbon flux to the associated with MEG and / or IPA production . In one embodi- desired pathway, three specific genes that could divert ment, the problem of a difficult to implement C3 pathway in 20 carbon flux were identified and deleted : xylB gene coding production of MEG from xylose is solved . In another for a xylulokinase ( this enzyme can divert carbon flux into embodiment, the problem of ATP shortage in production of the pentose phosphate pathway ), the aldA gene coding for MEG from xylose is solved . In another embodiment, the aldehyde dehydrogenase A ( can divert carbon flux from problem of loss of yield potential in production of MEG glycolaldehyde to glycolate instead of to MEG ) and the from glucose is solved . In another embodiment, the problem 25 IdhA gene coding for lactate dehydrogenase ( this enzyme of ATP shortage in production of MEG from glucose is can divert carbon flux from pyruvate to lactate instead of to solved . In another embodiment, the problem of excess acetyl - CoA ). NADH production in production of MEG from glucose is The first step of the pathway ( FIG . 2 ) is the natural solved . In another embodiment, the problem of loss of yield conversion of D - xylose into D - xylulose . D - xylulose nor potential in production of IPA from glucose is solved . In 30 mally enters the pentose phosphate pathway for energy and another embodiment, the problem of excess NADH produc- biomass generation , which is inhibited by the deletion of the tion in production of IPA from glucose is solved . xylB gene . In the engineered pathway, all carbon will be In one mbodiment, the pathway for MEG + IPA co - pro- re - directed to D -xylulose - 1 - phosphate by the D - xylulose duction in E. coli comprises the following enzymes for IPA 1 - kinase enzyme. D - xylulose - 1 -phosphate is then cleaved production : thiolase , acetate :acetoacetyl - CoA transferase or 35 into glycolaldehyde and dihydroxy acetone phosphate hydrolase , acetoacetate decarboxylase and secondary alco- ( DHAP ) by D - xylulose - 1 - phosphate aldolase . Production of hol dehydrogenase. The MEG pathway via ribulose- 1 -phos- MEG from glycolaldehyde and a three carbon compound phate comprises the following enzymes : D - tagatose 3 -epi- from DHAP ( for example , acetone , IPA and / or propene ) merase , D - ribulokinase , D - ribulose -phosphate aldolase and proceeds as described for FIG . 1 . glycolaldehyde reductase . In order to increase carbon flux to 40 In another embodiment, the pathway for MEG + IPA co the desired pathway, three specific genes that could divert production in E. coli comprises the following enzymes for carbon flux were identified and deleted : xylB gene coding IPA production: thiolase, acetate : acetoacetyl -CoA transfer for a xylulokinase ( this enzyme can divert carbon flux into ase or hydrolase , acetoacetate decarboxylase and secondary the pentose phosphate pathway ), the aldA gene coding for alcohol dehydrogenase. The MEG pathway via D - xylonate aldehyde dehydrogenase A ( can divert carbon flux from 45 comprises the following enzymes : xylose dehydrogenase , glycolaldehyde to glycolate instead of to MEG ) and the optionally xylonolactonase , xylonate dehydratase , 2 -keto - 3 IdhA gene coding for lactate dehydrogenase ( this enzyme deoxy - D -xylonate aldolase and glycolaldehyde reductase . In can divert carbon flux from pyruvate to lactate instead of to order to increase carbon flux to the desired pathway, three acetyl -CoA ). specific genes that could divert carbon flux were identified The first step of the pathway ( FIG . 1 ) is the natural 50 and deleted : xylA gene coding for a D - xylose isomerase conversion of D - xylose into D -xylulose . D - xylulose nor- ( this enzyme can divert carbon flux from D - xylose to mally enters the pentose phosphate pathway for energy and D - xylulose instead of to D - xylonate or D -xylonolactone ), biomass generation , which is inhibited by the deletion of the the aldA gene coding for aldehyde dehydrogenase A ( can xylB gene . In the engineered pathway, all carbon will be divert carbon flux from glycolaldehyde to glycolate instead re - directed to D - ribulose by the D - tagatose 3 - epimerase 55 of to MEG ) and the IdhA gene coding for lactate dehydro enzyme. D - ribulose is them converted to D -Ribulose - 1- genase ( this enzyme can divert carbon flux from pyruvate to phosphate by the native E. coli enzyme D - ribulokinase . lactate instead of to acetyl- CoA ). D - Ribulose - 1 - phosphate is cleaved into glycolaldehyde and The first step of the pathway ( FIG . 3 ) is the conversion of dihydroxy acetone phosphate ( DHAP ) by D -ribulose - phos- D - xylose into D - xylonate , either by a two - step process using phate aldolase . The further degradation of DHAP is termed 60 a xylose dehydrogenase to convert D - xylose to D -xylono the C3 branch , leading to IPA production . Degradation of lactone followed by conversion of D - xylonolactone to D -xy glycolaldehyde , termed the C2 -branch , can lead to ethylene lonate with a xylonolactonase enzyme, or by a one - step glycol or glycolate formation . Glycolate is the undesired process using a xylose dehydrogenase to convert D - xylose by -product that can be produced by the aldA gene product. directly to D -xylonate . The conversion of D - xylose to D -xy Ethylene glycol can be produced from glycolaldehyde using 65 lulose is inhibited by the deletion of the xylA gene . D -xy the enzyme glycolaldehyde reductase . The conversion of lonate is then converted to 2 -keto - 3 -deoxy -xylonate by a DHAP to acetyl -CoA ( through glyceraldehyde - 3 - phosphate xylonate dehydratase . 2 -keto - 3 -deoxy -xylonate is then US 10,941,424 B2 35 36 cleaved into glycolaldehyde and pyruvate by 2 -keto - 3 -de- mented in E. coli or other hosts . Acetogens, such as oxy - D - xylonate aldolase. Production of MEG from glyco- Clostridium ljungdahlii, can naturally utilize excess NADH laldehyde and a three carbon compound from pyruvate ( for generated in the presented xylose fermentation pathway example, acetone , IPA and / or propene ) proceeds as especially efficient to re - capture released CO2 in the Wood described for FIG . 1 . 5 Ljungdahl pathway to produce the intermediate acetyl -CoA , The pathway for MEG + IPA co - production in S. cerevisiae which can then be used to produce more acetone or related ( FIG . 5 ) comprises the following enzymes for IPA produc- products. CO , is released for instance in the pyruvate + CoA + tion : thiolase, acetate : acetoacetyl -CoA transferase or hydro- NAD + -acetyl -CoA + CO2 + 2 NADH or acetoacetone- > ac lase , acetoacetate decarboxylase and secondary alcohol tone + CO2 reactions . Furthermore, adding a second feed dehydrogenase . The MEG pathway via D - ribulose - 1 - phos- 10 stock , such as hydrogen gas ( Hz ) or syngas ( a composition phate comprises the following enzymes : D - tagatose 3 -epi- of H2 , CO , CO2 ) or methanol, can provide more reducing merase , D - ribulokinase, D - ribulose - phosphate aldolase and agents and even allow acetogens or similarly enabled organ glycolaldehyde reductase . Besides the two main pathways , isms to re - capture all CO2 released in the xylose fermenta S. cerevisiae is not capable of consuming xylose, so two tion pathway or CO2 present in the second feedstock . Such different pathways were tested for xylose consumption . 15 a mixotrophic fermentation can thus further increase yield Pathway 1 comprises 2 genes: Xyll converts D - Xylose to potential. In the case of MEG + acetone from xylose , CO2 xylitol , and Xyl2 converts Xylitol to D - xylulose . Pathway 2 fixation can lead to an increase of 25 % relative acetone or comprises only one gene: XylA that directly converts D -xy- 8 % total MEG + acetone product yield . With externally lose to D - xylulose . In order to increase carbon flux to the added reducing agents, calculated for full capture of all desired pathway, two specific genes that could divert carbon 20 xylose carbon , the yield potential is + 100 % for acetone flux were identified and deleted : XKS1 gene coding for a which equals + 32 % total product yield . xylulokinase ( this enzyme can divert carbon flux into the Yield potentials without CO2 fixation : pentose phosphate pathway ) and PHO13 gene coding for alkaline phosphatase ( can divert carbon from pentose phos 1 xylose- > 1 MEG + 1 / 2 acetone + 3 / 2 CO2 + 1 NADH phate pathway ). 25 1 xylose-> 1 MEG + 1 / 2 IPA + 3 / 2 CO2 + 1 / 2 NADH The first step of the pathway is the conversion of D - xylose into D - xylulose , directly or via the intermediate xylitol . Yield potentials with CO , fixation : D - xylulose is converted to D - ribulose by the D - tagatose 1 xylose - 1 MEG + 5 / 8 acetone + 9 / 8 CO2 3 - epimerase enzyme. D - ribulose is then converted to D -Ribulose - 1 - phosphate by D - ribulokinase . D - Ribulose - 1- 30 1 xylose ~ 1 MEG + 10 / 18 IPA + 4 / 3 CO2 phosphate is cleaved into glycolaldehyde and DHAP by Yield potentials with externally added reducing agents , D -ribulose - phosphate aldolase . DHAP enters the C3 branch for IPA production and glycolaldehyde can be converted calculated for fixation of CO2 equivalent to all CO2 released ethylene glycol using glycolaldehyde reductase . The con during xylose fermentation : version of DHAP to acetyl- CoA ( through glyceraldehyde- 35 1 xylose - 1 MEG + 1 acetone 3 -phosphate and pyruvate ) is part of the natural S. cerevisiae metabolism . One molecule of acetyl -CoA is condensed to 1 xylose-> 1 MEG + 1 IPA another molecule of acetyl - CoA by thiolase, producing While this present disclosure is theoretically sound and acetoacetyl - CoA . The CoA from acetoacetyl -CoA is synergistic, it surprisingly also avoids the biggest metabolic recycled to a molecule of acetate by acetate : acetoacetyl- 40 engineering and technical challenges of both MEG and IPA COA transferase or hydrolase, generating one molecule of fermentation processes: C3 - stream MEG fermentation and acetyl - CoA and one of acetoacetate . Acetoacetate is further carbon fixation for IPA process . decarboxylated by acetoacetate decarboxylase to acetone , In one embodiment, MEG is produced through the con which is further converted to IPA by a secondary alcohol version of glycolaldehyde in a C - 2 branch pathway and dehydrogenase enzyme. IPA can further be converted to 45 acetone is produced through the conversion of DHAP or propene by a dehydratase - isomerase ( FIG . 4 ) . pyruvate in a C - 3 branch pathway. In another embodiment, Surprisingly, the main problem of the IPA pathway, excess MEG is produced through the conversion of glycolaldehyde NADH production, is highly synergistic with a C2 -stream in a C - 2 branch pathway and IPA is produced through the for MEG production by complementing the NADH require- conversion of DHAP or pyruvate in a C - 3 branch pathway . ment of the C2 branch , while leaving just enough NADH to 50 In a further embodiment, MEG is produced through the generate required ATP in an aerobic process, without excess conversion of glycolaldehyde in a C - 2 branch pathway and ATP production. propene is produced through the conversion of DHAP or The described IPA process of US 2010/0311135 and other pyruvate in a C - 3 branch pathway. applications , without carbon fixation , can only achieve 34 wt In one embodiment, at least a portion of the excess NADH % versus the energetic maximum yield potential of 47 wt % . 55 produced in the C - 3 branch is used as a source of reducing Thus, this IPA pathway, even if implemented perfectly, can equivalents in the C - 2 branch . In another embodiment, at only achieve 72 % of the energetic maximum yield . In the least a portion of the excess NADH produced in the C - 3 present disclosure, the synergy of coupling IPA with MEG branch is used to produce ATP . production is such that, without necessity of CO2 fixation , In one embodiment, the co - produced MEG and acetone the combined products' yield potential of 61 wt % is very 60 comprise a yield potential greater than 90 % of the theoreti close ( 94 % ) to the energetic ( = theoretic , pathway indepen- cal maximum yield potential without carbon fixation . In dent) maximum yield potential of 65 wt % . another embodiment, the co -produced MEG and IPA com In a further embodiment, the inventive co -production prise a yield potential greater than 90 % of the theoretical pathway from xylose is implemented in an organism with maximum yield potential without carbon fixation . In a natural or added capability to fix CO , using excess reducing 65 further embodiment, the co - produced MEG and propene agents , thereby providing even higher yield potential. Vari- comprise a yield potential greater than 90 % of the theoreti ous CO2 fixation pathways are known and have been imple- cal maximum yield potential without carbon fixation . US 10,941,424 B2 37 38 In one embodiment, excess biomass formation is mini- Acetone is miscible with water and serves as an important mized and production of MEG and acetone is maximized . In solvent, typically for cleaning purposes in the laboratory. another embodiment, excess biomass formation is mini- Over 6.7 million tonnes are produced worldwide, mainly for use as a solvent and production of methyl methacrylate and mizedfurther andembodiment production, excess of MEG biomass and IPAformation is maximized is minimized . In a 5 bisphenol A. It is a common building block in organic chemistry . Familiar household uses of acetone are as the and production of MEG and propene is maximized . active ingredient in nail polish remover and as paint thinner . Monoethylene Glycol (MEG ) Isopropanol Monoethylene glycol ( MEG ) is an important raw material Isopropyl alcohol ( IUPAC name 2 - propanol) , also called for industrial applications. A primary use of MEG is in the isopropanol , is a compound with the chemical formula manufacture of polyethylene terephthalate ( PET ) resins, 10 C3H2O or C3H , OH or CH3CHOHCH3 . It is a colorless , films and fibers. In addition, MEG is important in the flammable chemical compound with a strong odor. It is the production of antifreezes , coolants, aircraft anti - icer and simplest example of a secondary alcohol, where the alcohol deicers and solvents . MEG is also known as ethane - 1,2 - diol. carbon atom is attached to two other carbon atoms some times shown as ( CH3 ) 2CHOH . It is a structural isomer of Ethylene glycol is also used as a medium for convective 15 propanol. It has a wide variety of industrial and household heat transfer in , for example, automobiles and liquid cooled uses . computers . Propene, also known as propylene or methyl ethylene, is Because of its high boiling point and affinity for water, an unsaturated organic compound having the chemical for ethylene glycol is a useful desiccant. Ethylene glycol is mula CzH .. It has one double bond , and is the second widely used to inhibit the formation of natural gas clathrates 20 simplest member of the alkene class of hydrocarbons. ( hydrates) in long multiphase pipelines that convey natural Propene is produced from fossil fuelspetroleum , natural gas from remote gas fields to a gas processing facility . gas , and , to a much lesser extent, coal . Propene is a byprod Ethylene glycol can be recovered from the natural gas and uct of oil refining and natural gas processing . reused as an inhibitor after purification treatment that Propene is the second most important starting product in removes water and inorganic salts . 25 the petrochemical industry after ethylene. It is the raw Minor uses of ethylene glycol include in the manufacture material for a wide variety of products. Manufacturers of the of capacitors , as a chemical intermediate in the manufacture plastic polypropylene account for nearly two thirds of all of 1,4 - dioxane, and as an additive to prevent corrosion in demand . Polypropylene is , for example, needed for the liquid cooling systems for personal computers. Ethylene production of films, packaging, caps and closures as well as glycol is also used in the manufacture of some vaccines ; as 30 for other applications. Propene is also used for the produc a minor ingredient in shoe polish , inks and dyes; as a rot and tion of important chemicals such as propylene oxide, acry fungal treatment for wood ; and as a preservative for bio- lonitrile, cumene , butyraldehyde, and acrylic acid . Over 85 logical specimens. million tonnes of propene is processed worldwide . Acetone Enzymes Acetone ( also known as propanone) is an organic com- 35 Exemplary enzymes that may be used in the MEG and pound with the formula ( CH3 ) 2CO . It is a colorless, volatile, three - carbon compound co - production pathways disclosed flammable liquid , and is the simplest ketone . herein are listed in Table 1 . TABLE 1 Required Natural/ enzyme Gene Source annotated Described Reaction EC no . activity candidate Organism function Isomerases that may be used in all xylulose dependent MEG pathways D - xylose + NAD ( P ) H <=> 1.1.1.307 xylose reductase xyli Scheffersomyces D - xylose Xylitol + NAD ( P ) + stipitis reductase D - xylose + NAD ( P ) H <=> 1.1.1.307 xylose reductase GRE3 Saccharomyces aldose Xylitol + NAD ( P ) + cerevisiae reductase Xylitol + NAD + <=> D 1.1.1.9 xylitol xyl2 Scheffersomyces D - xylulose xylulose + NADH dehydrogenase stipitis reductase Xylitol + NAD + <=> D 1.1.1.9 xylitol xdh1 Trichoderma Xylitol xylulose + NADH dehydrogenase reesei dehydrogenase D - xylopyranose <=> D 5.3.1.5 xylose isomerase xylA Pyromyces sp . xylose xylulose isomerase Glycolaldehyde reductases that may be used in all MEG pathways glycolaldehyde + 1.1.1. glycolaldehyde gldA Escherichia glycerol NAD (P ) H <=> reductase coli dehydrogenase monoethylene glycol + NAD ( P ) + glycolaldehyde + 1.1.1. glycolaldehyde GRE2 Saccharomyces methylglyoxal NAD ( P ) H <=> reductase cerevisiae reductase monoethylene glycol + NAD ( P ) + glycolaldehyde + 1.1.1. glycolaldehyde GRE3 Saccharomyces aldose NAD ( P ) H reductase cerevisiae reductase monoethylene glycol + NAD ( P ) + US 10,941,424 B2 39 40 TABLE 1 - continued glycolaldehyde + 1.1.1. glycolaldehyde yqhD * Escherichia Alcohol NAD ( P ) H <=> reductase coli dehydrogenase monoethylene glycol + NAD ( P ) + glycolaldehyde + 1.1.1. glycolaldehyde yqhD Escherichia Alcohol NAD ( P ) H reductase coli dehydrogenase monoethylene glycol + NAD ( P ) + glycolaldehyde + 1.1.1. glycolaldehyde ydjg Escherichia methylglyoxal NAD ( P ) H <=> reductase coli reductase monoethylene glycol + NAD ( P ) + glycolaldehyde + 1.1.1. glycolaldehyde fuco Escherichia lactaldehyde NAD ( P ) H <=> reductase coli reductase monoethylene glycol + NAD ( P ) + glycolaldehyde + 1.1.1. glycolaldehyde yafB Escherichia methylglyoxal NAD ( P ) H <=> reductase ( dkgB ) coli reductase monoethylene glycol + [multifunctional ] NAD ( P ) + glycolaldehyde + 1.1.1. glycolaldehyde yqhE Escherichia 2,5 -diketo - D NAD ( P ) H <=> reductase ( dkgA ) coli gluconic acid monoethylene glycol + reductase A NAD ( P ) + Enzymes that may be used in D - ribulose- 1 -phosphate pathway to MEG D - xylulose <=> D 5.1.3.- D - ribulose - 3 DTE Pseudomonas D - tagatose 3 ribulose epimerase cichorii epimerase D - xylulose <=> D 5.1.3.- D - ribulose - 3 CIKKR1 Rhodobacter D - tagatose 3 ribulose epimerase sphaeroides epimerase D - ribulose + ATP <=> D- 2.7.1.- D - ribulose - 1 fuck Escherichia L - fuculokinase ribulose - 1 -phosphate + kinase coli ADP D - ribulose - 1 -phosphate <=> 4.1.2.- D - ribulose - 1 fucA Escherichia L - fuculose glyceraldehyde + phosphate coli phosphate dihydroxyacetonephosphate aldolase aldolase Enzymes that may be used in D - xylulose - 1 - phosphate pathway to MEG D - xylulose + ATP <=> 2.7.1. D - xylulose 1 khk - C Homo sapiens ketohexokinase D - xylulose - 1 - phosphate + kinase ( cDNA ) C? ADP D - xylulose - 1 - phosphate <=> 4.1.2.- D - xylulose - 1- aldos Homo sapiens Fructose glyceraldehyde + phosphate ( cDNA ) bisphosphate dihydroxyacetonephosphate aldolase aldolase B Enzymes that may be used in xylonate pathway to MEG D - xylose + NAD + <=> 1.1.1.175 xylose xylB Caulobacter D - xylose 1 D - xylonolactone + NADH , dehydrogenase crescentus dehydrogenase or D - xylose + NAD + <=> D - xylonate + NADH D - xylose + NADP + <=> 1.1.1.179 xylose xdh1 , Haloferax D - xylose 1 D - xylonolactone + dehydrogenase HVO_B0028 volcanii dehydrogenase NADPH , or D - xylose + NADP + <=> D xylonate + NADPH D - xylose + NADP + <=> 1.1.1.179 xylose xyd1 Trichoderma D - xylose 1 D - xylonolactone + dehydrogenase reesei dehydrogenase NADPH , or D -xylose + NADP + <=> D xylonate + NADPH D - xylonolactone + 3.1.1.68 xylonolactonase xylc Caulobacter Xylonolactonase H20 <=> D - xylonate crescentus D - xylonate <=> 2 -keto- 4.2.1.82 xylonate xylD Caulobacter xylonate 3 -deoxy - xylonate + H2O dehydratase crescentus dehydratase D - xylonate <=> 2 -keto- 4.2.1.82 xylonate yjhG Escherichia xylonate 3 -deoxy - xylonate + H2O dehydratase coli dehydratase D - xylonate <=> 2 -keto- 4.2.1.82 xylonate yagF Escherichia xylonate 3 -deoxy -xylonate + H2O dehydratase coli dehydratase 2 -keto - 3 - deoxy 4.1.2.- 2 - keto - 3 -deoxy yjhH Escherichia Uncharacterized xylonate <=> D - pentonate coli lyase glycolaldehyde + aldolase pyruvate 2 -keto - 3 - deoxy 4.1.2.- 2- keto - 3 -deoxy yagE Escherichia Probable 2 - keto - 3 xylonate <=> D - pentonate coli deoxy - galactonate glycolaldehyde + aldolase aldolase pyruvate Enzymes that may be used in pathway to produce one or more three - carbon compounds 2 acetyl - Coa - > 2.3.1.9 acetyl coenzyme A thlA Clostridium acetyl coenzyme A acetoacetyl - CoA + COA acetyltransferase acetobutylicum acetyltransferase US 10,941,424 B2 41 42 TABLE 1 - continued 2 acetyl - Coa - > 2.3.1.9 acetyl coenzyme A atoB Escherichia acetyl coenzyme A acetoacetyl - CoA + COA acetyltransferase coli acetyltransferase 2 acetyl - Coa - > 2.3.1.9 acetyl coenzyme A ERG10 Saccharomyces acetyl coenzyme A acetoacetyl- CoA + COA acetyltransferase cerevisiae acetyltransferase acetoacetyl- CoA + 2.8.3.8 Acetyl - CoA : atoA Escherichia Acetyl- CoA : acetate - > acetoacetate + acetoacetate - COA coli acetoacetate acetyl- CoA transferase COA transferase subunit subunit acetoacetyl - CoA + 2.8.3.8 Acetyl - CoA : atoD Escherichia Acetyl - CoA : acetate - > acetoacetate + acetoacetate - COA coli acetoacetate acetyl - CoA transferase COA transferase subunit subunit acetoacetate - > 4.1.1.4 acetoacetate adc Clostridium acetoacetate acetone + CO2 decarboxylase acetobutylicum decarboxylase acetoacetate - > 4.1.1.4 acetoacetate adc Clostridium acetoacetate acetone + CO2 decarboxylase beijerinckii decarboxylase acetone + NAD ( P ) H 1.1.1.2 secondary adh Clostridium secondary 2 -propanol + NAD ( P ) + alcohol beijerinckii alcohol dehydrogenase dehydrogenase acetone + NAD ( P ) H - > 2- 1.1.1.2 secondary adh Clostridium alcohol propanol + NAD ( P ) + alcohol carboxidivorans dehydrogenase dehydrogenase NADH + NADP + < -- > 1.6.1.1 . Soluble pyridine udhA Escherichia Soluble pyridine NAD + + NADPH nucleotide coli nucleotide transhydrogenase transhydrogenase Hydrolases that may be used in pathway to produce one or more three -carbon compounds Acetoacetyl - CoA + 3.1.2.11 acetate : ctfA Clostridium butyrate H ( 2 ) 0 <=> COA + acetoacetyl acetobutylicum acetoacetate COA acetoacetate COA hydrolase transferase, complex A Acetoacetyl - CoA + 3.1.2.11 acetate : ctfB Clostridium butyrate H ( 2 ) 0 <=> COA + acetoacetyl acetobutylicum acetoacetate COA acetoacetate COA hydrolase transferase , subunit B Acetoacetyl - CoA + 3.1.2.11 acetate : atoA Escherichia Acetyl - CoA : H ( 2 ) 0 < > COA + acetoacetyl coli acetoacetate acetoacetate COA hydrolase ( strain K12 ) COA transferase subunit Acetoacetyl - CoA + 3.1.2.11 acetate : atoD Escherichia Acetyl - CoA : H ( 2 ) O <=> COA + acetoacetyl coli acetoacetate acetoacetate COA hydrolase ( strain K12 ) COA transferase subunit Gene Identifier SEQ ID SEQ ID Described Reaction ( nt) NO ( nt) Uniprot ID NO (AA ) Isomerases that may be used in all xylulose dependent MEG pathways D - xylose + NAD ( P ) H <=> GeneID : 82 , 83 P31867 84 Xylitol + NAD ( P ) + 4839234 D - xylose + NAD ( P ) H <=> GeneID : 85 , 86 P38715 87 Xylitol + NAD ( P ) + 856504 Xylitol + NAD + <=> D- GeneID : 88 , 89 P22144 90 xylulose + NADH 4852013 Xylitol + NAD + <=> D- ENA Nr.: 91 Q876R2 92 xylulose + NADH AF428150.1 D - xylopyranose < > D ENA Nr.: 93 , 94 Q9P8C9 95 xylulose CAB76571.1 Glycolaldehyde reductases that may be used in all MEG pathways glycolaldehyde + GeneID : 12 POA9S5 13 NAD ( P ) H <=> 12933659 monoethylene glycol + NAD ( P ) + glycolaldehyde + GeneID : 14 Q12068 15 NAD ( P ) H <=> 854014 monoethylene glycol + NAD ( P ) + glycolaldehyde + GeneID : 16 P38715 17 NAD ( P ) H <=> 856504 monoethylene glycol + NAD ( P ) + glycolaldehyde + GeneID : 18 , 19 Modified 20 NAD ( P ) H <=> 947493 version of monoethylene glycol + Q46856 ; NAD ( P ) + G149E US 10,941,424 B2 43 44 TABLE 1 - continued glycolaldehyde + GeneID : 21 , 22 046856 23 NAD ( P ) H <=> 947493 monoethylene glycol + NAD ( P ) + glycolaldehyde + GeneID : 24 P77256 25 NAD ( P ) H <=> 12930149 monoethylene glycol + NAD ( P ) + glycolaldehyde + GeneID : 26 , 27 POA9S1 28 NAD ( P ) H <=> 947273 monoethylene glycol + NAD ( P ) + glycolaldehyde + 545778205 29 P30863 30 NAD ( P ) H <=> monoethylene glycol + NAD ( P ) + glycolaldehyde + GeneID : 31 Q46857 32 NAD ( P ) H <=> 947495 monoethylene glycol + NAD ( P ) + Enzymes that may be used in D - ribulose - 1 - phosphate pathway to MEG D - xylulose <=> D ENA Nr.: 1 , 2 050580 3 ribulose BAA24429.1 D -xylulose <=> D ENA Nr.: 4 CIKKR1 5 ribulose FJ851309.1 D - ribulose + ATP <=> D- GeneID : 6 , 7 P11553 8 ribulose - 1 - phosphate + 946022 ADP D - ribulose - 1 - phosphate <=> GeneID : 9 , 10 POAB87 11 glyceraldehyde + 947282 dihydroxyacetonephosphate Enzymes that may be used in D - xylulose - 1 - phosphate pathway to MEG D -xylulose + ATP <=> GenBank : 53 , 54 P50053 55 D - xylulose - 1 - phosphate + CR456801.1 ADP D - xylulose - 1 -phosphate <=> CCDS6756.1 56 , 57 P05062 58 glyceraldehyde + dihydroxyacetonephosphate Enzymes that may be used in xylonate pathway to MEG D - xylose + NAD + <=> GeneID : 59 , 60 B8H1Z0 61 D - xylonolactone + NADH , 7329904 or D - xylose + NAD + <=> D -xylonate + NADH D - xylose + NADP + <=> GeneID : 62 D4GP29 63 D - xylonolactone + NADPH , 8919161 or D - xylose + NADP + <=> D xylonate + NADPH D - xylose + NADP + <=> ENA Nr.: 64 A0AO24SMV2 65 D - xylonolactone + EF136590.1 NADPH , or D - xylose + NADP + <=> D xylonate + NADPH D - xylonolactone + GeneID : 66 AOAOH3C6P8 67 H20 <=> D - xylonate 7329903 D -xylonate <=> 2 - keto- GeneID : 68 A0AOH3C6H6 69 3 - deoxy -xylonate + H2O 7329902 D -xylonate <=> 2 -keto- GeneID : 70 , 71 P39358 72 3 - deoxy - xylonate + H2O 946829 D -xylonate <=> 2 -keto- GeneID : 73 , 74 P77596 75 3 - deoxy - xylonate + H2O 944928 2 -keto - 3- deoxy GeneID : 76 , 77 P39359 78 xylonate <=> 948825 glycolaldehyde + pyruvate 2 - keto - 3 -deoxy GeneID : 79 , 80 P75682 81 xylonate <=> 944925 glycolaldehyde + pyruvate Enzymes that may be used in pathway to produce one or more three - carbon compounds 2 acetyl - Coa - > 3309200 33 , 34 P45359 35 acetoacetyl - CoA + COA 2 acetyl - Coa - > GeneID : 36 P76461 37 acetoacetyl - CoA + COA 946727 2 acetyl- Coa - > 856079 38 P41338 39 acetoacetyl- CoA + COA US 10,941,424 B2 45 46 TABLE 1 - continued acetoacetyl - CoA + 48994873 41 , 42 P76459 43 acetate - > acetoacetate + acetyl - CoA acetoacetyl- CoA + 48994873 44 , 45 P76458 46 acetate - > acetoacetate + acetyl- CoA acetoacetate - > 6466901 47 , 48 P23670 49 acetone + CO2 acetoacetate - > 149901357 50 , 51 A6M020 52 acetone + CO2 acetone + NAD ( P ) H - > 60592972 104 , 105 P25984 106 2 - propanol + NAD ( P ) + acetone + NAD ( P ) H - > 2- 308066805 107 COPZV5 108 propanol + NAD ( P ) + NADH + NADP + < -- > GeneID : 109 P27306 110 NAD + + NADPH 948461 Hydrolases that may be used in pathway to produce one or more three - carbon compounds Acetoacetyl - CoA + NCBI 96 P33752 97 H ( 2 ) 0 <=> COA + GeneID : acetoacetate 1116168 Acetoacetyl - CoA + NCBI 98 P23673 99 H ( 2 ) 0 <=> COA + GeneID : acetoacetate 1116169 Acetoacetyl - CoA + GeneID : 100 P76459 101 H ( 2 ) O <=> COA + 946719 acetoacetate Acetoacetyl - CoA + GeneID : 102 P76458 103 H ( 2 ) 0 <=> COA + 947525 acetoacetate

D - Tagatose 3 - Epimerase ( EC 5.1.3.31 ) FJ851309.1 , or homolog thereof. In a further embodiment, The present disclosure describes enzymes that can cata- 30 the D - tagatose 3 - epimerase comprises an amino acid lyze the epimerization of various ketoses at the C - 3 position , sequence selected from the group consisting of SEQ ID interconverting D - fructose and D - psicose , D - tagatose and NOs : 3 and 5. In yet a further embodiment, the D - tagatose D - sorbose , D - ribulose and D - xylulose , and L - ribulose and 3 - epimerase is encoded by a nucleic acid sequence selected L - xylulose . The specificity depends on the species. The from the group consisting of SEQ ID NOs : 1 , 2 and 4 . enzymes from Pseudomonas cichorii and Rhodobacter 35 D - tagatose 3 - epimerase may also be known as L - ribulose sphaeroides require Mn2 + . In one embodiment, the enzyme 3 - epimerase or ketose 3 - epimerase . is D - tagatose 3 - epimerase ( dte ). In another embodiment, the D - Ribulokinase ( EC 2.7.1.16 ) D - tagatose 3 - epimerase catalyzes the conversion of D -xy- The present disclosure describes enzymes that can cata lulose to D - ribulose . lyze the following reactions : 40 L - fuculose + ATPL- fuculose 1 - phosphate + ADP + H + OH OH HO . OH D - ribulose + ATPD - ribulose 1 - phosphate + ADP + H + OH 45 D - ribulokinase may also be known as L - fuculokinase , OH HO OH fuculokinase , ATP : L - fuculose 1 -phosphotransferase or D - ribulose L - fuculose kinase . D - xylulose Thus, in some embodiments, the disclosure provides for an enzyme that plays roles in the fucose degradation path In some embodiments, the D - tagatose 3 - epimerase is 50 way, the super pathway of fucose and rhamnose degradation from Pseudomonas spp . In another embodiment, the D - ta- and /or the D - arabinose degradation I pathway. gatose 3 - epimerase is from Pseudomonas cichorii . In In some embodiments , the enzyme can function as both an another embodiment, the D - tagatose 3 - epimerase is from L - fucolokinase and a D - ribulokinase , the second enzyme of Pseudomonas sp . ST - 24 . In another embodiment, the D - ta- the L - fucose and D - arabinose degradation pathways, respec gatose 3 - epimerase is from Mesorhizobium loti . In another 55 tively . embodiment, the D - tagatose 3 -epimerase is from Rhodo- In particular embodiments , the enzyme converts D - ribu bacter sphaeroides ( CIKKR1 ) . lose to D - ribulose - 1 - phosphate. In some embodiments, the In one embodiment, the D - tagatose 3 -epimerase is D - ribulokinase is from Escherichia coli . In some embodi encoded by one or more nucleic acid molecules obtained ments , the D - ribulokinase is encoded by the fuck gene . In from a microorganism selected from the group consisting of 60 one embodiment, the D - ribulokinase is encoded by one or Pseudomonas sp . , Mesorhizobium sp . and Rhodobacter sp . more nucleic acid molecules obtained from E. coli . In some In some embodiments, the D - tagatose 3 - epimerase is embodiments, the one or more nucleic acid molecules is encoded by one or more nucleic acid molecules obtained fuck , or homolog thereof. In a further embodiment, the from a microorganism selected from the group consisting of D - ribulokinase comprises an amino acid sequence set forth Pseudomonas cichorii, Pseudomonas sp . ST - 24 , Mesorhizo- 65 in SEQ ID NO : 8. In yet a further embodiment, the D - ribu bium loti and Rhodobacter sphaeroides. In some embodi- lokinase is encoded by a nucleic acid sequence selected from ments , the one or more nucleic acid molecules is dte and /or the group consisting of SEQ ID NOs : 6 and 7 . US 10,941,424 B2 47 48 D - Ribulose - 1 - Phosphate Aldolase ( EC 4.1.2.17 ) dehyde , a product of both the L - fucose and L - rhamnose The present disclosure describes enzymes that can cata- catabolic pathways, to L - 1,2 -propanediol , which is then lyze the following reversible reactions: excreted from the cell . Crystal structures of the enzyme have been solved , show L - fuculose 1 - phosphates ( S ) -lactaldehyde + dihydroxy 5 ing a domain - swapped dimer in which the metal, cofactor acetone phosphate ( DHAP ) and substrate binding sites could be located . An aspartate and three conserved histidine residues are required for Fe2 + D - ribulose 1 -phosphatessglycolaldehyde + dihydroxy binding and enzymatic activity . acetone phosphate (DHAP ) In vitro , the enzyme can be reactivated by high concen D - ribulose - 1 -phosphate aldolase may also be known as 10 trations of NAD + and efficiently inactivated by a mixture of L - fuculose -phosphate aldolase , L - fuculose 1 - phosphate Fe3 + and ascorbate or Fe2 + and H2O2 . Metal - catalyzed oxi aldolase or L - fuculose - 1 -phosphate ( S ) -lactaldehyde - lyase . dation of the conserved His277 residue is proposed to be the Thus, in some embodiments , the disclosure provides for cause of the inactivation . an enzyme that plays roles in the fucose degradation path Expression of Fuco enables engineered one - turn reversal way, the super pathway of fucose and rhamnose degradation 15 of the B - oxidation cycle . Fuc activity contributes to the and / or the D - arabinose degradation I pathway. In one conversion of isobutyraldehyde to isobutanol in an engi embodiment, the enzyme may use Zn2 + as a cofactor . In neered strain . another embodiment, an inhibitor of this enzyme may be In particular embodiments , the enzyme converts glycola phosphoglycolohydroxamate . ldehyde to MEG . In some embodiments, the glycolaldehyde 20 reductase is from Escherichia coli . In some embodiments , In some embodiments, the enzyme can function as both an the glycolaldehyde reductase is encoded by the fuco gene . L - fuculose - phosphate aldolase and a D - ribulose -phosphate In one embodiment, the glycolaldehyde reductase is aldolase , the third enzyme of the L - fucose and D - arabinose encoded by one or more nucleic acid molecules obtained degradation pathways, respectively . from a microorganism selected from E. coli and S. cerevi withThe a partiallysubstrate purifiedspecificity preparation of the enzyme from an has E. beencoli straintested . 25 siae . In another embodiment, the one or more nucleic acid Crystal structures of the enzyme and a number of point molecules is selected from gldA , GRE2 , GRE3 , yqhD , ydjG , mutants have been solved . The combination of structural fuco , yafB ( dkgB ), and / or yqhE ( dkgA ), or homolog data and enzymatic activity of mutants allowed modelling thereof. In another embodiment, the one or more nucleic and refinement of the catalytic mechanism of the enzyme. acid molecules is yqhD . In some embodiments , the yqhD The enantiomeric selectivity of the enzyme has been stud- 30 comprisesglycolaldehyde a G149E reductase mutation comprises . In a further an amino embodiment acid sequence, the ied . selected from the group consisting of SEQ ID NOs : 13 , 15 , In particular embodiments, the enzyme converts D - ribu 17 , 23 , 25 , 28 , 30 and 32. In yet a further embodiment, lose - 1 -phosphate to glycolaldehyde and DHAP. In some the glycolaldehyde reductase is encoded by a nucleic acid Escherichiaembodiments coli, the . InD -some ribulose embodiments - 1 -phosphate , the aldolase D - ribulose is from - 1- 35 sequence selected from the group consisting of SEQ ID phosphate aldolase is encoded by the fucA gene. In one NOs : 12 , 14 , 16 , 18 , 19 , 21 , 22 , 24 , 26 , 27 , 29 and 31 . embodiment, the D - ribulose - 1 - phosphate aldolase is Aldehyde Reductases encoded by one or more nucleic acid molecules obtained A number of aldehyde reductases may be used to convert from E. coli . In some embodiments , the one or more nucleic glycolaldehyde to MEG . acid molecules is fucA , or homolog thereof. In a further 40 An NADPH -dependent aldehyde reductase ( YqhD ) can embodiment, the D - ribulose - 1 -phosphate aldolase com catalyze the following reactions : prises an amino acid sequence set forth in SEQ ID NO : 11 . acetol+ NADP + Smethylglyoxal +NADPH + H + ( re In yet a further embodiment, the D - ribulose - 1 - phosphate versible , EC 1.1.1.- ) aldolase is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs : 9 and 10 . 45 an alcohol + NADP + san aldehyde + NADPH + H + (re Glycolaldehyde Reductase ( EC 1.1.1.77 ) versibility unspecified , EC 1.1.1.2 ) The present disclosure describes enzymes that can cata an aldehyde + NADP ++ H20 - a carboxylate +NA lyze the following reversible reactions: DPH + 2 H + ( EC 1.2.1.4 ) ethylene glycol + NAD + Sglycolaldehyde + NADH + H + 50 1,3 -propanediol + NADP +S3 - hydroxypropionalde hyde + NADPH + H + ( reversibility unspecified , EC 1.1.1.- ) ( S ) -propane - 1,2 - diol + NAD + 55 ( S ) -lactaldehyde + NADH + H + D - 3,4 -dihydroxybutanal + NADPHS1,3,4 -butanet Glycolaldehyde reductase may also be known as lactal- 55 riol + NADP + ( reversibility unspecified ) dehyde reductase , propanediol oxidoreductase , ( R ) [ or( S ) ]- YqhD is an NADPH - dependent aldehyde reductase that propane - 1,2 -diol :NAD + oxidoreductase or L -1,2 -propane- may be involved in glyoxal detoxification and /or be part of diol oxidoreductase . a glutathione - independent response to lipid peroxidation . Thus, in some embodiments, the disclosure provides for It has been reported that various alcohols , aldehydes, an enzyme that plays roles in the ethylene glycol degrada- 60 amino acids , sugars and a -hydroxy acids have been tested as tion pathway, the super pathway of glycol metabolism and substrates for YqhD . The purified protein only shows degradation , the anaerobic L - lactaldehyde degradation path- NADP - dependent alcohol dehydrogenase activity, with a way and / or the super pathway of fucose and rhamnose preference for alcohols longer than C ( 3 ) , but with Km values degradation . In one embodiment, the enzyme may use Fe2 + in the millimolar range , suggesting that they are not the as a cofactor. 65 physiological substrates. In contrast, YqhD does exhibit L - 1,2 - propanediol oxidoreductase is an iron -dependent short - chain aldehyde reductase activity with substrates such group III dehydrogenase . It anaerobically reduces L - lactal- as propanaldehyde, acetaldehyde, and butanaldehyde, as US 10,941,424 B2 49 50 well as acrolein and malondialdehyde . In a metabolically In particular embodiments, a multi - functional methylgly engineered strain , phenylacetaldehyde and 4 - hydroxyphe- oxal reductase converts glycolaldehyde to MEG . In some nylacetaldehyde are reduced to 2 - phenylethanol and 2- (4- embodiments , the multi - functional methylglyoxal reductase hydroxyphenyl ) ethanol by the endogenous aldehyde is from Escherichia coli . In some embodiments, the multi reductases YqhD , YjgB , and YahK . 5 functional methylglyoxal reductase is encoded by the dkgA Overexpression of YqhD increases 1,3 -propanediol oxi- gene . doreductase activity of the cell . E. coli has been engineered A multi - functional methylglyoxal reductase ( DkgB ) can to express YqhD for the industrial production of 1,3 -pro- catalyze the following reactions : panediol. YqhD activity contributes to the production of acetol + NADP + Smethylglyoxal + NADPH + H + (the isobutanol, 1,2 -propanediol , 1,2,4 - butanetriol and acetol as 10 reaction is physiologically favored in the oppo well . Mutation of yqhD enables production of butanol by an site direction , EC 1.1.1.- ) engineered one - turn reversal of the B - oxidation cycle . YqhD has furfural reductase activity , which appears to 4 - nitrobenzyl alcohol + NADP +S4 - nitrobenzalde cause growth inhibition due to depletion of NADPH in hyde + NADPH + H + ( reversibility unspecified , EC metabolically engineered strains that produce alcohol from 15 1.1.1.91 ) lignocellulosic biomass . 2 -keto - L - gulonate + NADP + 2,5 - didehydro- D - glu The crystal structure of YqhD has been solved at 2 Å conate + NADPH + H + ( the reaction is favored in resolution . YqhD is an asymmetric dimer of dimers, and the the opposite direction, EC 1.1.1.346 ) active site contains a Zn2 + ion . The NADPH cofactor is DkgB ( YafB ) is a member of the aldo - keto reductase nicotinamidemodified by hydroxylring. groups at positions 5 and 6 in the 20 (AKR ) subfamily 3F . DkgB was shown to have 2,5 - diketo Overexpression of yqhD leads to increased resistance to D - gluconate reductase, methylglyoxal reductase and 4 -ni reactive oxygen - generating compounds such as hydrogen trobenzaldehyde reductase activities . peroxide, paraquat , chromate and potassium tellurite . A dkgB is reported to encode 2,5 - diketo - D - gluconate yqhD deletion mutant shows increased sensitivity to these 25 reductasecoli . The (enzyme 25DKGR uses ) B , NADPHone of two as 25DKG the preferred reductases electron in E. compounds and to glyoxal , and contains increased levels of donor and is thought to be involved in ketogluconate reactive aldehydes that are generated during lipid peroxida metabolism . However, the specific activity of the enzyme tion . Conversely, yqhD deletion leads to increased furfural towards 2,5 - diketo - D - gluconate is reported to be almost tolerance . 1000 - fold lower than its activity towards methylglyoxal. In particular embodiments, an NADPH - dependent alde- 30 In particular embodiments, a multi - functional methylgly hyde reductase converts glycolaldehyde to MEG . In some oxal reductase converts glycolaldehyde to MEG . In some embodiments , the NADPH - dependent aldehyde reductase is embodiments, the multi- functional methylglyoxal reductase from Escherichia coli . In some embodime the NADPH is from Escherichia coli . In some embodiments , the multi dependent aldehyde reductase is encoded by the yqhD gene . functional methylglyoxal reductase is encoded by the dkgB A multi - functional methylglyoxal reductase ( DkgA ) can 35 gene . catalyze the following reactions : A methylglyoxal reductase ( YeaE ) can catalyze the fol acetol + NADP + Smethylglyoxal + NADPH + H + ( the lowing reaction: reaction is physiologically favored in the oppo site direction , EC 1.1.1.- ) acetol+ NADP + Smethylglyoxal +NADPH + H + ( the 40 reaction is physiologically favored in the oppo isobutanol + NADP + sisobutanal + NADPH + H + (re site direction , EC 1.1.1.- ) versibility unspecified , EC 1.1.1.- ) YeaE has been shown to have methylglyoxal reductase activity . ethyl- ( 2R )-methyl- (3S ) -hydroxybutanoate + NADP + Sethyl- 2- methylacetoacetate +NADPH + H + ( re The subunit structure of YeaE has not been determined , 45 but its amino acid sequence similarity to the aldo - keto versibility unspecified , EC 1.1.1.- ) reductases DkgA ( YqhE ) and DkgB ( YafB ) suggests that it 2 -keto -L -gulonate +NADP + 2,5 -didehydro - D - glu may be monomeric . conate + NADPH + H + ( the reaction is favored in In particular embodiments , a methylglyoxal reductase the opposite direction , EC 1.1.1.346 ) converts glycolaldehyde to MEG . In some embodiments, the DkgA ( YqhE ) belongs to the aldo - keto reductase ( AKR ) 50 methylglyoxal reductase is from Escherichia coli . In some family and has been shown to have methylglyoxal reductase embodiments, the methylglyoxal reductase is encoded by and beta - keto ester reductase activity . the yeaE gene. dkgA is reported to encode a 2,5 - diketo - D - gluconate A L - glyceraldehyde 3 -phosphate reductase (yghZ ) can reductase ( 25DKGR ) A , one of two 25DKG reductases in E. catalyze the following reactions : coli . The enzyme uses NADPH as the preferred electron 55 L - glyceraldehyde 3 -phosphate +NADPH + H + - > sn donor and is thought to be involved in ketogluconate glycerol 3 - phosphate + NADP + ( EC 1.1.1.- ) metabolism . The specific activity of the enzyme towards 2,5 - diketo - D - gluconate is reported to be almost 1000 - fold acetol + NADP +Smethylglyoxal + NADPH + H + ( the lower than its activity towards methylglyoxal. reaction is physiologically favored in the oppo Due to its low Km for NADPH , reduction of furans by 60 site direction , EC 1.1.1.- ) DkgA may deplete NADPH pools and thereby limit cellular Yghz is an L - glyceraldehyde 3 - phosphate ( L -GAP ) biosynthesis. A broad survey of aldehyde reductases showed reductase . The enzyme is also able to detoxify methylgly that DkgA was one of several endogenous aldehyde oxal at a low rate . YghZ defines the AKR14 ( aldo -keto reductases that contribute to the degradation of desired reductase 14 ) protein family . aldehyde end products of metabolic engineering. 65 L -GAP is not a natural metabolite and is toxic to E. coli . A crystal structure of DkgA has been solved at 2.16 Å L -GAP is a substrate of both the glycerol - 3 - phosphate and resolution . hexose phosphate transport systems of E. coli K - 12 . It has US 10,941,424 B2 51 52 been postulated that the physiological role of YghZ is the Gre2 is a versatile enzyme that catalyzes the stereoselec detoxification of L -GAP , which may be formed by non- tive reduction of a broad range of substrates including enzymatic racemization of GAP or by an unknown cellular aliphatic and aromatic ketones, diketones, as well as alde process . hydes, using NADPH as the cofactor. The crystal structure of the E. coli enzyme has been 5 The crystal structures of Gre2 from S. cerevisiae in an determined and is suggested to be a tetramer. However, apo - form at 2.00 Å and NADPH - complexed form at 2.40 Å others have found that the protein forms an octamer based on resolution have been solved . Gre2 forms a homodimer, each gel filtration and electron microscopy studies. subunit of which contains an N - terminal Rossmann - fold In particular embodiments , a L - glyceraldehyde 3 -phos- domain and a variable C - terminal domain , which partici phate reductase converts glycolaldehyde to MEG . In some 10 pates in substrate recognition . The induced fit upon binding embodiments, the L - glyceraldehyde 3 - phosphate reductase to the cofactor NADPH makes the two domains shift toward is from Escherichia coli . In some embodiments , the L - glyc- each other, producing an interdomain cleft that better fits the eraldehyde 3 - phosphate reductase is encoded by the yghZ substrate . Computational simulation combined with site gene . directed mutagenesis and enzymatic activity analysis An L - 1,2 -propanediol dehydrogenase / glycerol dehydro- 15 enabled characterization of a potential substrate -binding genase ( GIDA ) can catalyze the following reactions : pocket that determines the stringent substrate stereoselec tivity for catalysis . ( S )-propane - 1,2 -diol + NAD + Gacetol + NADH + H + Gre2 catalyzes the irreversible reduction of the cytotoxic ( reversible reaction ) compound methylglyoxal ( MG ) to ( S ) -lactaldehyde as an 20 alternative to detoxification of MG by glyoxalase I GLO1 . aminoacetone +NADH + H + ( R )-1 - aminopropan - 2 MG is synthesized via a bypath of glycolysis from dihy ol + NAD + ( EC 1.1.1.75 ) droxyacetone phosphate and is believed to play a role in cell glycerol + NAD + Ssdihydroxyacetone + NADH + H + cycle regulation and stress adaptation . GRE2 also catalyzes ( reversible reaction , EC 1.1.1.6 ) the reduction of isovaleraldehyde to isoamylalcohol. The The physiological function of the GIDA enzyme has long 25 enzyme serves to suppress isoamylalcohol- induced filamen been unclear . The enzyme was independently isolated as a tation by modulating the levels of isovaleraldehyde, the glycerol dehydrogenase and a D - 1 - amino - 2 - propanol: signal to which cells respond by filamentation . GRE2 is also NAD + oxidoreductase . At that time , D - 1 - amino - 2 - propanol involved in ergosterol metabolism . was thought to be an intermediate for the biosynthesis of In particular embodiments , an NADPH - dependent meth vitamin B12 , and although E. coli is unable to synthesize 30 ylglyoxal reductase converts glycolaldehyde to MEG . In vitamin B12 de novo , enzymes catalyzing the synthesis of some embodiments, the NADPH -dependent methylglyoxal this compound were sought. It was later found that GIDA reductase is from S. cerevisiae . In some embodiments , the was responsible for both activities . NADPH -dependent methylglyoxal reductase is encoded by The primary in vivo role of GIDA was recently proposed the GRE2 gene . to be the removal of dihydroxyacetone by converting it to 35 Thiolase / Acetyl Coenzyme A Acetyltransferase ( EC 2.3.1.9 ) glycerol. However, a dual role in the fermentation of glyc The present disclosure describes enzymes that can cata erol has also recently been established . Glycerol dissimila lyze the following reaction : tion in E. coli can be accomplished by two different path 2 acetyl - CoAsacetoacetyl - CoA + coenzyme A ( re ways . The glycerol and glycerophosphodiester degradation 40 versible reaction ) pathway requires the presence of a terminal electron accep Thiolase / Acetyl coenzyme A acetyltransferase may also tor and utilizes an ATP -dependent kinase of the Glp system , be known as acetyl -CoA - C - acetyltransferase , acetoacetyl which phosphorylates glycerol to glycerol - 3 - phosphate. COA thiolase , acetyl -CoA : acetyl -CoA C - acetyltransferase or However, upon inactivation of the kinase and selection for thiolase II . growth on glycerol, it was found that an NAD + -linked 45 Thus, in some embodiments, the disclosure provides for dehydrogenase, GIDA , was able to support glycerol fermen an enzyme that plays a role in acetoacetate degradation ( to tation . Recently, it was shown that GIDA was involved in acetyl CoA ) . In one embodiment, an inhibitor of this enzyme glycerol fermentation both as a glycerol dehydrogenase , may be acetoacetyl - CoA . producing dihydroxyacetone, and as a 1,2 - propanediol In particular embodiments, the enzyme converts acetyl panedioldehydrogenase from ,acetol regenerating. NAD + by producing 1,2 - pro- 50 CoA to acetoacetyl- CoA . In some embodiments, the thio The enzyme is found in two catalytically active forms, a lase /acetyl coenzyme A acetyltransferase is from large form of eight subunits and a small form of two Clostridium spp . In some embodiments , the thiolase / acetyl subunits . The large form appears to be the major species . coenzyme A acetyltransferase is from Clostridium aceto In particular embodiments, an L - 1,2 -propanediol dehy butylicum . In some embodiments , the thiolase /acetyl coen drogenase / glycerol dehydrogenase converts glycolaldehyde 55 zyme A acetyltransferase is from Clostridium thermosac to MEG . In some embodiments, the L - 1,2 - propanediol charolyticum . In some embodiments, the thiolase / acetyl dehydrogenase / glycerol dehydrogenase is from Escherichia coenzyme A acetyltransferase is from Bacillus cereus . In coli . In some embodiments , the L - 1,2 - propanediol dehydro some embodiments, the thiolase / acetyl coenzyme A acetyl genase / glycerol dehydrogenase is encoded by the gldA gene. 60 transferaseATCC 49840. is Infrom some Marinobacter embodiments hydrocarbonoclasticus, the thiolase / acetyl An NADPH - dependent methylglyoxal reductase ( GRE2 ) coenzyme A acetyltransferase is encoded by the thlA gene. from Saccharomyces cerevisiae can catalyze the following In some embodiments, the thiolase / acetyl coenzyme A reactions : acetyltransferase is from Escherichia coli . In some embodi ( S )-lactaldehyde + NADP + < smethylglyoxal + NADPH ments , the thiolase / acetyl coenzyme A acetyltransferase is 65 encoded by the atoB gene. 3 -methylbutanol + NAD ( P ) + < s -methylbutanal + NAD In one embodiment, the thiolase or acetyl coenzyme A ( P ) H acetyltransferase is encoded by one or more nucleic acid US 10,941,424 B2 53 54 molecules obtained from a microorganism selected from the acetate : acetoacetyl - CoA hydrolase is from Clostridium spp . group consisting of Clostridium sp . , Bacillus sp . , E. coli , In some embodiments, the acetate : acetoacetyl - CoA hydro Saccharomyces sp . and Marinobacter sp . In some embodi- lase is from Clostridium acetobutylicum . In another embodi ments , the thiolase or acetyl coenzyme A acetyltransferase is ment, the Acetoacetyl- CoA hydrolase is encoded by the ctfA encoded by one or more nucleic acid molecules obtained 5 ( subunit A ) and / or ctfB ( subunit B ) genes . from a microorganism selected from the group consisting of In a further embodiment, the acetyl- CoA :acetoacetate Clostridium acetobutylicum , Clostridium thermosaccharo- CoA transferase or acetate : acetoacetyl -CoA hydrolase com lyticum , Bacillus cereus , E. coli , Saccharomyces cerevisiae prises an amino acid sequence selected from the group and Marinobacter hydrocarbonoclasticus. In some embodi- consisting of SEQ ID NOs : 43 , 46 , 97 , 99 , 101 and 103. In ments, the one or more nucleic acid molecules is th1A , atoB 10 yet a further embodiment, the acetyl - CoA : acetoacetate - CoA and / or ERG10 , or homolog thereof. In a further embodi- transferase or acetate :acetoacetyl - CoA hydrolase is encoded ment, the thiolase or acetyl coenzyme A acetyltransferase by a nucleic acid sequence selected from the group consist comprises an amino acid sequence selected from the group ing of SEQ ID NOs : 41 , 42 , 44 , 45 , 96 , 98 , 100 and 102 . consisting of SEQ ID NO : 35 , 37 and 40. In yet a further Acetoacetate Decarboxylase ( EC 4.1.1.4 ) embodiment, the thiolase or acetyl coenzyme A acetyltrans- 15 The present disclosure describes enzymes that can cata ferase is encoded by a nucleic acid sequence selected from lyze the following reaction : the group consisting of SEQ ID NOs : 33 , 34 , 36 , 38 and 39 . Acetate : Acetoacetyl- CoA Transferase ( EC 2.8.3.- ) acetoacetate + H + acetone + CO2 The present disclosure describes enzymes that can cata- Acetoacetate decarboxylase may also be known as ADC , lyze the following reaction : 20 AADC or acetoacetate carboxy - lyase. Thus, in some embodiments, the disclosure provides for acetoacetate + acetyl - CoAsacetoacetyl - CoA + acetate an enzyme that plays roles in isopropanol biosynthesis, ( reversible reaction , EC 2.8.3.- ) pyruvate fermentation to acetone , the super pathway of Acetate : Acetoacetyl -CoA transferase may also be known Clostridium acetobutylicum acidogenic and solventogenic as acetoacetyl- CoA transferase or acetyl - CoA : acetoacetate- 25 fermentation and /or the super pathway of Clostridium aceto COA transferase . butylicum solventogenic fermentation . Thus, in some embodiments, the disclosure provides for Acetoacetate decarboxylase ( ADC ) plays a key role in an enzyme that plays a role in acetoacetate degradation ( to solvent production in Clostridium acetobutylicum . During acetyl COA ) . In one embodiment, inhibitors of this enzyme the acidogenic phase of growth , acids accumulate causing a may include acetyl- CoA and coenzyme A. 30 metabolic shift to solvent production . In this phase acids are The growth of E. coli on short - chain fatty acids ( C3 - C6 ) re - assimilated and metabolized to produce acetone , butanol requires the activation of the acids to their respective thio- and ethanol. esters . This activation is catalyzed by acetoacetyl - CoA trans- Preliminary purification and crystallization of the enzyme ferase . The reaction takes place in two half -reactions which has revealed that a lysine residue is implicated in the active involves a covalent enzyme -CoA . The enzyme undergoes 35 site . The enzyme is a large complex composed of 12 copies two detectable conformational changes during the reaction . of a single type of subunit. It is thought likely that the reaction proceeds by a ping - pong The enzyme of Clostridium acetobutylicum ATCC 824 mechanism . The enzyme can utilize a variety of short - chain has been purified and the adc gene encoding it cloned . The acyl - CoA and carboxylic acid substrates but exhibits maxi enzyme has also been purified from the related strain mal activity with normal and 3 - keto substrates . 40 Clostridium acetobutylicum DSM 792 and the gene cloned In particular embodiments, the enzyme converts and sequenced . The decarboxylation reaction proceeds by acetoacetyl -CoA to acetoacetate . In some embodiments, the the formation of a Schiff base intermediate . acetate :acetoacetyl - CoA transferase is from Clostridium ADC is a key enzyme in acid uptake, effectively pulling spp . In some embodiments, the acetate : acetoacetyl - CoA the COA - transferase reaction in the direction of acetoacetate transferase is from Clostridium acetobutylicum . In some 45 formation . embodiments, the acetate : acetoacetyl - CoA transferase is In particular embodiments, the enzyme converts acetoac from Escherichia coli . In some embodiments, the acetate : etate to acetone . In some embodiments , the acetoacetate acetoacetyl - CoA transferase is encoded by the atoA and decarboxylase is from Clostridium spp . In some embodi ato D genes . In another embodiment, the subunit composi- ments , the acetoacetate decarboxylase is from Clostridium tion of acetoacetyl -CoA transferase is [ (AtoA ) 2 ][ (AtoD )2 ], 50 acetobutylicum . In some embodiments, the acetoacetate with ( AtoA ) , being the B complex and ( AtoD ) , being the a decarboxylase is from Clostridium beijerinckii. In some complex . In one embodiment, the acetate : acetoacetyl - CoA embodiments, the acetoacetate decarboxylase is from transferase is a fused acetate : acetoacetyl -CoA transferase : a Clostridium cellulolyticum . In some embodiments , the subunit/ B subunit . In another embodiment, the acetate :ac- acetoacetate decarboxylase is from Bacillus polymyxa. In etoacetyl- CoA transferase is encoded by the ydiF gene . 55 some embodiments , the acetoacetate decarboxylase is from Acetate : Acetoacetyl- CoA Hydrolase ( EC 3.1.2.11 ) Chromobacterium violaceum . In some embodiments, the The present disclosure describes enzymes that can cata- acetoacetate decarboxylase is from Pseudomonas putida. In lyze the following reaction : another embodiment, the acetoacetate decarboxylase is encoded by the adc gene . acetoacetyl - CoA + H2O < COA + acetoacetate 60 In one embodiment, the acetoacetate decarboxylase is Acetoacetyl -CoA hydrolase may also be known as encoded by one or more nucleic acid molecules obtained acetoacetyl coenzyme A hydrolase, acetoacetyl CoA deacy- from a microorganism selected from the group consisting of lase or acetoacetyl coenzyme A deacylase. Clostridium sp . , Bacillus sp . , Chromobacterium sp . and This enzyme belongs to the family of hydrolases , specifi- Pseudomonas sp . In another embodiment, the acetoacetate cally those acting on thioester bonds . 65 decarboxylase is encoded by one or more nucleic acid In particular embodiments, the enzyme converts molecules obtained from a microorganism selected from the acetoacetyl - CoA to acetoacetate . In some embodiments, the group consisting of Clostridium acetobutylicum , US 10,941,424 B2 55 56 Clostridium beijerinckii, Clostridium cellulolyticum , Bacil- Ketohexokinase, or fructokinase , phosphorylates fructose lus polymyxa , Chromobacterium violaceum and Pseudomo- to fructose - 1 - phosphate. The enzyme is involved in fructose nas putida. In some embodiments, the one or more nucleic metabolism , which is part of carbohydrate metabolism . It is acid molecules encoding the acetoacetate decarboxylase is found in the liver, intestine and kidney cortex . ade , or homolog thereof. In a further embodiment, the 5 In human liver, purified fructokinase , when coupled with acetoacetate decarboxylase comprises an amino acid aldolase , has been discovered to contribute to an alternative sequence selected from the group consisting of SEQ ID mechanism to produce oxalate from xylitol . In coupled NOs : 49 and 52. In yet another embodiment, the acetoac- sequence , fructokinase and aldolase produce glycolalde etate decarboxylase is encoded by a nucleic acid sequence hyde , a precursor to oxalate, from D - xylulose via D - xylulose selected from the group consisting of SEQ ID NOs : 47 , 48 , 10 l - phosphate. 50 and 51 . In particular embodiments, the enzyme converts D -xylu Alcohol Dehydrogenase ( EC 1.1.1.- ) lose to D -xylulose - 1 - phosphate . In some embodiments , the The present disclosure describes enzymes that can cata- D - xylulose 1 - kinase is a ketohexokinase C. In some embodi lyze the reversible oxidation of primary or secondary alco- ments, the ketohexokinase C is from Homo sapiens. In some hols to aldehydes or ketones , respectively . In one embodi- 15 embodiments , the human ketohexokinase C is encoded by ment, the enzyme is a secondary alcohol dehydrogenase the khk - C gene . ( S - ADH ) and catalyzes the reduction of ketones such as In one embodiment, the D - xylulose 1 - kinase is encoded acetone into secondary alcohols such as 2 - propanol ( isopro- by one or more nucleic acid molecules obtained from Homo panol) . sapiens. In some embodiments, the one or more nucleic acid In some embodiments the S - ADH is from Burkholderia 20 molecules encoding the D - xylulose 1 - kinase is ketohexoki sp . In some embodiments , the S - ADH is from Burkholderia nase C ( khk - C ), or homolog thereof. In another embodi sp . AIU 652. In some embodiments, the S - ADH is from ment, the one or more nucleic acid molecules encoding the Alcaligenes sp . In some embodiments, the S - ADH is from D - xylulose 1 - kinase comprises an amino acid sequence set Alcaligenes eutrophus. In some embodiments, the S - ADH is forth in SEQ ID NO : 55. In a further embodiment, the one from Clostridium sp . In some embodiments, the S - ADH is 25 or more nucleic acid molecules encoding the D - xylulose from Clostridium ragsdalei. In some embodiments , the 1 - kinase is encoded by a nucleic acid sequence selected S - ADH is from Clostridium beijerinckii. In some embodi- from the group consisting of SEQ ID NOs : 53 and 54 . ments, the S - ADH is from Thermoanaerobacter sp . In some D -Xylulose - 1 -Phosphate Aldolase ( EC 4.1.2.- ) embodiments , the S - ADH is from Thermoanaerobacter The present disclosure describes enzymes that can cata brockii. In some embodiments, the S - ADH is from Ther- 30 lyze the conversion of D - xylulose - 1 - phosphate to glycolal moanaerobacter ethanolicus ( Clostridium thermohydrosul- dehyde and DHAP . In some embodiments , the conversion furicum ). In some embodiments, the S - ADH is encoded by can be catalyzed by a human aldolase B , which is also the adhB gene . In some embodiments, the S - ADH is from known as fructose - bisphosphate aldolase B or liver - type the trypanosomatid Phytomonas sp . In some embodiments, aldolase . the S - ADH is from Rhodococcus sp . In some embodiments , 35 Aldolase B is one of three isoenzymes ( A , B , and C ) of the the S - ADH is from Rhodococcus ruber . In some embodi- class I fructose 1,6 - bisphosphate aldolase enzyme ( EC ments , the S - ADH is from Methanobacterium palustre . In 4.1.2.13 ) , and plays a key role in both glycolysis and some embodiments, the S - ADH is from methanogenic gluconeogenesis. The generic fructose 1,6 - bisphosphate archaea Methanogenium liminatans. In some embodiments, aldolase enzyme catalyzes the reversible cleavage of fruc the S - ADH is from the parasitic protist Entamoeba his- 40 tose 1,6 -bisphosphate ( FBP ) into glyceraldehyde 3 - phos tolytica ( EhAdh1) . In some embodiments, the S - ADH is phate and dihydroxyacetone phosphate ( DHAP ) as well as from parasitic protozoan Tritrichomonas foetus. In some the reversible cleavage of fructose 1 -phosphate ( F1P ) into embodiments, the S - ADH is from human parasite Trichomo- glyceraldehyde and dihydroxyacetone phosphate . In mam nas vaginalis. mals , aldolase B is preferentially expressed in the liver, In some embodiments, the S - ADH is predicted from 45 while aldolase A is expressed in muscle and erythrocytes and homology and can be from Thermoanaerobacter mathranii , aldolase C is expressed in the brain . Slight differences in Micrococcus luteus, Nocardiopsis alba, Mycobacterium isozyme structure result in different activities for the two hassiacum , Helicobacter suis, Candida albicans, Candida substrate molecules : FBP and fructose 1 - phosphate. Aldo parapsilosis, Candida orthopsilosis, Candida metapsilosis, lase B exhibits no preference and thus catalyzes both reac Grosmannia clavigera and Scheffersomyces stipitis. 50 tions , while aldolases A and C prefer FBP . In a further embodiment, the alcohol dehydrogenase com Aldolase B is a homotetrameric enzyme, composed of prises an amino acid sequence selected from the group four subunits . Each subunit has a molecular weight of 36 consisting of SEQ ID NOs : 106 and 108. In yet another kDa and contains an eight - stranded aß barrel, which embodiment, the alcohol dehydrogenase is encoded by a encloses lysine 229 ( the Schiff -base forming amino acid that nucleic acid sequence selected from the group consisting of 55 is key for catalysis ). SEQ ID NOs : 104 , 105 and 107 . In particular embodiments, the enzyme converts D - xylu Dehydratase ( EC 4.2.1.- ) lose - 1 - phosphate to glycolaldehyde and DHAP. In some The present disclosure describes enzymes that can cata- embodiments , the D -xylulose - 1 - phosphate aldolase is an lyze the following reactions: aldolase B. In some embodiments, the aldolase B is from 60 Homo sapiens. In some embodiments, the human aldolase B isopropanolsspropene + H20 is encoded by the ALDOB gene . D - Xylulose 1 -Kinase ( EC 2.7.1.- ) In one embodiment, the D - xylulose - 1 - phosphate aldolase The present disclosure describes enzymes that can cata- is encoded by one or more nucleic acid molecules obtained lyze the conversion of D - xylulose to D - xylulose - 1 -phos- from Homo sapiens. In another embodiment, the one or phate . In some embodiments, the conversion can be cata- 65 more nucleic acid molecules encoding the D - xylulose -1 lyzed by a human ketohexokinase C ( khk - C ), also known as phosphate aldolase is aldolase B ( ALDOB ) , or homolog fructokinase . thereof. In some embodiments , the one or more nucleic acid US 10,941,424 B2 57 58 molecules encoding the D - xylulose - 1 - phosphate aldolase phorylation of 1 -deoxy - D - xylulose. This would allow a comprises an amino acid sequence set forth in SEQ ID NO : potential salvage pathway for generating 1- deoxy -D -xylu 58. In some embodiments , the one or more nucleic acid lose 5 - phosphate for use in the biosynthesis of terpenoids, molecules encoding the D - xylulose - 1 -phosphate aldolase is thiamine and pyridoxal. The rate of phosphorylation of encoded by a nucleic acid sequence selected from the group 5 1 - deoxy - D - xylulose is 32 - fold lower than the rate of phos consisting of SEQ ID NOs : 56 and 57 . phorylation of D - xylulose . D - Xylose Isomerase ( EC 5.3.1.5 ) The kinetic mechanism of the bacterial enzyme has been The present disclosure describes enzymes that can cata studied , suggesting a predominantly ordered reaction lyze the following reversible reaction : mechanism . The enzyme undergoes significant conforma D - xylopyranoses D - xylulose 10 tional changes upon binding of the substrate and of ATP . D - xylose isomerase may also be known as xylose Two conserved aspartate residues , D6 and D233 , were found isomerase or D - xylose ketol - isomerase . to be essential for catalytic activity, and a catalytic mecha Thus, in some embodiments, the disclosure provides for nism has been proposed . an enzyme that plays a role in xylose degradation . 15 Crystal structures of bacterial xylulokinase in the apo Xylose isomerase catalyzes the first reaction in the form and bound to D - xylulose have been determined at 2.7 catabolism of D - xylose . and 2.1 X resolution , respectively . Two conserved histidine residues, H101 and H271 , were In particular embodiments, the enzyme converts D - xylu shown to be essential for catalytic activity . The fluorescence lose to D - xylulose - 5 -phosphate . In some embodiments, the of two conserved tryptophan residues , W49 and W188 , is 20 D - xylulose - 5 - kinase is from Escherichia coli . In some quenched during binding of xylose , and W49 was shown to embodiments , the D - xylulose - 5 - kinase is encoded by the be essential for catalytic activity . The presence of Mg2 + , xylB gene. In some embodiments, the D - xylulose - 5 - kinase Mn2 + or Co2 + protects the enzyme from thermal denatur- is from Saccharomyces cerevisiae . In some embodiments ation . the D - xylulose - 5 - kinase is encoded by the XKS1 gene . In The subunit composition has not been established experi- 25 some embodiments, the D - xylulose - 5 -kinase is from Pichia mentally. stipitis . In some embodiments the D - xylulose - 5 - kinase is In particular embodiments, the enzyme converts D - xylose encoded by the XYL3 gene . to D - xylulose. In some embodiments , the D - xylose In some embodiments , a recombinant microorganism isomerase is from Escherichia coli . In some embodiments, producing MEG and a three - carbon compound comprises a the D - xylose isomerase is encoded by the xylA gene . 30 deletion , insertion , or loss of function mutation in a gene In some embodiments, a recombinant microorganism encoding a D -xylulose - 5 - kinase to prevent the conversion of producing MEG and a three -carbon compound comprises a D - xylulose to D -xylulose - 5 - phosphate and instead shunt the deletion , insertion , or loss of function mutation in a gene reaction toward conversion of D - xylulose to D - xylulose - 1 encoding a D - xylose isomerase to prevent conversion of phosphate . D -xylose to D - xylulose and instead shunt the reaction 35 Xylose Dehydrogenase ( EC 1.1.1.175 or EC 1.1.1.179 ) toward the conversion of D - xylose to D - xylonate. The present disclosure describes enzymes that can cata In one embodiment, the recombinant microorganism lyze the following reactions: comprises an endogenous or exogenous xylose isomerase that catalyzes the conversion of D - xylose to D - xylulose. In aldehydo - D - xylose + NAD ++ H20-> D - xylonate + NADH + 2 H + one embodiment, the xylose isomerase is exogenous . In 40 another embodiment, the xylose isomerase is encoded by one or more nucleic acid molecules obtained from Pyromy a - D -xylopyranose + NAD + SD - xylonolactone + ces sp . In another embodiment, the one or more nucleic acid NADH + H + ( reversibility unspecified , EC molecules encoding the xylose isomerase is xyl? , or 1.1.1.175 ) homolog thereof. In yet another embodiment, the one or 45 Xylose dehydrogenase may also be known as D - xylose more nucleic acid molecules encoding the xylose isomerase dehydrogenase, D - xylose 1 - dehydrogenase, (NAD + ) - linked comprises an amino acid sequence set forth in SEQ ID NO : D - xylose dehydrogenase , NAD + -D - xylose dehydrogenase , 95. In a further embodiment, the one or more nucleic acid D - xylose : NAD + 1 - oxidoreductase molecules encoding the xylose isomerase is encoded by a D - Xylose dehydrogenase catalyzes the NAD + -dependent nucleic acid sequence selected from the group consisting of 50 oxidation of D - xylose to D -xylonolactone . This is the first SEQ ID NOs : 93 and 94 . reaction in the oxidative , non - phosphorylative pathway for D - Xylulose - 5 - Kinase / Xylulokinase the degradation of D - xylose in Caulobacter crescentus . This The present disclosure describes enzymes that can cata pathway is similar to the pathway for L - arabinose degrada lyze the following reactions: tion in Azospirillum brasilense . The amino acid sequence of D - xylulose + ATP-> D - xylulose 5 - phosphate + ADP + H + 55 the C. crescentus enzyme is unrelated to that of xylose ( EC 2.7.1.17 ) dehydrogenase from the archaeon Haloarcula marismortui , or the L - arabinose 1 - dehydrogenase of Azospirillum brasi ATP + 1 -deoxy - D - xylulose - 1 - deoxy - D - xylulose lense . 5 - phosphate + ADP + H + (EC 2.7.1.- ) D - xylose is the preferred substrate for recombinant D -xy D - xylulose - 5 - kinase may also be known as xylulose 60 lose dehydrogenase from Caulobacter crescentus . The kinase or xylulokinase. enzyme can use L - arabinose , but it is a poorer substrate . The Xylulokinase catalyzes the phosphorylation of D - xylu- Km for L - arabinose is 166 mM . Other substrates such as lose , the second step in the xylose degradation pathway, D - arabinose , L - xylose , D - ribose , D - galactose , D - glucose producing D - xylulose - 5 - phosphate, an intermediate of the and D - glucose - 6 - phosphate showed little or no activity in pentose phosphate pathway . 65 the assay , as measured by NADH production . C. crescentus In the absence of substrate, xylulokinase has weak D - xylose dehydrogenase can convert D - xylose to D -xy ATPase activity . Xylulokinase can also catalyze the phos- lonate directly . US 10,941,424 B2 59 60 Partially purified , native D - xylose dehydrogenase from C. organism selected from Caulobacter sp . and Haloferax sp . crescentus had a Km of 70 uM for D - xylose . This value was In another embodiment, the xylonolactonase is encoded by lower than the Km of 760 uM for the recombinant, His- one or more nucleic acid molecules obtained from a micro tagged enzyme. organism selected from the group consisting of Caulobacter In some embodiments, the D - Xylose dehydrogenase is 5 crescentus, Haloferax volcanii and Haloferax gibbonsii . In from the halophilic archaeon Haloferax volcanii . The some embodiments, the one or more nucleic acid molecules Haloferax volcanii D - Xylose dehydrogenase catalyzes the encoding the xylonolactonase is xylc , or homolog thereof. first reaction in the oxidative xylose degradation pathway of In a further embodiment, the one or more nucleic acid the halophilic archaeon Haloferax volcanii . The H. volcanii molecules encoding the xylonolactonase comprises an D - Xylose dehydrogenase shows 59 % amino acid sequence 10 amino acid sequence set forth in SEQ ID NO : 67. In yet identity to a functionally characterized xylose dehydroge- another embodiment, the one or more nucleic acid mol nase from Haloarcula marismortui and 56 % identity to an ecules encoding the xylonolactonase is encoded by a nucleic ortholog in Halorubrum lacusprofundi, but is only 11 % acid sequence set forth in SEQ ID NO : 66 . identical to the bacterial NAD + -dependent xylose dehydro- Xylonate Dehydratase ( EC 4.2.1.82 ) genase from Caulobacter crescentus CB15 . 15 The present disclosure describes enzymes that can cata In particular embodiments, the enzyme converts D - xylose lyze the following reaction : to D - xylonolactone . In some embodiments, the D - Xylose D - xylonates2- keto - 3 - deoxy - D -xylonate + H2O dehydrogenase is from Caulobacter crescentus. In some embodiments, the D - Xylose dehydrogenase is encoded by This enzyme belongs to the family of lyases , specifically the xylB gene . In some embodiments, the D - Xylose dehy- 20 the hydro - lyases, which cleave carbon - oxygen bonds . This drogenase is from Haloferax volcanii. In some embodi enzyme participates in pentose and glucuronate interconver ments , the D - Xylose dehydrogenase is from Haloarcula sions . marismortui. In some embodiments , the D - Xylose dehydro Xylonate dehydratase may also be known as D - xylonate genase is from Halorubrum lacusprofundi. In some embodi hydro - lyase, D -xylo - aldonate dehydratase or D - xylonate ments , the D - Xylose dehydrogenase is encoded by the xdh 25 dehydratase . gene . In particular embodiments, the enzyme converts D - xy In one embodiment, the xylose dehydrogenase is encoded lonate to 2 -keto - 3 -deoxy - D - xylonate . In some embodi by one or more nucleic acid molecules obtained from a ments, the xylonate dehydratase is from Caulobacter cres microorganism selected from the group consisting of Cau centus. In some embodiments, the xylonate dehydratase is lobacter sp . , Haloarcula sp . , Haloferax sp . , Halorubrum sp . 30 encoded by the xylD gene . In some embodiments , the and Trichoderma sp . In another embodiment, the xylose xylonate dehydratase is from Escherichia coli . In some dehydrogenase is encoded by one or more nucleic acid embodiments, the xylonate dehydratase is encoded by the molecules obtained from a microorganism selected from the yjhG gene . In some embodiments , the xylonate dehydratase group consisting of Caulobacter crescentus, Haloarcula is encoded by the yagF gene . In some embodiments , the marismortui, Haloferax volcanii, Halorubrum lacuspro- 35 xylonateembodiments dehydratase, the xylonate is from dehydratase Haloferax isvolcanii encoded. In by some the fundi and Trichoderma reesei. In some embodiments, the xad gene . In some embodiments, the xylonate dehydratase is one or more nucleic acid molecules encoding the xylose from Sulfolobus solfataricus. dehydrogenase is selected from xylB , xdh1 ( HVO_B0028 ) In one embodiment, the xylonate dehydratase is encoded theand one/ or xyd? or more , or nucleichomolog acid thereof molecules. In a furtherencoding embodiment the xylose, 40 by one or more nucleic acid molecules obtained from a dehydrogenase comprises an amino acid sequence selected microorganism selected from the group consisting of Cau from the group consisting of SEQ ID NOs : 61 , 63 and 65 . lobacter sp . , Sulfolobus sp . and E. coli . In another embodi In yet another embodiment, the one or more nucleic acid ment, the xylonate dehydratase is encoded by one or more molecules encoding the xylose dehydrogenase is encoded by nucleic acid molecules obtained from a microorganism a nucleic acid sequence selected from the group consisting 45 selected from the group consisting of Caulobacter crescen of SEQ ID NOs : 59 , 60 , 62 and 64 . tus , Sulfolobus solfataricus and E. coli . In some embodi Xylonolactonase ( 3.1.1.68 ) ments, the one or more nucleic acid molecules encoding the The present disclosure describes enzymes that can cata xylonate dehydratase is selected from xylD , yjhG and / or lyze the following reaction : yagF , or homolog thereof. In a further embodiment, the one 50 or more nucleic acid molecules encoding the xylonate D - xylono - 1,4 - lactone + H20 GD - xylonate dehydratase comprises an amino acid sequence selected This enzyme belongs to the family of hydrolases , specifi from the group consisting of SEQ ID NOs : 69 , 72 and 75 . cally those acting on carboxylic ester bonds. This enzyme In yet another embodiment, the one or more nucleic acid participates in pentose and glucuronate interconversions. molecules encoding the xylonate dehydratase is encoded by Xylonolactonase may also be known as D -xylonolacto- 55 a nucleic acid sequence selected from the group consisting nase , xylono - 1,4 - lactonase, xylono - gamma -lactonase or of SEQ ID NOs : 68 , 70 , 71 , 73 and 74 . D -xylono - 1,4 - lactone lactonohydrolase. 2 -Keto - 3 - Deoxy - D -Pentonate Aldolase ( 4.1.2.28 ) In particular embodiments, the enzyme converts D - xylo The present disclosure describes enzymes that can cata nolactone to D - xylonate . In some embodiments, the D -xy lyze the following reaction : lonolactonase is from Haloferax sp . In some embodiments, 60 2 - dehydro - 3 - deoxy - D -pentonatessglycolaldehyde + the D - xylonolactonase is from Haloferax volcanii . In some pyruvate ( reversibility unspecified ) embodiments, the D -xylonolactonase is from Haloferax This enzyme belongs to the family of lyases , specifically gibbonsii . In some embodiments, the D - xylonolactonase is the aldehyde - lyases, which cleave carbon - carbon bonds . from Caulobacter crescentus . In some embodiments, the This enzyme participates in pentose and glucuronate inter D -xylonolactonase is encoded by the xylC gene . 65 conversions. In one embodiment, the xylonolactonase is encoded by 2 -keto - 3 - deoxy - D -pentonate aldolase may also be known one or more nucleic acid molecules obtained from a micro- as 2 - dehydro - 3 -deoxy - D -pentonate glycolaldehyde -lyase US 10,941,424 B2 61 62 ( pyruvate - forming ), 2 - dehydro - 3 -deoxy - D - pentonate aldo- In parallel pathways utilizing the same enzymes, D - ara lase , 3 -deoxy - D -pentulosonic acid aldolase, and 2 -dehydro- binose and L - xylose can be metabolized to dihydoxy -ac 3 - deoxy - D - pentonate glycolaldehyde - lyase . etone phosphate and glycolaldehyde, which is oxidized to YjhH appears to be a 2 - dehydro - 3 -deoxy - D -pentonate glycolate by aldehyde dehydrogenase A. aldolase . Genetic evidence suggests that YagE may also 5 Crystal structures of the enzyme alone and in ternary and function as a 2 -dehydro - 3 -deoxy - D -pentonate aldolase . binary complexes have been solved . yagE is part of the prophage CP4-6 . Aldehyde dehydrogenase A is only present under aerobic A yjhH yagE double mutant cannot use D - xylonate as the conditions and is most highly induced by the presence of sole source of carbon , and crude cell extracts do not contain fucose, rhamnose or glutamate . The enzyme is inhibited by 2 - dehydro - 3 - deoxy - D - pentonate aldolase activity. Both phe- 10 NADH , which may act as a switch to shift from oxidation of notypes are complemented by providing yjhH on a plasmid . lactaldehyde to its reduction by propanediol oxidoreductase . ArcA appears to activate yjhH gene expression under AldA is upregulated during short - term adaptation to glucose anaerobiosis. Two putative ArcA binding sites were identi- limitation . fied 211 and 597 bp upstream of this gene , but no promoter Based on sequence similarity, AldA was predicted to be a upstream of it has been identified. 15 succinate -semialdehyde dehydrogenase. The crystal structure of YagE suggests that the protein is Regulation of aldA expression has been investigated . The a homotetramer. Co - crystal structures of YagE in the pres- gene is regulated by catabolite repression, repression under ence of pyruvate and 2 -keto - 3 - deoxygalactonate have been anaerobic conditions via ArcA , and induction by the carbon solved . source . In particular embodiments , the enzyme converts 2 - keto- 20 In particular embodiments , the enzyme converts glycola 3 - deoxy - xylonate to glycolaldehyde and pyruvate . In some ldehyde to glycolate . In some embodiments, the glycolal embodiments , the 2 -keto - 3 -deoxy - D - pentonate aldolase is dehyde dehydrogenase is from Escherichia coli . In some from Pseudomonas sp . In some embodiments, the 2 -keto- embodiments, the glycolaldehyde dehydrogenase is encoded 3 -deoxy - D -pentonate aldolase is from Escherichia coli . In by the aldA gene . some embodiments, the 2 -keto - 3 - deoxy - D - pentonate aldo- 25 In some embodiments , a recombinant microorganism lase is encoded by the yjhH gene . In some embodiments , the producing MEG and a three - carbon compound comprises a 2 - keto - 3 - deoxy - D - pentonate aldolase is encoded by the deletion , insertion , or loss of function mutation in a gene yagE gene . encoding a glycolaldehyde dehydrogenase to prevent the In one embodiment, the 2 -keto - 3 - deoxy - D - pentonate production of glycolic acid from glycolaldehyde and instead aldolase is encoded by one or more nucleic acid molecules 30 shunt the reaction toward conversion of glycolaldehyde to obtained from a microorganism selected from Pseudomonas MEG . sp . and E. coli . In another embodiment, the 2 - keto - 3 -deoxy- Lactate Dehydrogenase ( 1.1.1.28 ) D -pentonate aldolase is encoded by one or more nucleic acid The present disclosure describes enzymes that can cata molecules obtained from E. coli . In some embodiments, the lyze the following reaction : one or more nucleic acid molecules encoding the 2 - keto - 3- 35 deoxy - D -pentonate aldolase is selected from yjhH and / or ( R ) -lactate + NAD + < pyruvate + NADH + H + yagE , or homolog thereof. In a further embodiment, the one Lactate dehydrogenase ( LDH ) is an enzyme found in or more nucleic acid molecules encoding the 2 -keto - 3- nearly all living cells such as in animals , plants and pro deoxy - D -pentonate aldolase comprises an amino acid karyotes. LDH catalyzes the conversion of lactate to pyruvic sequence selected from the group consisting of SEQ ID 40 acid and back , as it converts NADH to NAD + and back . A NOs : 78 and 81. In yet another embodiment, the one or more dehydrogenase is an enzyme that transfers a hydride from nucleic acid molecules encoding the 2- keto -3 - deoxy - D- one molecule to another. pentonate aldolase is encoded by a nucleic acid sequence LDH exist in four distinct enzyme classes . The most selected from the group consisting of SEQ ID NOs : 76 , 77 , common one is NAD ( P ) -dependent L - lactate dehydroge 79 and 80 . 45 nase . Other LDHs act on D - lactate and / or are dependent on Glycolaldehyde Dehydrogenase ( 1.2.1.21 ) cytochrome c : D - lactate dehydrogenase ( cytochrome) and The present disclosure describes enzymes that can cata- L - lactate dehydrogenase ( cytochrome ) . lyze the following reaction : LDH has been of medical significance because it is found extensively in body tissues , such as blood cells and heart glycolaldehyde + NAD ++ H20 ssglycolate + NADH + 50 muscle . Because it is released during tissue damage , it is a 2H + marker of common injuries and disease such as heart failure . This enzyme belongs to the family of oxidoreductases, Lactate dehydrogenase may also be known as lactic acid specifically those acting on the aldehyde or oxo group of dehydrogenase , ( R ) -lactate :NAD + oxidoreductase or D - lac donor with NAD + or NADP + s acceptor. This enzyme tate dehydrogenase - fermentative. participates in glyoxylate and dicarboxylate metabolism . 55 In E. coli , lactate dehydrogenase (LdhA ) is a soluble Glycolaldehyde dehydrogenase may also be known as NAD - linked lactate dehydrogenase (LDH ) that is specific glycolaldehyde :NAD + oxidoreductase or glycol aldehyde for the production of D - lactate . LdhA is a homotetramer and dehydrogenase . shows positive homotropic cooperativity under higher pH In E. coli aldehyde dehydrogenase A ( AldA ) is an enzyme conditions . of relatively broad substrate specificity for small a -hydroxy- 60 E. coli contains two other lactate dehydrogenases : D -lac aldehyde substrates. It is thus utilized in several metabolic tate dehydrogenase and L - lactate dehydrogenase . Both are pathways. membrane - associated flavoproteins required for aerobic L - fucose and L - rhamnose are metabolized through par- growth on lactate . allel pathways which converge after their corresponding LdhA is present under aerobic conditions but is induced aldolase reactions yielding the same products: dihydoxy- 65 when E. coli is grown on a variety of sugars under anaerobic acetone phosphate and L - lactaldehyde. Aerobically , alde- conditions at acidic pH . Unlike most of the genes involved hyde dehydrogenase A oxidizes L - lactaldehyde to L - lactate . in anaerobic respiration , IdhA is not activated by Fnr; rather US 10,941,424 B2 63 64 the ArcAB system and several genes involved in the control tinguishes the P. stipitis and the C. shehatae enzymes from of carbohydrate metabolism ( csrAB and mlc ) appear to most other ALRs so far isolated from mammalian or micro regulate expression . The expression of IdhA is negatively bial sources . The yeast Candida tenuis CBS 4435 produces affected by the transcriptional regulator ArcA . IdhA belongs comparable NADH- and NADPH -linked aldehyde -reducing to the o32 regulon . 5 activities during growth on D - xylose . The IdhA gene is a frequent target for mutations in In particular embodiments, the enzyme converts D - xylose metabolic engineering, most often to eliminate production of to xylitol. In some embodiments, the xylose reductase or undesirable fermentation side products , but also to specifi- aldose reductase is from Hypocrea jecorina . In some cally produce D - lactate. embodiments, the xylose reductase or aldose reductase is In particular embodiments, the enzyme converts pyruvate 10 encoded by the xyll gene . In some embodiments, the xylose to lactate . In some embodiments , the lactate dehydrogenase reductase or aldose reductase is from Saccharomyces cere is from Escherichia coli . In some embodiments , the lactate visiae . In some embodiments, the xylose reductase or aldose dehydrogenase is encoded by the IdhA gene . reductase is encoded by the GRE3 gene . In some embodi In some embodiments, a recombinant microorganism ments , the xylose reductase or aldose reductase is from producing MEG and a three - carbon compound comprises a 15 Pachysolen tannophilus. In some embodiments, the xylose deletion , insertion , or loss of function mutation in a gene reductase or aldose reductase is from Pichia sp . In some encoding a lactate dehydrogenase to prevent the production embodiments, the xylose reductase or aldose reductase is of lactate from pyruvate and instead shunt the reaction from Pichia stipitis. In some embodiments, the xylose toward production of a three - carbon compound . reductase or aldose reductase is from Pichia quercuum . In Xylose Reductase or Aldose Reductase ( EC 1.1.1.21 ) 20 some embodiments , the xylose reductase or aldose reductase The present disclosure describes enzymes that can cata- is from Candida sp . In some embodiments , the xylose lyze the following reactions: reductase or aldose reductase is from Candida shehatae . In some embodiments, the xylose reductase or aldose reductase a - D - xylose + NADPH + H + S > xylitol + NADP is from Candida tenuis. In some embodiments, the xylose 25 reductase or aldose reductase is from Candida tropicalis. In an alditol + NAD ( P ) + SNAD ( P ) H + aldose some embodiments, the xylose reductase or aldose reductase Aldose reductase may also be known as alditol: NAD ( P ) + is from Aspergillus niger . In some embodiments, the xylose 1 - oxidoreductase , polyol dehydrogenase or aldehyde reductase or aldose reductase is from Neurospora crassa . In reductase . some embodiments, the xylose reductase or aldose reductase Aldose reductase is a cytosolic oxidoreductase that cata- 30 is from Cryptococcus lactativorus . lyzes the reduction of a variety of aldehydes and carbonyls, In some embodiments , the xylose reductase or aldose including monosaccharides . reductase is encoded by one or more nucleic acid molecules Aldose reductase may be considered a prototypical obtained from a microorganism selected from the group enzyme of the aldo - keto reductase enzyme superfamily. The consisting of Hypocrea sp . , Scheffersomyces sp . , Saccharo enzyme comprises 315 amino acid residues and folds into a 35 myces sp . , Pachysolen sp . , Pichia sp . , Candida sp . , Asper Bla - barrel structural motif composed of eight parallel B gillus sp . , Neurospora sp . , and Cryptococcus sp . In some strands. Adjacent strands are connected by eight peripheral embodiments, the xylose reductase or aldose reductase is a - helical segments running anti- parallel to the ß sheet . The encoded by one or more nucleic acid molecules obtained catalytic active site is situated in the barrel core . The from a microorganism selected from the group consisting of NADPH cofactor is situated at the top of the Bla barrel, with 40 Hypocrea jecorina , Scheffersomyces stipitis, Saccharomyces the nicotinamide ring projecting down in the center of the cerevisiae , Pachysolen tannophilus, Pichia stipitis, Pichia barrel and pyrophosphate straddling the barrel lip . quercuum , Candida shehatae, Candida tenuis, Candida The reaction mechanism of aldose reductase in the direc- tropicalis, Aspergillus niger, Neurospora crassa and Cryp tion of aldehyde reduction follows a sequential ordered path tococcus lactativorus. In another embodiment, the one or where NADPH binds, followed by the substrate. Binding of 45 more nucleic acid molecules encoding the xylose reductase NADPH induces a conformational change or aldose reductase is xyll and / or GRE3 or homolog thereof. ( Enzyme •NADPH- > Enzyme * •NADPH ) that involves In some embodiments , the one or more nucleic acid mol hinge - like movement of a surface loop ( residues 213-217 ) so ecules encoding the xylose reductase or aldose reductase as to cover a portion of the NADPH in a manner similar to comprises an amino acid sequence selected from the group that of a safety belt . The alcohol product is formed via a 50 consisting of SEQ ID NOs : 84 and 87. In some embodi transfer of the pro - R hydride of NADPH to the face of the ments, the one or more nucleic acid molecules encoding the substrate's carbonyl carbon . Following release of the alco- xylose reductase or aldose reductase is encoded by a nucleic hol product, another conformational change occurs acid sequence selected from the group consisting of SEQ ID ( E * •NAD ( P ) + - > E •NAD ( P ) + ) in order to release NADP + . NOs : 82 , 83 , 85 and 86 . Kinetic studies have shown that reorientation of this loop to 55 Xylitol Dehydrogenase ( 1.1.1.9 ) permit release of NADP + appears to represent the rate- The present disclosure describes enzymes that can cata limiting step in the direction of aldehyde reduction . As the lyze the following reaction : rate of coenzyme release limits the catalytic rate , it can be seen that perturbation of interactions that stabilize coenzyme xylitol + NAD + SD -xylulose + NADH + H + binding can have dramatic effects on the maximum velocity 60 Xylitol dehydrogenase may also be known as D - xylulose ( Vmax ). reductase , NAD + -dependent xylitol dehydrogenase, eryth D -xylose - fermenting Pichia stipitis and Candida sheha- ritol dehydrogenase, 2,3 - cis - polyol ( DPN ) dehydrogenase tae were shown to produce one single aldose reductase ( C3-5 ) , pentitol- DPN dehydrogenase , xylitol- 2 - dehydroge ( ALR ) that is active both with NADPH and NADH . Other nase or xylitol : NAD + 2 - oxidoreductase ( D - xylulose - form yeasts such as Pachysolen tannophilus and C. tropicalis 65 ing ) . synthesize multiple forms of ALR with different coenzyme Xylitol dehydrogenase (XDH ) is one of several enzymes specificities. The significant dual coenzyme specificity dis- responsible for assimilating xylose into eukaryotic metabo US 10,941,424 B2 65 66 lism and is useful for fermentation of xylose contained in Soluble Pyridine Nucleotide Transhydrogenase ( EC agricultural byproducts to produce ethanol. For efficient 1.6.1.1 . ) xylose utilization at high flux rates, cosubstrates should be The present disclosure describes enzymes that can cata recycled between the NAD + -specific XDH and the lyze the following reaction : NADPH - preferring xylose reductase , another enzyme in the 5 pathway. NADH + NADP + SNAD ++ NADPH In particular embodiments , the enzyme converts xylitol to Soluble pyridine nucleotide transhydrogenase may also D -xylulose . In some embodiments , the xylitol dehydroge be known as NAD ( P ) + transhydrogenase ( B -specific ), STH , nase is from yeast . In some embodiments , the xylitol dehy pyridine nucleotide transhydrogenase, or transhydrogenase . 10 E. coli contains both a soluble and a membrane - bound drogenase is from Pichia sp . , Saccharomyces sp . , Glucono pyridine nucleotide transhydrogenase . The soluble pyridine bacter sp . , Galactocandida sp . , Neurospora sp . or Serratia nucleotide transhydrogenase is the sthA or udhA gene prod sp . In some embodiments, the xylitol dehydrogenase is from uct ; its primary physiological role appears to be the reoxi Pichia stipitis, S. cerevisiae, Gluconobacter oxydans, dation of NADPH ( Canonaco F. et al . ( 2001 ) Metabolic flux Galactocandida mastotermitis , Neurospora crassa or Ser 15 response to phosphoglucose isomerase knock - out in ratia marcescens . In some embodiments, the xylitol dehy Escherichia coli and impact of overexpression of the soluble drogenase is encoded by xyl2 or xdh1 . transhydrogenase UdhA . FEMS Microbiol Lett 204 ( 2 ) : 247 In one embodiment, the xylitol dehydrogenase is encoded 252 ; Sauer U. et al . ( 2004 ) The soluble and membrane by one or more nucleic acid molecules obtained from a bound transhydrogenases UdhA and PntAB have divergent microorganism selected from the group consisting of Schef- 20 functions in NADPH metabolism of Escherichia coli . J Biol fersomyces sp . , Trichoderma sp . , Pichia sp . , Saccharomyces Chem 279 ( 8 ) : 6613-6619 ) . The membrane - bound proton sp . , Gluconobacter sp . , Galactocandida sp . , Neurospora sp ., translocating transhydrogenase is the pntAB gene product; and Serratia sp . In another embodiment, the xylitol dehy- PntAB is a major source of NADPH ( Sauer et al . 2004 ) . drogenase is encoded by one or more nucleic acid molecules UdhA contains noncovalently bound FAD and is present obtained from a microorganism selected from the group 25 in a form consisting of seven or eight monomers ( Boonstra consisting of Scheffersomyces stipitis, Trichoderma reesei, B. et al . ( 1999 ) The udhA gene of Escherichia coli encodes Pichia stipitis , Saccharomyces cerevisiae, Gluconobacter a soluble pyridine nucleotide transhydrogenase . J Bacteriol oxydans, Galactocandida mastotermitis, Neurospora crassa 181 ( 3 ) : 1030-1034 ) . and Serratia marcescens . In another embodiment, the one or Moderate overexpression of UdhA ( SthA ) allows an more nucleic acid molecules encoding the xylitol dehydro- 30 increased maximal growth rate of a phosphoglucose genase is xy12 and / or xdhl , or homolog thereof. In some isomerase mutant (Canonaco et al . 2001 ) , and a pgi sthA embodiments , the one or more nucleic acid molecules double mutant is not viable ( Sauer et al . 2004 ) . These encoding the xylitol dehydrogenase comprises an amino phenotypes may be due to the ability of UdhA to restore the acid sequence selected from the group consisting of SEQ ID cellular redox balance under conditions of excess NADPH NOs : 90 and 92. In some embodiments, the one or more 35 formation ( Canonaco et al . 2001 ; Sauer et al . 2004 ) . Muta nucleic acid molecules encoding the xylitol dehydrogenase tions in sthA appear during adaptation of a pgi mutant strain is encoded by a nucleic acid sequence selected from the to growth on glucose minimal medium ( Charusanti P. et al . group consisting of SEQ ID NOs : 88 , 89 and 91 . ( 2010 ) Genetic basis of growth adaptation of Escherichia Alkaline Phosphatase ( EC 3.1.3.1 ) coli after deletion of pgi , a major metabolic gene. ” PLOS Alkaline phosphatase is a hydrolase enzyme responsible 40 Genet 6 ( 11 ) : e1001186 ) . for removing phosphate groups from many types of mol- Transcription of sthA is downregulated by growth on ecules , including nucleotides , proteins, and alkaloids . As the glycerol ( Sauer et al . 2004 ) . name suggests , alkaline phosphatases are most effective in In some embodiments, expression of a transhydrogenase an alkaline environment. It is sometimes used synony- can increase activity of a NADPH -dependent alcohol dehy mously as basic phosphatase. 45 drogenase, leading to improved acetone to 2 -propanol con The S. cerevisiae Pho13 alkaline phosphatase enzyme is version . In one embodiment, the soluble pyridine nucleotide a monomeric protein with molecular mass of 60 kDa and transhydrogenase is encoded by one or more nucleic acid hydrolyzes p -nitrophenyl phosphate with maximal activity molecules obtained from E. coli . In another embodiment, the at pH 8.2 with strong dependence on Mg2 + ions and an one or more nucleic acid molecules encoding the soluble apparent Km of 3.6x10 ( -5 ) M. No other substrates tested 50 pyridine nucleotide transhydrogenase is udhA , or homolog except phosphorylated histone II - A and casein were hydro- thereof. In some embodiments , the one or more nucleic acid lyzed at any significant rate . These data suggest that the molecules encoding the soluble pyridine nucleotide trans physiological role of the p - nitrophenyl phosphate - specific hydrogenase comprises an amino acid sequence set forth in phosphatase may involve participation in reversible tein SEQ ID NO : 110. In some embodiments, the one or more phosphorylation. 55 nucleic acid molecules encoding the soluble pyridine In particular embodiments, the enzyme converts D -xylu- nucleotide transhydrogenase is encoded by a nucleic acid lose - 5 - phosphate to D - xylulose . In some embodiments , the sequence set forth in SEQ ID NO : 109 . alkaline phosphatase is from yeast . In some embodiments, Biosynthesis of MEG and One or More Three - Carbon the alkaline phosphatase is from Saccharomyces sp . In some Compound Using a Recombinant Microorganism embodiments , the alkaline phosphatase is from S. cerevisiae. 60 As discussed above , the present application provides a In some embodiments, the alkaline phosphatase is encoded recombinant microorganism co - producing monoethylene by the PHO13 gene. glycol ( MEG ) and one or more three - carbon compounds. In In some embodiments , a recombinant microorganism one embodiment, the MEG and one or more three - carbon producing MEG and a three -carbon compound comprises a compounds are co -produced from xylose . In another deletion , insertion , or loss of function mutation in a gene 65 embodiment, the recombinant microorganism comprises a encoding an alkaline phosphatase to prevent the conversion deletion, insertion , or loss of function mutation in a gene of D -xylulose - 5 -phosphate to D - xylulose . encoding a D - xylulose - 5 - kinase and / or in a gene encoding a US 10,941,424 B2 67 68 glycoaldehyde dehydrogenase. In some embodiments, the the D - tagatose 3 - epimerase comprises an amino acid gene encoding the D - xylulose - 5 -kinase is xylB . In some sequence selected from the group consisting of SEQ ID embodiments , the gene encoding the glycoaldehyde dehy- NOs : 3 and 5. In yet a further embodiment, the D - tagatose drogenase is aldA . 3 -epimerase is encoded by a nucleic acid sequence selected In one embodiment, MEG is produced from xylose via 5 from the group consisting of SEQ ID NOs: 1 , 2 and 4 . ribulose - 1 - phosphate . In another embodiment, MEG is pro- In one embodiment, the D - ribulokinase is encoded by one duced from xylose via xylulose - 1 -phosphate . In a further or more nucleic acid molecules obtained from E. coli . In embodiment, MEG is produced from xylose via xylonate. some embodiments, the one or more nucleic acid molecules In one embodiment, one or more three - carbon compounds is fuck , or homolog thereof. In a further embodiment, the isthe produced one or morefrom DHAPthree - carbon or pyruvate compounds. In one is embodiment acetone . In, 10 D - ribulokinase comprises an amino acid sequence set forth another embodiment, the one or more three - carbon com in SEQ ID NO : 8. In yet a further embodiment, the D - ribu pounds is isopropanol. In a further embodiment, the one or lokinase is encoded by a nucleic acid sequence selected from more three - carbon compounds is propene . the group consisting of SEQ ID NOs : 6 and 7 . In one preferred embodiment, MEG and one or more 15 In one embodiment, the D - ribulose - 1 - phosphate aldolase three -carbon compounds are produced from xylose using a is encoded by one or more nucleic acid molecules obtained ribulose - 1 - phosphate pathway for the conversion of xylose from E. coli . In some embodiments , the one or more nucleic to MEG and dihydroxyacetone - phosphate ( DHAP ) , and acid molecules is fucA , or homolog thereof. In a further using a C3 branch pathway for the conversion of DHAP to embodiment, the D -ribulose - 1 -phosphate aldolase com one or more three - carbon compounds. 20 prises an amino acid sequence set forth in SEQ ID NO : 11 . As discussed above , in a first aspect , the present disclo- In yet a further embodiment, the D - ribulose - 1 -phosphate sure relates to a recombinant microorganism capable of aldolase is encoded by a nucleic acid sequence selected from co - producing monoethylene glycol (MEG ) and acetone the group consisting of SEQ ID NOs : 9 and 10 . from exogenous D - xylose , wherein the recombinant micro- In one embodiment, the recombinant microorganism fur organism expresses one or more of the following: 25 ther comprises at least one endogenous or exogenous nucleic ( a ) at least one endogenous or exogenous nucleic acid acid molecule encoding a secondary alcohol dehydrogenase molecule encoding a D - tagatose 3 - epimerase that cata- that catalyzes the conversion of acetone from ( g ) to isopro lyzes the conversion of D - xylulose to D - ribulose ; panol. ( b ) at least one endogenous or exogenous nucleic acid In another embodiment, the recombinant microorganism molecule encoding a D - ribulokinase that catalyzes the 30 further comprises at least one endogenous or exogenous conversion of D - ribulose from ( a ) to D - ribulose - 1- nucleic acid molecule encoding a dehydratase that catalyzes phosphate; the conversion of isopropanol to propene. ( c ) at least one endogenous or exogenous nucleic acid In another embod ent , the recombinant microorganism molecule encoding a D - ribulose - 1 - phosphate aldolase further comprises one or more modifications selected from that catalyzes the conversion of D -ribulose - 1 -phos- 35 the group consisting of: phate from ( b ) to glycolaldehyde and dihydroxyaceto- ( a ) a deletion , insertion , or loss of function mutation in a nephosphate ( DHAP ) ; gene encoding a D - xylulose - 5 - kinase that catalyzes the ( d ) at least one endogenous or exogenous nucleic acid conversion of D - xylulose to D - xylulose - 5 - phosphate ; molecule encoding a glycolaldehyde reductase that ( b ) a deletion , insertion, or loss of function mutation in a catalyzes the conversion of glycolaldehyde from ( c ) to 40 gene encoding a glycolaldehyde dehydrogenase that MEG ; catalyzes the conversion of glycolaldehyde to glycolic ( e ) at least one exogenous nucleic acid molecule encoding acid ; and a thiolase that catalyzes the conversion of acetyl- CoA ( c ) a deletion , insertion , or loss of function mutation in a to acetoacetyl - CoA ; gene encoding a lactate dehydrogenase that catalyzes ( f) at least one endogenous or exogenous nucleic acid 45 the conversion of pyruvate to lactate . molecule encoding an acetate :acetoacetyl -CoA trans- In one embodiment, an endogenous D -xylose isomerase ferase or hydrolase that catalyzes the conversion of catalyzes the conversion of D - xylose to D -xylulose . In one acetoacetyl- CoA from ( e ) to acetoacetate ; and / or embodiment, the xylose isomerase is exogenous . In another ( g ) at least one endogenous or exogenous nucleic acid embodiment, the xylose isomerase is encoded by one or molecule encoding an acetoacetate decarboxylase that 50 more nucleic acid molecules obtained from Pyromyces sp . In catalyzes the conversion of acetoacetate from ( f) to another embodiment, the one or more nucleic acid mol acetone ; ecules encoding the xylose isomerase is xyl? , or homolog wherein the produced intermediate DHAP is converted to thereof. In yet another embodiment, the one or more nucleic acetyl- CoA through the endogenous glycolysis pathway in acid molecules encoding the xylose isomerase comprises an the microorganism , and wherein MEG and acetone are 55 amino acid sequence set forth in SEQ ID NO : 95. In a further co -produced embodiment, the one or more nucleic acid molecules encod In one embodiment, the D - tagatose 3 - epimerase is ing the xylose isomerase is encoded by a nucleic acid encoded by one or more nucleic acid molecules obtained sequence selected from the group consisting of SEQ ID from a microorganism selected from the group consisting of NOs : 93 and 94 . Pseudomonas sp . , Mesorhizobium sp . and Rhodobacter sp . 60 In one preferred embodiment, MEG and acetone are In some embodiments , the D -tagatose 3 - epimerase is co - produced from xylose using a xylulose - 1 - phosphate encoded by one or more nucleic acid molecules obtained pathway for the conversion of xylose to MEG and a C3 from a microorganism selected from the group consisting of branch pathway for the conversion of dihydroxyacetone Pseudomonas cichorii, Pseudomonas sp . ST - 24 , Mesorhizo- phosphate ( DHAP ) to acetone . bium loti and Rhodobacter sphaeroides. In some embodi- 65 As discussed above , in a second aspect , the present ments , the one or more nucleic acid molecules is dte and /or disclosure relates to a recombinant microorganism capable FJ851309.1 , or homolog thereof. In a further embodiment, of co - producing monoethylene glycol ( MEG ) and acetone US 10,941,424 B2 69 70 from exogenous D - xylose , wherein the recombinant micro- ( a ) a deletion , insertion , or loss of function mutation in a organism expresses one or more of the following: gene encoding a D - xylulose - 5 -kinase that catalyzes the ( a ) at least one endogenous or exogenous nucleic acid conversion of D -xylulose to D -xylulose - 5 - phosphate; molecule encoding a D - xylulose 1 - kinase that catalyzes ( b ) a deletion, insertion, or loss of function mutation in a the conversion of D - xylulose to D -xylulose -1 -phos 5 gene encoding a glycolaldehyde dehydrogenase that phate ; catalyzes the conversion of glycolaldehyde to glycolic ( b ) at least one endogenous or exogenous nucleic acid acid ; and molecule encoding a D -xylulose - 1 - phosphate aldolase ( c ) a deletion, insertion , or loss of function mutation in a that catalyzes the conversion of D - xylulose - 1 -phos- gene encoding a lactate dehydrogenase that catalyzes phate from ( a ) to glycolaldehyde and dihydroxyaceto 10 the conversion of pyruvate to lactate . nephosphate ( DHAP ) ; In some embodiments of any aspect disclosed above , a ( c ) at least one endogenous or exogenous nucleic acid recombinant microorganism producing MEG and one or molecule encoding a glycolaldehyde reductase that more three - carbon compounds comprises a deletion , inser catalyzes the conversion of glycolaldehyde from ( b ) to 15 tion , or loss of function mutation in a gene encoding a MEG ; D - xylulose - 5 - kinase to prevent the conversion of D -xylu ( d ) at least one endogenous or exogenous nucleic acid lose to D - xylulose - 5 -phosphate and instead shunt the reac molecule encoding a thiolase that catalyzes the con- tion toward conversion of D -xylulose to D - xylulose - 1 -phos version of acetyl - CoA to acetoacetyl - CoA ; phate . In some embodiments , the D -xylulose - 5 -kinase is ( e ) at least one endogenous or exogenous nucleic acid 20 from Escherichia coli . In some embodiments, the D - xylu molecule encoding an acetate :acetoacetyl -CoA trans- lose - 5 - kinase is encoded by the xylB gene , or homolog ferase or hydrolase that catalyzes the conversion of thereof. acetoacetyl- CoA from ( d ) to acetoacetate ; and / or In some embodiments of any aspect disclosed above , a ( f) at least one endogenous or exogenous nucleic acid recombinant microorganism producing MEG and one or molecule encoding an acetoacetate decarboxylase that 25 more three - carbon compounds comprises a deletion , inser catalyzes the conversion of acetoacetate from ( e ) to tion , or loss of function mutation in a gene encoding a acetone ; glycolaldehyde dehydrogenase to prevent the production of wherein the produced intermediate DHAP is converted to glycolic acid from glycolaldehyde and instead shunt the acetyl - CoA through the endogenous glycolysis pathway in reaction toward conversion of glycolaldehyde to MEG . In the microorganism , and wherein MEG and acetone are 30 some embodiments , the glycolaldehyde dehydrogenase is co - produced from Escherichia coli . In some embodiments, the glycola In one embodiment, the D - xylulose 1 - kinase is encoded ldehyde dehydrogenase is encoded by the aldA gene , or by one or more nucleic acid molecules obtained from Homo homolog thereof. sapiens. In some embodiments, the one or more nucleic acid In some embodiments of any aspect disclosed above , a molecules encoding the D - xylulose 1 - kinase is ketohexoki- 35 recombinant microorganism producing MEG and one or nase C ( khk - C ), or homolog thereof. In another embodi- more three - carbon compounds comprises a deletion , inser ment, the one or more nucleic acid molecules encoding the tion , or loss of function mutation in a gene encoding a lactate D - xylulose 1 -kinase comprises an amino acid sequence set dehydrogenase to prevent the production of lactate from forth in SEQ ID NO : 55. In a further embodiment, the one 40 pyruvate and instead shunt the reaction toward production of or more nucleic acid molecules encoding the D - xylulose one or more three -carbon compounds. In some embodi 1 - kinase is encoded by a nucleic acid sequence selected ments , the lactate dehydrogenase is from Escherichia coil . from the group consisting of SEQ ID NOs : 53 and 54 . In some embodiments, the lactate dehydrogenase is encoded In one embodiment, the D - xylulose - 1 - phosphate aldolase by the IdhA gene , or homolog thereof . is encoded by one or more nucleic acid molecules obtained 45 In one embodiment, an endogenous D - xylose isomerase from Homo sapiens. In another embodiment, the one or catalyzes the conversion of D - xylose to D -xylulose . In one more nucleic acid molecules encoding the D -xylulose - 1- embodiment, the xylose isomerase is exogenous. In another phosphate aldolase is aldolase B ( ALDOB ) , or homolog embodiment, the xylose isomerase is encoded by one or thereof. In some embodiments, the one or more nucleic acid more nucleic acid molecules obtained from Pyromyces sp . In molecules encoding the D -xylulose - 1 -phosphate aldolase 50 another embodiment, the one or more nucleic acid mol comprises an amino acid sequence set forth in SEQ ID NO : ecules encoding the xylose isomerase is xyl? , or homolog 58. In some embodiments, the one or more nucleic acid thereof. In yet another embodiment, the one or more nucleic molecules encoding the D - xylulose - 1 -phosphate aldolase is acid molecules encoding the xylose isomerase comprises an encoded by a nucleic acid sequence selected from the group amino acid sequence set forth in SEQ ID NO : 95. In a further consisting of SEQ ID NOs: 56 and 57 . 55 embodiment, the one or more nucleic acid molecules encod In one embodiment, the recombinant microorganism fur- ing the xylose isomerase is encoded by a nucleic acid ther comprises at least one endogenous or exogenous nucleic sequence selected from the group consisting of SEQ ID acid molecule encoding a secondary alcohol dehydrogenase NOs : 93 and 94 . that catalyzes the conversion of acetone from ( f) to isopro- As discussed above , in a third aspect , the present appli panol. 60 cation relates to a recombinant microorganism capable of In another embodiment, the recombinant microorganism co - producing monoethylene glycol ( MEG ) and acetone further comprises at least one endogenous or exogenous from exogenous D - xylose and glucose , wherein the recom nucleic acid molecule encoding a dehydratase - isomerase binant microorganism expresses one or more of the follow that catalyzes the conversion of isopropanol to propene . ing : In another embodiment, the recombinant microorganism 65 ( a ) at least one exogenous nucleic acid molecule encoding further comprises one or more modifications selected from a xylose reductase or aldose reductase that catalyzes the the group consisting of: conversion of D - xylose to xylitol and at least one US 10,941,424 B2 71 72 exogenous nucleic acid molecule encoding a xylitol fersomyces sp . , Trichoderma sp . , Pichia sp ., Saccharomyces dehydrogenase that catalyzes the conversion of xylitol sp . , Gluconobacter sp . , Galactocandida sp . , Neurospora sp . , to D - xylulose ; and Serratia sp . In another embodiment, the xylitol dehy ( b ) at least one exogenous nucleic acid molecule encoding drogenase is encoded by one or more nucleic acid molecules a D - xylose isomerase that catalyzes the conversion of 5 obtained from a microorganism selected from the group D - xylose to D - xylulose, and consisting of Scheffersomyces stipitis, Trichoderma reesei, wherein the microorganism further expresses one or more of Pichia stipitis , Saccharomyces cerevisiae, Gluconobacter the following : oxydans, Galactocandida mastotermitis , Neurospora crassa ( c ) at least one endogenous or exogenous nucleic acid and Serratia marcescens . In another embodiment, the one or molecule encoding a D - tagatose 3 - epimerase that cata- 10 more nucleic acid molecules encoding the xylitol dehydro lyzes the conversion of D - xylulose from ( a ) or ( b ) to genase is xy12 and / or xdhl, or homolog thereof. In some D - ribulose ; embodiments, the one or more nucleic acid molecules ( d ) at least one endogenous or exogenous nucleic acid encoding the xylitol dehydrogenase comprises an amino molecule encoding a D - ribulokinase that catalyzes the acid sequence selected from the group consisting of SEQ ID conversion of D - ribulose from ( c ) to D - ribulose - 1- 15 NOs : 90 and 92. In some embodiments, the one or more phosphate ; nucleic acid molecules encoding the xylitol dehydrogenase ( e ) at least one endogenous or exogenous nucleic acid is encoded by a nucleic acid sequence selected from the molecule encoding a D - ribulose - 1 -phosphate aldolase group consisting of SEQ ID NOs : 88 , 89 and 91 . that catalyzes the conversion of D - ribulose - 1 -phos- In one embodiment, an endogenous D - xylose isomerase phate from ( d ) to glycolaldehyde and dihydroxyaceto- 20 catalyzes the conversion of D - xylose to D - xylulose . In one nephosphate ( DHAP ) ; embodiment, the xylose isomerase is exogenous . In another ( f) at least one endogenous or exogenous nucleic acid embodiment, the xylose isomerase is encoded by one or molecule encoding a glycolaldehyde reductase or more nucleic acid molecules obtained from Pyromyces sp . In methylglyoxal reductase that catalyzes the conversion another embodiment, the one or more nucleic acid mol of glycolaldehyde from ( e ) to MEG ; 25 ecules encoding the xylose isomerase is xyla , or homolog ( g ) at least one endogenous or exogenous nucleic acid thereof. In yet another embodiment, the one or more nucleic molecule encoding a thiolase that catalyzes the con- acid molecules encoding the xylose isomerase comprises an version of acetyl -CoA to acetoacetyl -CoA ; amino acid sequence set forth in SEQ ID NO : 95. In a further ( h ) at least one endogenous or exogenous nucleic acid embodiment, the one or more nucleic acid molecules encod molecule encoding an acetate : acetoacetyl- CoA trans- 30 ing the xylose isomerase is encoded by a nucleic acid ferase or hydrolase that catalyzes the conversion of sequence selected from the group consisting of SEQ ID acetoacetyl- CoA from ( g ) to acetoacetate; and / or NOs : 93 and 94 . ( i ) at least one endogenous or exogenous nucleic acid In one embod ent, the recombinant microorganism fur molecule encoding an acetoacetate decarboxylase that ther comprises at least one endogenous or exogenous nucleic catalyzes the conversion of acetoacetate from ( h ) to 35 acid molecule encoding a secondary alcohol dehydrogenase acetone ; that catalyzes the conversion of acetone from ( i ) to isopro wherein the produced intermediate DHAP is converted to panol. acetyl- CoA through the endogenous glycolysis pathway in In another embodiment, the recombinant microorganism the microorganism , and wherein MEG and acetone are further comprises at least one endogenous or exogenous co -produced 40 nucleic acid molecule encoding a dehydratase that catalyzes In some embodiments , the xylose reductase or aldose the conversion of isopropanol to propene . reductase is encoded by one or more nucleic acid molecules In another embodiment, the recombinant microorganism obtained from a microorganism selected from the group further comprises one or more modifications selected from consisting of Hypocrea sp . , Scheffersomyces sp . , Saccharo- the group consisting of: myces sp . , Pachysolen sp . , Pichia sp . , Candida sp . , Asper- 45 ( a ) a deletion , insertion , or loss of function mutation in a gillus sp . , Neurospora sp . , and Cryptococcus sp . In some gene encoding a D - xylulose - 5 - kinase that catalyzes the embodiments, the xylose reductase or aldose reductase is conversion of D - xylulose to D - xylulose - 5 - phosphate ; encoded by one or more nucleic acid molecules obtained and from a microorganism selected from the group consisting of ( b ) a deletion , insertion, or loss of function mutation in a Hypocrea jecorina, Scheffersomyces stipitis, Saccharomyces 50 gene encoding an alkaline phosphatase that catalyzes cerevisiae, Pachysolen tannophilus, Pichia stipitis, Pichia the conversion of D -xylulose - 5 - phosphate to D - xylu quercuum , Candida shehatae , Candida tenuis, Candida lose . tropicalis, Aspergillus niger, Neurospora crassa and Cryp- In one embodiment, the enzyme that catalyzes the con tococcus lactativorus. In another embodiment, the one or version of D - xylulose to D - xylulose - 5 -phosphate is a D -xy more nucleic acid molecules encoding the xylose reductase 55 lulose - 5 - kinase . In some embodiments , the D - xylulose - 5 or aldose reductase is xyll and / or GRE3 or homolog thereof. kinase is from Saccharomyces cerevisiae. In some In some embodiments, the one or more nucleic acid mol- embodiments the D - xylulose - 5 - kinase is encoded by the ecules encoding the xylose reductase or aldose reductase XKS1 gene , or homolog thereof. In some embodiments, the comprises an amino acid sequence selected from the group D - xylulose - 5 - kinase is from Pichia stipitis. In some embodi consisting of SEQ ID NOs : 84 and 87. In some embodi- 60 ments the D - xylulose - 5 - kinase is encoded by the XYL3 ments, the one or more nucleic acid molecules encoding the gene , or homolog thereof. xylose reductase or aldose reductase is encoded by a nucleic In a further embodiment, the microorganism is a fungus . acid sequence selected from the group consisting of SEQ ID In one preferred embodiment, MEG and acetone are NOs : 82 , 83 , 85 and 86 . co - produced from xylose using a xylonate pathway for the In one embodiment, the xylitol dehydrogenase is encoded 65 conversion of xylose to MEG and a C3 branch pathway for by one or more nucleic acid molecules obtained from a the conversion of dihydroxyacetone - phosphate ( DHAP ) to microorganism selected from the group consisting of Schef- acetone . US 10,941,424 B2 73 74 As discussed above, in a fourth aspect , the present appli- In a further embodiment, the one or more nucleic acid cation relates to a recombinant microorganism capable of molecules encoding the xylonolactonase comprises an co - producing monoethylene glycol ( MEG ) and acetone amino acid sequence set forth in SEQ ID NO : 67. In yet from exogenous D - xylose , wherein the recombinant micro- another embodiment, the one or more nucleic acid mol organism expresses one or more of the following: 5 ecules encoding the xylonolactonase is encoded by a nucleic ( a ) at least one endogenous or exogenous nucleic acid acid sequence set forth in SEQ ID NO : 66 . molecule encoding a xylose dehydrogenase that cata- In one embodiment, the xylonate dehydratase is encoded lyzes the conversion of D - xylose to D - xylonolactone ; by one or more nucleic acid molecules obtained from a ( b ) at least one endogenous or exogenous nucleic acid microorganism selected from the group consisting of Cau molecule encoding a xylonolactonase that catalyzes the 10 lobacter sp . , Sulfolobus sp . and E. coli . In another embodi conversion of D -xylonolactone from ( a ) to D -xylonate ; ment, the xylonate dehydratase is encoded by one or more ( c ) at least one endogenous or exogenous nucleic acid nucleic acid molecules obtained from a microorganism molecule encoding a xylonate dehydratase that cata- selected from the group consisting of Caulobacter crescen lyzes the conversion of D - xylonate from ( b ) to 2 -keto- tus, Sulfolobus solfataricus and E. coli . In some embodi 3 -deoxy - xylonate; 15 ments, the one or more nucleic acid molecules encoding the ( d ) at least one endogenous or exogenous nucleic acid xylonate dehydratase is selected from xylD , yjhG and / or molecule encoding a 2 -keto - 3 -deoxy - D - pentonate yagF , or homolog thereof. In a further embodiment, the one aldolase that catalyzes the conversion of 2 -keto - 3- or more nucleic acid molecules encoding the xylonate deoxy - xylonate from ( c ) to glycolaldehyde and pyru- dehydratase comprises an amino acid sequence selected vate ; 20 from the group consisting of SEQ ID NOs : 69 , 72 and 75 . ( e ) at least one endogenous or exogenous nucleic acid In yet another embodiment, the one or more nucleic acid molecule encoding a glycolaldehyde reductase that molecules encoding the xylonate dehydratase is encoded by catalyzes the conversion of glycolaldehyde from ( d ) to a nucleic acid sequ ce selected from the group consisting MEG : of SEQ ID NOs : 68 , 70 , 71 , 73 and 74 . ( f) at least one exogenous nucleic acid molecule encoding 25 In one embodiment, the 2 -keto - 3 -deoxy - D -pentonate a thiolase that catalyzes the conversion of acetyl- CoA aldolase is encoded by one or more nucleic acid molecules to acetoacetyl - CoA ; obtained from a microorganism selected from Pseudomonas ( g ) at least one endogenous or exogenous nucleic acid sp . and E. coli . In another embodiment, the 2 -keto - 3 -deoxy molecule encoding an acetate : acetoacetyl - CoA trans- D - pentonate aldolase is encoded by one or more nucleic acid ferase or hydrolase that catalyzes the conversion of 30 molecules obtained from E. coli . In some embodiments, the acetoacetyl- CoA from ( f) to acetoacetate ; and / or one or more nucleic acid molecules encoding the 2 -keto - 3 ( h ) at least one exogenous nucleic acid molecule encoding deoxy - D - pentonate aldolase is selected from yjhH and /or an acetoacetate decarboxylase that catalyzes the con- yagE , or homolog thereof. In a further embodiment, the one version of acetoacetate from ( g ) to acetone ; or more nucleic acid molecules encoding the 2 -keto - 3 wherein the produced intermediate pyruvate is converted to 35 deoxy - D - pentonate aldolase comprises an amino acid acetyl -CoA through the endogenous glycolysis pathway in sequence selected from the group consisting of SEQ ID the microorganism , and wherein MEG and acetone are NOs : 78 and 81. In yet another embodiment, the one ormore co - produced . nucleic acid molecules encoding the 2 -keto - 3 -deoxy - D In one embodiment, the xylose dehydrogenase is encoded pentonate aldolase is encoded by a nucleic acid sequence by one or more nucleic acid molecules obtained from a 40 selected from the group consisting of SEQ ID NOs : 76 , 77 , microorganism selected from the group consisting of Cau- 79 and 80 . lobacter sp . , Haloarcula sp . , Haloferax sp . , Halorubrum sp . In one embodiment, the recombinant microorganism fur and Trichoderma sp . In another embodiment, the xylose ther comprises at least one endogenous or exogenous nucleic dehydrogenase is encoded by one or more nucleic acid acid molecule encoding a secondary alcohol dehydrogenase molecules obtained from a microorganism selected from the 45 that catalyzes the conversion of acetone from ( h ) to isopro group consisting of Caulobacter crescentus, Haloarcula panol. marismortui, Haloferax volcanii, Halorubrum lacuspro- In another embodiment, the recombinant microorganism fundi and Trichoderma reesei . In some embodiments , the further comprises at least one endogenous or exogenous one or more nucleic acid molecules encoding the xylose nucleic acid molecule encoding a dehydratase that catalyzes dehydrogenase is selected from xylB , xdh1 ( HVO_B0028 ) 50 the conversion of isopropanol to propene . and / or xyd? , or homolog thereof. In a further embodiment, In another embodiment, the recombinant microorganism the one or more nucleic acid molecules encoding the xylose further comprises one or more modifications selected from dehydrogenase comprises an amino acid sequence selected the group consisting of : from the group consisting of SEQ ID NOs : 61 , 63 and 65 . ( a ) a deletion , insertion , or loss of function mutation in a In yet another embodiment, the one or more nucleic acid 55 gene encoding a D - xylose isomerase that catalyzes the molecules encoding the xylose dehydrogenase is encoded by conversion of D - xylose to D -xylulose ; a nucleic acid sequence selected from the group consisting ( b ) a deletion , insertion , or loss of function mutation in a of SEQ ID NOs : 59 , 60 , 62 and 64 . gene encoding a glycolaldehyde dehydrogenase that In one embodiment, the xylonolactonase is encoded by catalyzes the conversion of glycolaldehyde to glycolic one or more nucleic acid molecules obtained from a micro- 60 acid ; and organism selected from Caulobacter sp . and Haloferax sp . ( c ) a deletion , insertion , or loss of function mutation in a In another embodiment, the xylonolactonase is encoded by gene encoding a lactate dehydrogenase that catalyzes one or more nucleic acid molecules obtained from a micro the conversion of pyruvate to lactate . organism selected from the group consisting of Caulobacter In some embodiments , a recombinant microorganism crescentus , Haloferax volcanii and Haloferax gibbonsii . In 65 producing MEG and one or more three - carbon compounds some embodiments, the one or more nucleic acid molecules comprises a deletion , insertion , or loss of function mutation encoding the xylonolactonase is xylC , or homolog thereof. in a gene encoding a D -xylose isomerase to prevent con US 10,941,424 B2 75 76 version of D - xylose to D - xylulose and instead shunt the lobacter sp . , Haloarcula sp . , Haloferax sp . , Halorubrum sp . reaction toward the conversion of D -xylose to D -xylonate . and Trichoderma sp . In another embodiment, the xylose In one embodiment, the enzyme that catalyzes the conver- dehydrogenase is encoded by one or more nucleic acid sion of D - xylose to D - xylulose is a D - xylose isomerase . In molecules obtained from a microorganism selected from the some embodiments, the D - xylose isomerase is from 5 group consisting of Caulobacter crescentus , Haloarcula Escherichia coli . In some embodiments, the D - xylose marismortui, Haloferax volcanii, Halorubrum lacuspro isomerase is encoded by the xylA gene, or homolog thereof. fundi and Trichoderma reesei. In some embodiments, the In some embodiments , a recombinant microorganism one or more nucleic acid molecules encoding the xylose producing MEG and one or more three - carbon compounds dehydrogenase is selected from xy1B , xdh1 ( HVO_B0028 ) comprises a deletion , insertion, or loss of function mutation 10 and / or xyd? , or homolog thereof. In a further embodiment, in a gene encoding a glycolaldehyde dehydrogenase to the one or more nucleic acid molecules encoding the xylose prevent the production of glycolic acid from glycolaldehyde dehydrogenase comprises an amino acid sequence selected and instead shunt the reaction toward conversion of glyco- from the group consisting of SEQ ID NOs : 61 , 63 and 65 . laldehyde to MEG . In one embodiment, the glycolaldehyde In yet another embodiment, the one or more nucleic acid dehydrogenase is from Escherichia coli . In some embodi- 15 molecules encoding the xylose dehydrogenase is encoded by ments , the glycolaldehyde dehydrogenase is encoded by the a nucleic acid sequence selected from the group consisting aldA gene , or homolog thereof. of SEQ ID NOs : 59 , 60 , 62 and 64 . In some embodiments, a recombinant microorganism In one embodiment, the xylonate dehydratase is encoded producing MEG and one or more three - carbon compounds by one or more nucleic acid molecules obtained from a comprises a deletion, insertion , or loss of function mutation 20 microorganism selected from the group consisting of Cau in a gene encoding a lactate dehydrogenase to prevent the lobacter sp . , Sulfolobus sp . and E. coli . In another embodi production of lactate from pyruvate and instead shunt the ment, the xylonate dehydratase is encoded by one or more reaction toward production of one or more three - carbon nucleic acid molecules obtained from a microorganism compounds. In one embodiment, the enzyme that catalyzes selected from the group consisting of Caulobacter crescen the conversion of pyruvate to lactate is a lactate dehydro- 25 tus, Sulfolobus solfataricus and E. coli . In some embodi genase . In particular embodiments, the enzyme converts ments, the one or more nucleic acid molecules encoding the pyruvate to lactate. In some embodiments, the lactate dehy- xylonate dehydratase is selected from xylD , yjhG and / or drogenase is from Escherichia coli . In some embodiments , yagF , or homolog thereof . In a further embodiment, the one the lactate dehydrogenase is encoded by the IdhA gene , or or more nucleic acid molecules encoding the xylonate homolog thereof. 30 dehydratase comprises an amino acid sequence selected As discussed above , in a fifth aspect , the present appli- from the group consisting of SEQ ID NOs : 69 , 72 and 75 . cation relates to a recombinant microorganism capable of In yet another embodiment, the one or more nucleic acid co - producing monoethylene glycol ( MEG ) and acetone molecules encoding the xylonate dehydratase is encoded by from exogenous D - xylose , wherein the recombinant micro- a nucleic acid sequence selected from the group consisting organism expresses one or more of the following: 35 of SEQ ID NOs : 68 , 70 , 71 , 73 and 74 . ( a ) at least one endogenous or exogenous nucleic acid In one embodiment, the 2 - keto - 3 - deoxy - D - pentonate molecule encoding a xylose dehydrogenase that cata- aldolase is encoded by one or more nucleic acid molecules lyzes the conversion of D - xylose to D -xylonate ; obtained from a microorganism selected from Pseudomonas ( b ) at least one endogenous or exogenous nucleic acid sp . and E. coli . In another embodiment, the 2 -keto - 3 -deoxy molecule encoding a xylonate dehydratase that cata- 40 D - pentonate aldolase is encoded by one or more nucleic acid lyzes the conversion of D -xylonate from ( a ) to 2 -keto- molecules obtained from E. coli . In some embodiments, the 3 -deoxy - xylonate ; one or more nucleic acid molecules encoding the 2 -keto - 3 ( c ) at least one endogenous or exogenous nucleic acid deoxy - D -pentonate aldolase is selected from yjhH and / or molecule encoding a 2 -keto - 3 -deoxy - D - pentonate yagE , or homolog thereof. In a further embodiment, the one aldolase that catalyzes the conversion of 2 - keto - 3- 45 or more nucleic acid molecules encoding the 2 -keto - 3 deoxy - xylonate from ( b ) to glycolaldehyde and pyru- deoxy - D -pentonate aldolase comprises an amino acid vate ; sequence selected from the group consisting of SEQ ID ( d ) at least one exogenous nucleic acid molecule encoding NOs : 78 and 81. In yet another embodiment, the one or more a glycolaldehyde reductase that catalyzes the conver- nucleic acid molecules encoding the 2- keto - 3- deoxy - D sion of glycolaldehyde from ( c ) to MEG ; 50 pentonate aldolase is encoded by a nucleic acid sequence ( e ) at least one exogenous nucleic acid molecule encoding selected from the group consisting of SEQ ID NOs : 76 , 77 , a thiolase that catalyzes the conversion of acetyl - CoA 79 and 80 . to acetoacetyl - CoA ; In one embodiment, the recombinant microorganism fur ( f) at least one endogenous or exogenous nucleic acid ther comprises at least one endogenous or exogenous nucleic molecule encoding an acetate : acetoacetyl -CoA trans- 55 acid molecule encoding a secondary alcohol dehydrogenase ferase or hydrolase that catalyzes the conversion of that catalyzes the conversion of acetone from ( g ) to isopro acetoacetyl- CoA from ( e ) to acetoacetate ; and / or panol. ( g ) at least one exogenous nucleic acid molecule encoding In another embodiment, the recombinant microorganism an acetoacetate decarboxylase that catalyzes the con- further comprises at least one endogenous or exogenous version of acetoacetate from ( f) to acetone ; 60 nucleic acid molecule encoding a dehydratase that catalyzes wherein the produced intermediate pyruvate is converted to the conversion of isopropanol to propene . acetyl- CoA through the endogenous glycolysis pathway in In another embodiment, the recombinant microorganism the microorganism , and wherein MEG and acetone are further comprises one or more modifications selected from co - produced the group consisting of: In one embodiment, the xylose dehydrogenase is encoded 65 ( a ) a deletion , insertion , or loss of function mutation in a by one or more nucleic acid molecules obtained from a gene encoding a D - xylose isomerase that catalyzes the microorganism selected from the group consisting of Cau- conversion of D -xylose to D - xylulose ; US 10,941,424 B2 77 78 ( b ) a deletion , insertion , or loss of function mutation in a cereus , E. coli, Saccharomyces cerevisiae and Marinobacter gene encoding a glycolaldehyde dehydrogenase that hydrocarbonoclasticus . In some embodiments, the one or catalyzes the conversion of glycolaldehyde to glycolic more nucleic acid molecules is th1A , atoB and / or ERG10 , or acid ; and homolog thereof . In a further embodiment, the thiolase or ( c ) a deletion , insertion , or loss of function mutation in a 5 acetyl coenzyme A acetyltransferase comprises an amino gene encoding a lactate dehydrogenase that catalyzes acid sequence selected from the group consisting of SEQ ID the conversion of pyruvate to lactate . NOs : 35 , 37 and 40. In yet a further embodiment, the In some embodiments, a recombinant microorganism thiolase or acetyl coenzyme A acetyltransferase is encoded producing MEG and one or more three - carbon compounds by a nucleic acid sequence selected from the group consist comprises a deletion , insertion, or loss of function mutation 10 ing of SEQ ID NOs : 33 , 34 , 36 , 38 and 39 . in a gene encoding a D - xylose isomerase to prevent con- In one embodiment of any aspect disclosed above , the version of D - xylose to D - xylulose and instead shunt the acetyl - CoA :acetoacetate - CoA transferase or acetate : ac reaction toward the conversion of D - xylose to D -xylonate . etoacetyl -CoA hydrolase is encoded by one or more nucleic In one embodiment, the enzyme that catalyzes the conver- acid molecules obtained from a microorganism selected sion of D - xylose to D - xylulose is a D - xylose isomerase . In 15 from Clostridium sp . and E. coli . In another embodiment, some embodiments, the D - xylose isomerase is from the acetyl -CoA :acetoacetate - CoA transferase or acetate :ac Escherichia coli . In some embodiments, the D - xylose etoacetyl- CoA hydrolase is encoded by one or more nucleic isomerase is encoded by the xylA gene, or homolog thereof. acid molecules obtained from E. coli . In some embodiments , In some embodiments, a recombinant microorganism the one or more nucleic acid molecules encoding the acetyl producing MEG and one or more three - carbon compounds 20 CoA : acetoacetate - CoA transferase is ato A and / or atoD , or comprises a deletion , insertion , or loss of function mutation homolog thereof. In another embodiment, the acetyl- CoA : in a gene encoding a glycolaldehyde dehydrogenase to acetoacetate - CoA transferase or acetate : acetoacetyl - CoA prevent the production of glycolic acid from glycolaldehyde hydrolase is encoded by one or more nucleic acid molecules and instead shunt the reaction toward conversion of glyco- obtained from Clostridium acetobutylicum . In some embodi laldehyde to MEG . In one embodiment, the glycolaldehyde 25 ments, the one or more nucleic acid molecules encoding the dehydrogenase is from Escherichia coli . In some embodi- acetate : acetoacetyl - CoA hydrolase is ctfA and / or ctfB , or ments , the glycolaldehyde dehydrogenase is encoded by the homolog thereof . In a further embodiment, the acetyl - CoA : aldA gene , or homolog thereof. acetoacetate - CoA transferase or acetate : acetoacetyl - CoA In some embodiments , a recombinant microorganism hydrolase comprises an amino acid sequence selected from producing MEG and one or more three - carbon compounds 30 the group consisting of SEQ ID NOs : 43 , 46 , 97 , 99 , 101 and comprises a deletion , insertion , or loss of function mutation 103. In yet a further embodiment, the acetyl- CoA : acetoac in a gene encoding a lactate dehydrogenase to prevent the etate - CoA transferase or acetate : acetoacetyl -CoA hydrolase production of lactate from pyruvate and instead shunt the is encoded by a nucleic acid sequence selected from the reaction toward production of one or more three -carbon group consisting of SEQ ID NOs : 41 , 42 , 44 , 45 , 96 , 98 , 100 compounds. In one embodiment, the enzyme that catalyzes 35 and 102 . the conversion of pyruvate to lactate is a lactate dehydro- In one embodiment of any aspect disclosed above , the genase . In particular embodiments, the enzyme converts acetoacetate decarboxylase is encoded by one or more pyruvate to lactate. In some embodiments, the lactate dehy- nucleic acid molecules obtained from a microorganism drogenase is from Escherichia coli . In some embodiments, selected from the group consisting of Clostridium sp . , Bacil the lactate dehydrogenase is encoded by the IdhA gene , or 40 lus sp . , Chromobacterium sp . and Pseudomonas sp . In homolog thereof. another embodiment, the acetoacetate decarboxylase is In one embodiment of any aspect disclosed above , the encoded by one or more nucleic acid molecules obtained glycolaldehyde reductase is encoded by one or more nucleic from a microorganism selected from the group consisting of acid molecules obtained from a microorganism selected Clostridium acetobutylicum , Clostridium beijerinckii, from E. coli and S. cerevisiae . In another embodiment, the 45 Clostridium cellulolyticum , Bacillus polymyxa , Chromobac one or more nucleic acid molecules is selected from gldA , terium violaceum and Pseudomonas putida. In some GRE2 , GRE3 , yqhD , ydjy , fuco , yafB ( dkgB ), and /or yqhE embodiments , the one or more nucleic acid molecules ( dkgA ), or homolog thereof . In another embodiment, the one encoding the acetoacetate decarboxylase is adc , or homolog or more nucleic acid molecules is yqhD . In some embodi- thereof. In a further embodiment, the acetoacetate decar ments, the yqhD comprises a G149E mutation . In a further 50 boxylase comprises an amino acid sequence selected from embodiment, the glycolaldehyde reductase comprises an the group consisting of SEQ ID NOs : 49 and 52. In yet amino acid sequence selected from the group consisting of another embodiment, the acetoacetate decarboxylase is SEQ ID NOs : 13 , 15 , 17 , 20 , 23 , 25 , 28 , 30 and 32. In yet encoded by a nucleic acid sequence selected from the group a further embodiment, the glycolaldehyde reductase is consisting of SEQ ID NOs : 47 , 48 , 50 and 51 . encoded by a nucleic acid sequence selected from the group 55 In one embodiment of any aspect disclosed above , the consisting of SEQ ID NOs : 12 , 14 , 16 , 18 , 19 , 21 , 22 , 24 , 26 , enzyme that catalyzes the conversion of acetone to isopro 27 , 29 and 31 . panol is a secondary alcohol dehydrogenase ( S - ADH ) . In In one embodiment of any aspect disclosed above , the another embodiment, the enzyme is a secondary alcohol thiolase or acetyl coenzyme A acetyltransferase is encoded dehydrogenase that is encoded by a nucleic acid molecule by one or more nucleic acid molecules obtained from a 60 obtained from a microorganism selected from Burkholderia microorganism selected from the group consisting of sp , Alcaligenes sp . , Clostridium sp . , Thermoanaerobacter Clostridium sp . ,Bacillus sp . , E. coli, Saccharomyces sp . and sp . , Phytomonas sp . , Rhodococcus sp . , Methanobacterium Marinobacter sp . In some embodiments, the thiolase or sp . , Methanogenium sp . , Entamoeba sp . , Trichomonas sp . , acetyl coenzyme A acetyltransferase is encoded by one or and Tritrichomonas sp . In some embodiments, the nucleic more nucleic acid molecules obtained from a microorganism 65 acid molecule encoding the secondary alcohol dehydroge selected from the group consisting of Clostridium aceto- nase is obtained from a microorganism selected from Bur butylicum , Clostridium thermosaccharolyticum , Bacillus kholderia sp . AIU 652 , Alcaligenes eutrophus, Clostridium US 10,941,424 B2 79 80 ragsdalei, Clostridium beijerinckii, Clostridium carboxidi- embodiments, the sugar is selected from the group consist vorans , Thermoanaerobacter brockii, Thermoanaerobacter ing of glucose , fructose , and sucrose . ethanolicus ( Clostridium thermohydrosulfuricum ), Rhodo- Methods of Producing a Recombinant Microorganism that coccus ruber , Methanobacterium palustre, methanogenic Produces or Accumulates MEG and One or More Three archaea Methanogenium liminatans, parasitic protist Enta- 5 Carbon Compounds moeba histolytica , parasitic protozoan Tritrichomonas foe As discussed above , the present application provides a tus and human parasite Trichomonas vaginalis. In some method of producing a recombinant microorganism that embodiments , the one or more nucleic acid molecule encod produces or accumulates MEG and one or more three ing secondary alcohol dehydrogenase is adh, adhB , EhAdh1, carbon compounds. In one embodiment, the MEG and one or homolog thereof. In some embodiments, the S - ADH is 10 xyloseor more . In threeanother - carbon embodiment compounds, a methodare co -of produced producing from a predicted from homology and can be from Thermoanaero recombinant microorganism that produces or accumulates bacter mathranii, Micrococcus luteus, Nocardiopsis alba , MEG and one or more three - carbon compounds from exog Mycobacterium hassiacum , Helicobacter suis, Candida enous D - xylose comprises introducing into the recombinant albicans, Candida parapsilosis, Candida orthopsilosis, 15 microorganism a deletion, insertion , or loss of function Candida metapsilosis, Grosmannia clavigera and Scheffer mutation in a gene encoding a D - xylulose - 5 - kinase and / or in somyces stipitis. In a further embodiment, the alcohol dehy a gene encoding a glycoaldehyde dehydrogenase. In some drogenase comprises an amino acid sequence selected from embodiments, the gene encoding the D - xylulose - 5 - kinase is the group consisting of SEQ ID NOs : 106 and 108. In yet xylB . In some embodiments, the gene encoding the glyco another embodiment, the alcohol dehydrogenase is encoded 20 aldehyde dehydrogenase is aldA . by a nucleic acid sequence selected from the group consist In one embodiment, MEG is produced from xylose via ing of SEQ ID NOs : 104 , 105 and 107 . ribulose - 1 - phosphate . In another embodiment, MEG is pro Recombinant Microorganism duced from xylose via xylulose - 1 -phosphate . In a further The disclosure provides microorganisms that can be engi- embodiment, MEG is produced from xylose via xylonate. neered to express various endogenous or exogenous 25 In one embodiment, one or more three - carbon compounds enzymes . is produced from DHAP or pyruvate . In one embodiment, In various embodiments described herein , the recombi- the one or more three - carbon compounds is acetone . In nant microorganism is a eukaryotic microorganism . In some another embodiment, the one or more three - carbon com embodiments, the eukaryotic microorganism is a yeast . In pounds is isopropanol. In a further embodiment, the one or exemplary embodiments, the yeast is a member of a genus 30 more three - carbon compounds is propene . selected from the group consisting of Yarrowia, Candida , As discussed above , in one aspect , the present disclosure Saccharomyces, Pichia , Hansenula , Kluyveromyces, provides a method of producing a recombinant microorgan Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizo- ism that produces or accumulates MEG and acetone from saccharomyces, Pachysolen, Cryptococcus, Trichosporon , exogenous D -xylose , comprising introducing into the Rhodotorula , and Myxozyma. 35 recombinant microorganism and / or overexpressing one or In some embodiments , the recombinant microorganism is more of the following : a prokaryotic microorganism . In exemplary embodiments , ( a ) at least one endogenous or exogenous nucleic acid the prokaryotic microorganism is a member of a genus molecule encoding a D - tagatose 3 - epimerase that cata selected from the group consisting of Escherichia , lyzes the conversion of D - xylulose to D - ribulose ; Clostridium , Zymomonas , Salmonella , Rhodococcus, 40 ( b ) at least one endogenous or exogenous nucleic acid Pseudomonas, Bacillus, Lactobacillus, Enterococcus, molecule encoding a D - ribulokinase that catalyzes the Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, conversion of D - ribulose from ( a ) to D - ribulose - 1 Corynebacterium , and Brevibacterium . phosphate ; In some embodiments , the recombinant microorganism is ( c ) at least one endogenous or exogenous nucleic acid used to produce monoethylene glycol ( MEG ) disclosed 45 molecule encoding a D - ribulose - 1 -phosphate aldolase herein . that catalyzes the conversion of D - ribulose - 1 -phos Accordingly , in another aspect , the present inventions phate from ( b ) to glycolaldehyde and dihydroxyaceto provide a method of producing MEG and one or more nephosphate ( DHAP ) ; three - carbon compounds using a recombinant microorgan- ( d ) at least one endogenous or exogenous nucleic acid ism described herein . In one embodiment, the method com- 50 molecule encoding a glycolaldehyde reductase that prises cultivating the recombinant microorganism in a cul- catalyzes the conversion of glycolaldehyde from ( c ) to ture medium containing a feedstock providing a carbon MEG ; source until MEG and one or more three - carbon compounds ( e ) at least one exogenous nucleic acid molecule encoding is produced . In a further embodiment, the MEG and one or a thiolase that catalyzes the conversion of acetyl -CoA more three -carbon compounds is recovered . Recovery can 55 to acetoacetyl -CoA ; be by methods known in the art , such as distillation , mem- ( f) at least one endogenous or exogenous nucleic acid brane - based separation gas stripping, solvent extraction , and molecule encoding an acetate : acetoacetyl - CoA trans expanded bed adsorption . In an exemplary embodiment, the ferase or hydrolase that catalyzes the conversion of three carbon compound is selected from acetone , isopropa- acetoacetyl - CoA from ( e ) to acetoacetate ; and / or nol , and propene . 60 ( g ) at least one endogenous or exogenous nucleic acid In some embodiments, the feedstock comprises a carbon molecule encoding an acetoacetate decarboxylase that source . In various embodiments described herein , the carbon catalyzes the conversion of acetoacetate from ( f) to source may be selected from sugars , glycerol, alcohols, acetone ; organic acids, alkanes , fatty acids , lignocellulose , proteins , wherein the produced intermediate DHAP is converted to carbon dioxide , and carbon monoxide. In an exemplary 65 acetyl- CoA through the endogenous glycolysis pathway in embodiment, the carbon source is a sugar . In a further the microorganism , and wherein MEG and acetone are exemplary embodiment, the sugar is D - xylose . In alternative co - produced US 10,941,424 B2 81 82 In one embodiment, the D - tagatose 3 - epimerase is ing the xylose isomerase is encoded by a nucleic acid encoded by one or more nucleic acid molecules obtained sequence selected from the group consisting of SEQ ID from a microorganism selected from the group consisting of NOs : 93 and 94 . Pseudomonas sp . , Mesorhizobium sp . and Rhodobacter sp . As discussed above , in another aspect , the present disclo In some embodiments , the D - tagatose 3 - epimerase is 5 sure provides a method of producing a recombinant micro encoded by one or more nucleic acid molecules obtained organism that produces or accumulates MEG and acetone from a microorganism selected from the group consisting of from exogenous D - xylose , comprising introducing into the Pseudomonas cichorii, Pseudomonas sp . ST - 24 , Mesorhizo- recombinant microorganism and / or overexpressing one or bium loti and Rhodobacter sphaeroides. In some embodi- more of the following : ments , the one or more nucleic acid molecules is dte and / or 10 ( a ) at least one endogenous or exogenous nucleic acid FJ851309.1 , or homolog thereof. In a further embodiment, molecule encoding a D - xylulose 1 - kinase that catalyzes the D -tagatose 3 -epimerase comprises an amino acid the conversion of D - xylulose to D -xylulose - 1 - phos sequence selected from the group consisting of SEQ ID phate, NOs : 3 and 5. In yet a further embodiment, the D - tagatose ( b ) at least one endogenous or exogenous nucleic acid 3 -epimerase is encoded by a nucleic acid sequence selected 15 molecule encoding a D -xylulose - 1 -phosphate aldolase from the group consisting of SEQ ID NOs : 1 , 2 and 4 . that catalyzes the conversion of D - xylulose - 1 -phos In one embodiment, the D - ribulokinase is encoded by one phate from ( a ) to glycolaldehyde and dihydroxyaceto or more nucleic acid molecules obtained from E. coli . In nephosphate ( DHAP ) ; some embodiments , the one or more nucleic acid molecules ( c ) at least one endogenous or exogenous nucleic acid is fuck , or homolog thereof. In a further embodiment, the 20 molecule encoding a glycolaldehyde reductase that D -ribulokinase comprises an amino acid sequence set forth catalyzes the conversion of glycolaldehyde from ( b ) to in SEQ ID NO : 8. In yet a further embodiment, the D - ribu MEG ; lokinase is encoded by a nucleic acid sequence selected from ( d ) at least one endogenous or exogenous nucleic acid the group consisting of SEQ ID NOs : 6 and 7 . molecule encoding a thiolase that catalyzes the con In one embodiment, the D - ribulose - 1 -phosphate aldolase 25 version of acetyl -CoA to acetoacetyl -CoA ; is encoded by one or more nucleic acid molecules obtained ( e ) at least one endogenous or exogenous nucleic acid from E. coli . In some embodiments, the one or more nucleic molecule encoding an acetate : acetoacetyl -CoA trans acid molecules is fucA , or homolog thereof. In a further ferase or hydrolase that catalyzes the conversion of embodiment, the D - ribulose - 1 - phosphate aldolase com acetoacetyl- CoA from ( d ) to acetoacetate ; and / or prises an amino acid sequence set forth in SEQ ID NO : 11. 30 ( f) at least one endogenous or exogenous nucleic acid In yet a further embodiment, the D - ribulose - 1 -phosphate molecule encoding an acetoacetate decarboxylase that aldolase is encoded by a nucleic acid sequence selected from catalyzes the conversion of acetoacetate from ( e ) to the group consisting of SEQ ID NOs : 9 and 10 . acetone ; In one embodiment, the method further comprises intro- wherein the produced intermediate DHAP is converted to ducing into the recombinant microorganism and / or overex- 35 acetyl -CoA through the endogenous glycolysis pathway in pressing at least one endogenous or exogenous nucleic acid the microorganism , and wherein MEG and acetone are molecule encoding a secondary alcohol dehydrogenase that co - produced . catalyzes the conversion of acetone from ( g ) to isopropanol. In one embodiment, the D - xylulose 1 - kinase is encoded In another embodiment, the method further comprises by one or more nucleic acid molecules obtained from Homo introducing into the recombinant microorganism and / or 40 sapiens. In some embodiments, the one or more nucleic acid overexpressing at least one endogenous or exogenous molecules encoding the D - xylulose 1 - kinase is ketohexoki nucleic acid molecule encoding a dehydratase that catalyzes nase C (khk - C ), or homolog thereof. In another embodi the conversion of isopropanol to propene . ment, the one or more nucleic acid molecules encoding the In another embodiment, the method further comprises D - xylulose 1 - kinase comprises an amino acid sequence set introducing into the recombinant microorganism one or 45 forth in SEQ ID NO : 55. In a further embodiment, the one more modifications selected from the group consisting of: or more nucleic acid molecules encoding the D - xylulose ( a ) a deletion , insertion , or loss of function mutation in a 1 - kinase is encoded by a nucleic acid sequence selected gene encoding a D -xylulose - 5 - kinase that catalyzes the from the group consisting of SEQ ID NOs : 53 and 54 . conversion of D - xylulose to D - xylulose - 5 - phosphate ; In one embodiment, the D - xylulose - 1 - phosphate aldolase ( b ) a deletion , insertion , or loss of function mutation in a 50 is encoded by one or more nucleic acid molecules obtained gene encoding a glycolaldehyde dehydrogenase that from Homo sapiens. In another embodiment, the one or catalyzes the conversion of glycolaldehyde to glycolic more nucleic acid molecules encoding the D - xylulose - 1 acid ; and phosphate aldolase is aldolase B ( ALDOB ) , or homolog ( c ) a deletion , insertion , or loss of function mutation in a thereof. In some embodiments, the one or more nucleic acid gene encoding a lactate dehydrogenase that catalyzes 55 molecules encoding the D - xylulose - 1 - phosphate aldolase the conversion of pyruvate to lactate . comprises an amino acid sequence set forth in SEQ ID NO : In one embodiment, an endogenous D - xylose isomerase 58. In some embodiments, the one or more nucleic acid catalyzes the conversion of D - xylose to D - xylulose . In one molecules encoding the D - xylulose - 1 -phosphate aldolase is embodiment , the xylose isomerase is exogenous. In another encoded by a nucleic acid sequence selected from the group embodiment, the xylose isomerase is encoded by one or 60 consisting of SEQ ID NOs : 56 and 57 . more nucleic acid molecules obtained from Pyromyces sp . In In one embodiment, the method further comprises intro another embodiment, the one or more nucleic acid mol- ducing into the recombinant microorganism and / or overex ecules encoding the xylose isomerase is xyl? , or homolog pressing at least one endogenous or exogenous nucleic acid thereof. In yet another embodiment, the one or more nucleic molecule encoding a secondary alcohol dehydrogenase that acid molecules encoding the xylose isomerase comprises an 65 catalyzes the conversion of acetone from ( f) to isopropanol. amino acid sequence set forth in SEQ ID NO : 95. In a further In another embodiment, the method further comprises embodiment, the one or more nucleic acid molecules encod- introducing into the recombinant microorganism and / or US 10,941,424 B2 83 84 overexpressing at least one endogenous or exogenous chia coli . In some embodiments , the lactate dehydrogenase nucleic acid molecule encoding a dehydratase that catalyzes is encoded by the IdhA gene , or homolog thereof . the conversion of isopropanol to propene . As discussed above , in another aspect , the present disclo In another embodiment, the method further comprises sure provides a method of producing a recombinant micro introducing into the recombinant microorganism one or 5 organism that produces or accumulates MEG and acetone more modifications selected from the group consisting of: from exogenous D - xylose and glucose , comprising intro ( a ) a deletion , insertion , or loss of function mutation in a ducing into the recombinant microorganism and / or overex gene encoding a D - xylulose - 5 - kinase that catalyzes the pressing one or more of the following: conversion of D - xylulose to D - xylulose - 5 - phosphate ; ( a ) at least one exogenous nucleic acid molecule encoding 10 a xylose reductase or aldose reductase that catalyzes the ( b ) a deletion , insertion, or loss of function mutation in a conversion of D - xylose to xylitol and at least one gene encoding a glycolaldehyde dehydrogenase that exogenous nucleic acid molecule encoding a xylitol catalyzes the conversion of glycolaldehyde to glycolic dehydrogenase that catalyzes the conversion of xylitol acid ; and to D - xylulose ; ( c ) a deletion , insertion , or loss of function mutation in a 15 ( b ) at least one exogenous nucleic acid molecule encoding gene encoding a lactate dehydrogenase that catalyzes a D - xylose isomerase that catalyzes the conversion of the conversion of pyruvate to lactate . D -xylose to D -xylulose , and In one embodiment, an endogenous D - xylose isomerase wherein the method further comprises introducing into the catalyzes the conversion of D - xylose to D -xylulose . In one recombinant microorganism and / or overexpressing one embodiment, the xylose isomerase is exogenous. In another 20 or more of the following: embodiment, the xylose isomerase is encoded by one or ( c ) at least one endogenous or exogenous nucleic acid more nucleic acid molecules obtained from Pyromyces sp . In molecule encoding a D - tagatose 3 - epimerase that cata another embodiment, the one or more nucleic acid mol- lyzes the conversion of D - xylulose from ( a ) or ( b ) to ecules encoding the xylose isomerase is xyl? , or homolog D -ribulose ; thereof. In yet another embodiment, the one or more nucleic 25 ( d ) at least one endogenous or exogenous nucleic acid acid molecules encoding the xylose isomerase comprises an molecule encoding a D - ribulokinase that catalyzes the amino acid sequence set forth in SEQ ID NO : 95. In a further conversion of D - ribulose from ( c ) to D - ribulose - 1 embodiment, the one or more nucleic acid molecules encod phosphate ; ing the xylose isomerase is encoded by a nucleic acid ( e ) at least one endogenous or exogenous nucleic acid sequence selected from the group consisting of SEQ ID 30 molecule encoding a D - ribulose - 1 -phosphate aldolase NOs : 93 and 94 . that catalyzes the conversion of D - ribulose - 1 -phos In some embodiments of any aspect disclosed above , a phate from ( d ) to glycolaldehyde and dihydroxyaceto method of producing a recombinant microorganism that nephosphate ( DHAP ) ; produces or accumulates MEG and one or more three- ( f) at least one endogenous or exogenous nucleic acid carbon compounds from exogenous D - xylose comprises 35 molecule encoding a glycolaldehyde reductase or introducing into the recombinant microorganism a deletion , methylglyoxal reductase that catalyzes the conversion insertion , or loss of function mutation in a gene encoding a of glycolaldehyde from ( e ) to MEG ; D -xylulose - 5 -kinase to prevent the conversion of D -xylu- ( g ) at least one endogenous or exogenous nucleic acid lose to D - xylulose - 5 -phosphate and instead shunt the reac- molecule encoding a thiolase that catalyzes the con tion toward conversion of D - xylulose to D - xylulose - 1 -phos- 40 version of acetyl -CoA to acetoacetyl -CoA ; phate. In some embodiments , the D - xylulose - 5 - kinase is ( h ) at least one endogenous or exogenous nucleic acid from Escherichia coli . In some embodiments, the D -xylu- molecule encoding an acetate : acetoacetyl - CoA trans lose - 5 - kinase is encoded by the xylB gene , or homolog ferase or hydrolase that catalyzes the conversion of thereof. acetoacetyl- CoA from ( g ) to acetoacetate ; and / or In some embodiments of any aspect disclosed above , a 45 ( i ) at least one endogenous or exogenous nucleic acid method of producing a recombinant microorganism that molecule encoding an acetoacetate decarboxylase that produces or accumulates MEG and one or more three- catalyzes the conversion of acetoacetate from ( h ) to carbon compounds from exogenous D - xylose comprises acetone ; introducing into the recombinant microorganism a deletion , wherein the produced intermediate DHAP is converted to insertion , or loss of function mutation in a gene encoding a 50 acetyl - CoA through the endogenous glycolysis pathway in glycolaldehyde dehydrogenase to prevent the production of the microorganism , and wherein MEG and acetone are glycolic acid from glycolaldehyde and instead shunt the co - produced reaction toward conversion of glycolaldehyde to MEG . In In some embodiments , the xylose reductase or aldose some embodiments, the glycolaldehyde dehydrogenase is reductase is encoded by one or more nucleic acid molecules from Escherichia coli . In some embodiments, the glycola- 55 obtained from a microorganism selected from the group ldehyde dehydrogenase is encoded by the aldA gene, or consisting of Hypocrea sp . , Scheffersomyces sp . , Saccharo homolog thereof. myces sp . , Pachysolen sp . , Pichia sp . , Candida sp . , Asper In some embodiments of any aspect disclosed above , a gillus sp . , Neurospora sp . , and Cryptococcus sp . In some method of producing a recombinant microorganism that embodiments, the xylose reductase or aldose reductase is produces or accumulates MEG and one or more three- 60 encoded by one or more nucleic acid molecules obtained carbon compounds from exogenous D - xylose comprises from a microorganism selected from the group consisting of introducing into the recombinant microorganism a deletion , Hypocrea jecorina , Scheffersomyces stipitis, Saccharomyces insertion , or loss of function mutation in a gene encoding a cerevisiae, Pachysolen tannophilus, Pichia stipitis, Pichia lactate dehydrogenase to prevent the production of lactate quercuum , Candida shehatae, Candida tenuis, Candida from pyruvate and instead shunt the reaction toward pro- 65 tropicalis , Aspergillus niger, Neurospora crassa and Cryp duction of one or more three - carbon compounds. In some tococcus lactativorus . In another embodiment, the one or embodiments, the lactate dehydrogenase is from Escheri- more nucleic acid molecules encoding the xylose reductase US 10,941,424 B2 85 86 or aldose reductase is xyll and / or GRE3 or homolog thereof. In one embodiment, the enzyme that catalyzes the con In some embodiments, the one or more nucleic acid mol- version of D -xylulose to D - xylulose - 5 -phosphate is a D -xy ecules encoding the xylose reductase or aldose reductase lulose - 5 - kinase . In some embodiments, the D -xylulose -5 comprises an amino acid sequence selected from the group kinase is from Saccharomyces cerevisiae. In some consisting of SEQ ID NOs : 84 and 87. In some embodi- 5 embodiments the D - xylulose - 5 -kinase is encoded by the ments, the one or more nucleic acid molecules encoding the XKS1 gene, or homolog thereof . In some embodiments , the xylose reductase or aldose reductase is encoded by a nucleic D - xylulose - 5 - kinase is from Pichia stipitis. In some embodi acid sequence selected from the group consisting of SEQ ID ments the D - xylulose - 5 - kinase is encoded by the XYL3 NOs : 82 , 83 , 85 and 86 . gene , or homolog thereof. In one embodiment of any aspect disclosed above , the 10 In a further embodiment , the microorganism is a fungus. xylitol dehydrogenase is encoded by one or more nucleic As discussed above , in another aspect , the present appli acid molecules obtained from a microorganism selected cation provides a method of producing a recombinant micro from the group consisting of Scheffersomyces sp . , Tricho organism that produces or accumulates MEG and acetone derma sp ., Pichia sp ., Saccharomyces sp . , Gluconobacter 15 recombinantfrom exogenous microorganism D - xylose , comprising and / or overexpressing introducing into one the or sp . , Galactocandida sp . , Neurospora sp . , and Serratia sp . In more of the following: another embodiment, the xylitol dehydrogenase is encoded ( a ) at least one endogenous or exogenous nucleic acid by one or more nucleic acid molecules obtained from a molecule encoding a xylose dehydrogenase that cata microorganism selected from the group consisting of Schef- lyzes the conversion of D - xylose to D -xylonolactone ; fersomyces stipitis, Trichoderma reesei, Pichia stipitis , Sac- 20 ( b ) at least one endogenous or exogenous nucleic acid charomyces cerevisiae, Gluconobacter oxydans, Galacto molecule encoding a xylonolactonase that catalyzes the candida mastotermitis, Neurospora crassa and Serratia conversion of D - xylonolactone from ( a ) to D - xylonate ; marcescens . In another embodiment, the one or more nucleic ( c ) at least one endogenous or exogenous nucleic acid acid molecules encoding the xylitol dehydrogenase is xy12 molecule encoding a xylonate dehydratase that cata and / or xdh1 , or homolog thereof. In some embodiments, the 25 lyzes the conversion of D - xylonate from ( b ) to 2 -keto one or more nucleic acid molecules encoding the xylitol 3 - deoxy - xylonate ; dehydrogenase comprises an amino acid sequence selected ( d ) at least one endogenous or exogenous nucleic acid from the group consisting of SEQ ID NOs : 90 and 92. In molecule encoding a 2 -keto - 3 -deoxy - D -pentonate some embodiments , the one or more nucleic acid molecules aldolase that catalyzes the conversion of 2 -keto - 3 encoding the xylitol dehydrogenase is encoded by a nucleic 30 deoxy - xylonate from ( c ) to glycolaldehyde and pyru acid sequence selected from the group consisting of SEQ ID vate ; NOs : 88 , 89 and 91 . ( e ) at least one endogenous or exogenous nucleic acid In one embodiment, an endogenous D - xylose isomerase molecule encoding a glycolaldehyde reductase that catalyzes the conversion of D - xylose to D - xylulose . In one catalyzes the conversion of glycolaldehyde from ( d ) to embodiment, the xylose isomerase is exogenous. In another 35 MEG ; embodiment, the xylose isomerase is encoded by one or ( f) at least one exogenous nucleic acid molecule encoding more nucleic acid molecules obtained from Pyromyces sp . In a thiolase that catalyzes the conversion of acetyl -CoA another embodiment, the one or more nucleic acid mol to acetoacetyl - CoA ; ecules encoding the xylose isomerase is xyl? , or homolog ( g ) at least one endogenous or exogenous nucleic acid thereof. In yet another embodiment, the one or more nucleic 40 molecule encoding an acetate : acetoacetyl- CoA trans acid molecules encoding the xylose isomerase comprises an ferase or hydrolase that catalyzes the conversion of amino acid sequence set forth in SEQ ID NO : 95. In a further acetoacetyl- CoA from ( f) to acetoacetate ; and / or embodiment, the one or more nucleic acid molecules encod- ( h ) at least one exogenous nucleic acid molecule encoding ing the xylose isomerase is encoded by a nucleic acid an acetoacetate decarboxylase that catalyzes the con sequence selected from the group consisting of SEQ ID 45 version of acetoacetate from ( g ) to acetone ; NOs : 93 and 94 . wherein the produced intermediate pyruvate is converted to In one embodiment, the method further comprises intro- acetyl - CoA through the endogenous glycolysis pathway in ducing into the recombinant microorganism and / or overex- the microorganism , and wherein MEG and acetone are pressing at least one endogenous or exogenous nucleic acid co -produced molecule encoding a secondary alcohol dehydrogenase that 50 In one embodiment, the xylose dehydrogenase is encoded catalyzes the conversion of acetone from ( i ) to isopropanol. by one or more nucleic acid molecules obtained from a In another embodiment, the method further comprises microorganism selected from the group consisting of Cau introducing into the recombinant microorganism and / or lobacter sp . , Haloarcula sp . , Haloferax sp . , Halorubrum sp . overexpressing at least one endogenous or exogenous and Trichoderma sp . In another embodiment, the xylose nucleic acid molecule encoding a dehydratase that catalyzes 55 dehydrogenase is encoded by one or more nucleic acid the conversion of isopropanol to propene . molecules obtained from a microorganism selected from the In another embodiment, the method further comprises group consisting of Caulobacter crescentus, Haloarcula introducing into the recombinant microorganism one or marismortui, Haloferax volcanii, Halorubrum lacuspro more modifications selected from the group consisting of: fundi and Trichoderma reesei. In some embodiments, the ( a ) a deletion , insertion , or loss of function mutation in a 60 one or more nucleic acid molecules encoding the xylose gene encoding a D -xylulose - 5 -kinase that catalyzes the dehydrogenase is selected from xylB , xdh1 ( HVO_B0028 ) conversion of D - xylulose to D - xylulose - 5 - phosphate ; and / or xyd? , or homolog thereof. In a further embodiment, and the one or more nucleic acid molecules encoding the xylose ( b ) a deletion , insertion , or loss of function mutation in a dehydrogenase comprises an amino acid sequence selected gene encoding an alkaline phosphatase that catalyzes 65 from the group consisting of SEQ ID NOs : 61 , 63 and 65 . the conversion of D - xylulose - 5 - phosphate to D -xylu- In yet another embodiment, the one or more nucleic acid lose . molecules encoding the xylose dehydrogenase is encoded by US 10,941,424 B2 87 88 a nucleic acid sequence selected from the group consisting ( a ) a deletion , insertion , or loss of function mutation in a of SEQ ID NOs : 59 , 60 , 62 and 64 . gene encoding a D - xylose isomerase that catalyzes the In one embodiment, the xylonolactonase is encoded by conversion of D -xylose to D - xylulose ; one or more nucleic acid molecules obtained from a micro- ( b ) a deletion, insertion, or loss of function mutation in a organism selected from Caulobacter sp . and Haloferax sp . 5 gene encoding a glycolaldehyde dehydrogenase that In another embodiment, the xylonolactonase is encoded by catalyzes the conversion of glycolaldehyde to glycolic one or more nucleic acid molecules obtained from a micro acid ; and organism selected from the group consisting of Caulobacter ( c ) a deletion, insertion , or loss of function mutation in a crescentus , Haloferax volcanii and Haloferax gibbonsii . In gene encoding a lactate dehydrogenase that catalyzes some embodiments , the one or more nucleic acid molecules 10 the conversion of pyruvate to lactate . encoding the xylonolactonase is xylc , or homolog thereof. In some embodiments, a method of producing a recom In a further embodiment, the one or more nucleic acid binant microorganism that produces or accumulates MEG molecules encoding the xylonolactonase comprises an and one or more three - carbon compounds from exogenous amino acid sequence set forth in SEQ ID NO : 67. In yet 15 D - xylose comprises introducing into the recombinant micro another embodiment, the one or more nucleic acid mol- organism a deletion , insertion , or loss of function mutation ecules encoding the xylonolactonase is encoded by a nucleic in a gene encoding a D - xylose isomerase to prevent con acid sequence set forth in SEQ ID NO : 66 . version of D - xylose to D - xylulose and instead shunt the In one embodiment, the xylonate dehydratase is encoded reaction toward the conversion of D - xylose to D - xylonate . by one or more nucleic acid molecules obtained from a 20 In one embodiment, the enzyme that catalyzes the conver microorganism selected from the group consisting of Cau- sion of D - xylose to D - xylulose is a D - xylose isomerase . In lobacter sp . , Sulfolobus sp . and E. coli . In another embodi- some embodiments, the D - xylose isomerase is from ment, the xylonate dehydratase is encoded by one or more Escherichia coli . In some embodiments , the D - xylose nucleic acid molecules obtained from a microorganism isomerase is encoded by the xylA gene , or homolog thereof. selected from the group consisting of Caulobacter crescen- 25 In some embodiments, a method of producing a recom tus, Sulfolobus solfataricus and E. coli . In some embodi- binant microorganism that produces or accumulates MEG ments, the one or more nucleic acid molecules encoding the and one or more three -carbon compounds from exogenous xylonate dehydratase is selected from xylD , yjhG and / or D - xylose comprises introducing into the recombinant micro yagF , or homolog thereof. In a further embodiment, the one organism a deletion , insertion, or loss of function mutation or more nucleic acid molecules encoding the xylonate 30 in a gene encoding a glycolaldehyde dehydrogenase to dehydratase comprises an amino acid sequence selected prevent the production of glycolic acid from glycolaldehyde from the group consisting of SEQ ID NOs : 69 , 72 and 75 . and instead shunt the reaction toward conversion of glyco In yet another embodiment, the one or more nucleic acid laldehyde to MEG . In one embodiment, the glycolaldehyde molecules encoding the xylonate dehydratase is encoded by dehydrogenase is from Escherichia coli . In some embodi a nucleic acid sequence selected from the group consisting 35 ments, the glycolaldehyde dehydrogenase is encoded by the of SEQ ID NOs : 68 , 70 , 71 , 73 and 74 . aldA gene, or homolog thereof. In one embodiment, the 2 - keto - 3 - deoxy - D - pentonate In some embodiments, a method of producing a recom aldolase is encoded by one or more nucleic acid molecules binant microorganism that produces or accumulates MEG obtained from a microorganism selected from Pseudomonas 40 and one or more three - carbon compounds from exogenous sp . and E. coli . In another embodiment, the 2 -keto - 3 -deoxy- D - xylose comprises introducing into the recombinant micro D - pentonate aldolase is encoded by one or more nucleic acid organism a deletion , insertion , or loss of function mutation molecules obtained from E. coli . In some embodiments , the in a gene encoding a lactate dehydrogenase to prevent the one or more nucleic acid molecules encoding the 2 -keto - 3- production of lactate from pyruvate and instead shunt the deoxy - D -pentonate aldolase is selected from yjhH and / or 45 reaction toward production of one or more three - carbon yagE , or homolog thereof. In a further embodiment, the one compounds. In one embodiment, the enzyme that catalyzes or more nucleic acid molecules encoding the 2 -keto - 3- the conversion of pyruvate to lactate is a lactate dehydro deoxy - D -pentonate aldolase comprises an amino acid genase . In particular embodiments, the enzyme converts sequence selected from the group consisting of SEQ ID pyruvate to lactate . In some embodiments, the lactate dehy NOs : 78 and 81. In yet another embodiment, the one or more 50 drogenase is from Escherichia coli . In some embodiments, nucleic acid molecules encoding the 2 -keto - 3 -deoxy - D- the lactate dehydrogenase is encoded by the IdhA gene , or pentonate aldolase is encoded by a nucleic acid sequence homolog thereof. selected from the group consisting of SEQ ID NOs : 76 , 77 , As discussed above , in another aspect , the present appli 79 and 80 . cation provides a method of producing a recombinant micro In one embodiment, the method further comprises intro- 55 organism that produces or accumulates MEG and acetone ducing into the recombinant microorganism and / or overex- from exogenous D - xylose , comprising introducing into the pressing at least one endogenous or exogenous nucleic acid recombinant microorganism and / or overexpressing one or molecule encoding a secondary alcohol dehydrogenase that more of the following: catalyzes the conversion of acetone from ( h ) to isopropanol. ( a ) at least one endogenous or exogenous nucleic acid In another embodiment, the method further comprises 60 molecule encoding a xylose dehydrogenase that cata introducing into the recombinant microorganism and / or lyzes the conversion of D - xylose to D -xylonate ; overexpressing at least one endogenous or exogenous ( b ) at least one endogenous or exogenous nucleic acid nucleic acid molecule encoding a dehydratase that catalyzes molecule encoding a xylonate dehydratase that cata the conversion of isopropanol to propene . lyzes the conversion of D - xylonate from ( a ) to 2 -keto In another embodiment, the method further comprises 65 3 -deoxy - xylonate ; introducing into the recombinant microorganism one or ( c ) at least one endogenous or exogenous nucleic acid more modifications selected from the group consisting of: molecule encoding a 2 - keto - 3 - deoxy - D - pentonate US 10,941,424 B2 89 90 aldolase that catalyzes the conversion of 2 -keto - 3- or more nucleic acid molecules encoding the 2 -keto - 3 deoxy -xylonate from ( b ) to glycolaldehyde and pyru- deoxy - D -pentonate aldolase comprises an amino acid vate ; sequence selected from the group consisting of SEQ ID ( d ) at least one exogenous nucleic acid molecule encoding NOs : 78 and 81. In yet another embodiment, the one or more a glycolaldehyde reductase that catalyzes the conver- 5 nucleic acid molecules encoding the 2 -keto - 3 - deoxy - D sion of glycolaldehyde from ( c ) to MEG ; pentonate aldolase is encoded by a nucleic acid sequence ( e ) at least one exogenous nucleic acid molecule encoding selected from the group consisting of SEQ ID NOs : 76 , 77 , a thiolase that catalyzes the conversion of acetyl- CoA 79 and 80 . to acetoacetyl - CoA ; In one embodiment, the method further comprises intro ( f) at least one endogenous or exogenous nucleic acid 10 ducing into the recombinant microorganism and / or overex molecule encoding an acetate : acetoacetyl -CoA trans- pressing at least one endogenous or exogenous nucleic acid ferase or hydrolase that catalyzes the conversion of molecule encoding a secondary alcohol dehydrogenase that acetoacetyl- CoA from ( e ) to acetoacetate; and / or catalyzes the conversion of acetone from ( g ) to isopropanol. ( g ) at least one exogenous nucleic acid molecule encoding In another embodiment, the method further comprises an acetoacetate decarboxylase that catalyzes the con- 15 introducing into the recombinant microorganism and / or version of acetoacetate from ( f ) to acetone ; overexpressing at least one endogenous or exogenous wherein the produced intermediate pyruvate is converted to nucleic acid molecule encoding a dehydratase that catalyzes acetyl - CoA through the endogenous glycolysis pathway in the conversion of isopropanol to propene . the microorganism , and wherein MEG and acetone are In another embodiment, the method further comprises co - produced . 20 introducing into the recombinant microorganism one or In one embodiment, the xylose dehydrogenase is encoded more modifications selected from the group consisting of: by one or more nucleic acid molecules obtained from a ( a ) a deletion , insertion , or loss of function mutation in a microorganism selected from the group consisting of Cau- gene encoding a D - xylose isomerase that catalyzes the lobacter sp . , Haloarcula sp . , Haloferax sp . , Halorubrum sp . conversion of D -xylose to D - xylulose ; and Trichoderma sp . In another embodiment, the xylose 25 ( b ) a deletion , insertion , or loss of function mutation in a dehydrogenase is encoded by one or more nucleic acid gene encoding a glycolaldehyde dehydrogenase that molecules obtained from a microorganism selected from the catalyzes the conversion of glycolaldehyde to glycolic group consisting of Caulobacter crescentus, Haloarcula acid ; and marismortui, Haloferax volcanii, Halorubrum lacuspro- ( c ) a deletion , insertion , or loss of function mutation in a fundi and Trichoderma reesei. In some embodiments, the 30 gene encoding a lactate dehydrogenase that catalyzes one or more nucleic acid molecules encoding the xylose the conversion of pyruvate to lactate . dehydrogenase is selected from xylB , xdh1 ( HVO_B0028 ) In some embodiments, a method of producing a recom and / or xyd? , or homolog thereof. In a further embodiment, binant microorganism that produces or accumulates MEG the one or more nucleic acid molecules encoding the xylose and one or more three - carbon compounds from exogenous dehydrogenase comprises an amino acid sequence selected 35 D - xylose comprises introducing into the recombinant micro from the group consisting of SEQ ID NOs : 61 , 63 and 65 . organism a deletion , insertion , or loss of function mutation In yet another embodiment, the one or more nucleic acid in a gene encoding a D - xylose isomerase to prevent con molecules encoding the xylose dehydrogenase is encoded by version of D - xylose to D - xylulose and instead shunt the a nucleic acid sequence selected from the group consisting reaction toward the conversion of D - xylose to D - xylonate . of SEQ ID NOs : 59 , 60 , 62 and 64 . 40 In one embodiment, the enzyme that catalyzes the conver In one embodiment, the xylonate dehydratase is encoded sion of D - xylose to D - xylulose is a D - xylose isomerase . In by one or more nucleic acid molecules obtained from a some embodiments, the D - xylose isomerase is from microorganism selected from the group consisting of Cau- Escherichia coli . In some embodiments, the D - xylose lobacter sp . , Sulfolobus sp . and E. coli . In another embodi- isomerase is encoded by the xylA gene , or homolog thereof. ment, the xylonate dehydratase is encoded by one or more 45 In some embodiments, a method of producing a recom nucleic acid molecules obtained from a microorganism binant microorganism that produces or accumulates MEG selected from the group consisting of Caulobacter crescen- and one or more three - carbon compounds from exogenous tus, Sulfolobus solfataricus and E. coli . In some embodi- D - xylose comprises introducing into the recombinant micro ments, the one or more nucleic acid molecules encoding the organism a deletion , insertion , or loss of function mutation xylonate dehydratase is selected from xylD , yjhG and / or 50 in a gene encoding a glycolaldehyde dehydrogenase to yagF , or homolog thereof. In a further embodiment, the one prevent the production of glycolic acid from glycolaldehyde or more nucleic acid molecules encoding the xylonate and instead shunt the reaction toward conversion of glyco dehydratase comprises an amino acid sequence selected laldehyde to MEG . In one embodiment, the glycolaldehyde from the group consisting of SEQ ID NOs : 69 , 72 and 75 . dehydrogenase is from Escherichia coli . In some embodi In yet another embodiment, the one or more nucleic acid 55 ments, the glycolaldehyde dehydrogenase is encoded by the molecules encoding the xylonate dehydratase is encoded by aldA gene , or homolog thereof. a nucleic acid sequence selected from the group consisting In some embodiments , a method of producing a recom of SEQ ID NOs : 68 , 70 , 71 , 73 and 74 . binant microorganism that produces or accumulates MEG In one embodiment, the 2 -keto - 3 - deoxy - D -pentonate and one or more three -carbon compounds from exogenous aldolase is encoded by one or more nucleic acid molecules 60 D - xylose comprises introducing into the recombinant micro obtained from a microorganism selected from Pseudomonas organism a deletion, insertion, or loss of function mutation sp . and E. coli . In another embodiment, the 2 -keto - 3 -deoxy- in a gene encoding a lactate dehydrogenase to prevent the D - pentonate aldolase is encoded by one or more nucleic acid production of lactate from pyruvate and instead shunt the molecules obtained from E. coli . In some embodiments, the reaction toward production of one or more three - carbon one or more nucleic acid molecules encoding the 2 - keto - 3- 65 compounds. In one embodiment, the enzyme that catalyzes deoxy - D -pentonate aldolase is selected from yjhH and / or the conversion of pyruvate to lactate is a lactate dehydro yagE , or homolog thereof. In a further embodiment, the one genase . In particular embodiments , the enzyme converts US 10,941,424 B2 91 92 pyruvate to lactate . In some embodiments, the lactate dehy- nucleic acid molecules obtained from a microorganism drogenase is from Escherichia coli . In some embodiments , selected from the group consisting of Clostridium sp . , Bacil the lactate dehydrogenase is encoded by the IdhA gene , or lus sp . , Chromobacterium sp . and Pseudomonas sp . In homolog thereof. another embodiment, the acetoacetate decarboxylase is In one embodiment of any aspect disclosed above , the 5 encoded by one or more nucleic acid molecules obtained glycolaldehyde reductase is encoded by one or more nucleic from a microorganism selected from the group consisting of acid molecules obtained from a microorganism selected Clostridium acetobutylicum , Clostridium beijerinckii, from E. coli and S. cerevisiae . In another embodiment, the one or more nucleic acid molecules is selected from gldA , Clostridium cellulolyticum , Bacillus polymyxa , Chromobac GRE2 , GRE3 , yqhD , ydjG , fuco , yafB ( dkgB ), and / or yqhE 10 terium violaceum and Pseudomonas putida. In some ( dkgA ), or homolog thereof. In another embodiment, the one embodiments, the one or more nucleic acid molecules or more nucleic acid molecules is yqhD . In some embodi encoding the acetoacetate decarboxylase is adc , or homolog ments , the yqhD comprises a G149E mutation . In a further thereof. In a further embodiment, the acetoacetate decar embodiment, the glycolaldehyde reductase comprises an boxylase comprises an amino acid sequence selected from amino acid sequence selected from the group consisting of 15 the group consisting of SEQ ID NOs : 49 and 52. In yet SEQ ID NOs : 13 , 15 , 17 , 20 , 23 , 25 , 28 , 30 and 32. In yet another embodiment, the acetoacetate decarboxylase is a further embodiment, the glycolaldehyde reductase is encoded by a nucleic acid sequence selected from the group encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs : 47 , 48 , 50 and 51 . consisting of SEQ ID NOs : 12 , 14 , 16 , 18 , 19 , 21 , 22 , 24 , 26 , In one embodiment of any aspect disclosed above , the 27 , 29 and 31 . 20 enzyme that catalyzes the conversion of acetone to isopro In one embodiment of any aspect disclosed above , the panol is a secondary alcohol dehydrogenase ( S - ADH ) . In thiolase or acetyl coenzyme A acetyltransferase is encoded another embodiment, the enzyme is a secondary alcohol by one or more nucleic acid molecules obtained from a dehydrogenase that is encoded by a nucleic acid molecule microorganism selected from the group consisting of obtained from a microorganism selected from Burkholderia Clostridium sp . , Bacillus sp . , E. coli , Saccharomyces sp . and 25 sp , Alcaligenes sp . , Clostridium sp . , Thermoanaerobacter Marinobacter sp . In some embodiments, the thiolase or sp . , Phytomonas sp . , Rhodococcus sp . , Methanobacterium acetyl coenzyme A acetyltransferase is encoded by one or sp . , Methanogenium sp . , Entamoeba sp . , Trichomonas sp . , more nucleic acid molecules obtained from a microorganism selected from the group consisting of Clostridium aceto and Tritrichomonas sp . In some embodiments, the nucleic butylicum , Clostridium thermosaccharolyticum , Bacillus 30 acid molecule encoding the secondary alcohol dehydroge cereus , E. coli, Saccharomyces cerevisiae and Marinobacter nase is obtained from a microorganism selected from Bur hydrocarbonoclasticus. In some embodiments , the one or kholderia sp . AIU 652 , Alcaligenes eutrophus, Clostridium more nucleic acid molecules is thlA , atoB and /or 10 , or ragsdalei , Clostridium beijerinckii, Clostridium carboxidi homolog thereof. In a further embodiment, the thiolase or vorans , Thermoanaerobacter brockii, Thermoanaerobacter acetyl coenzyme A acetyltransferase comprises an amino 35 ethanolicus ( Clostridium thermohydrosulfuricum ), Rhodo acid sequence selected from the group consisting of SEQ ID coccus ruber, Methanobacterium palustre , methanogenic NOs : 35 , 37 and 40. In yet a further embodiment, the archaea Methanogenium liminatans, parasitic protist Enta thiolase or acetyl coenzyme A acetyltransferase is encoded moeba histolytica , parasitic protozoan Tritrichomonas foe by a nucleic acid sequence selected from the group consist tus and human parasite Trichomonas vaginalis . In some ing of SEQ ID NOs : 33 , 34 , 36 , 38 and 39 . 40 embodiments , the one or more nucleic acid molecule encod In one embodiment of any aspect disclosed above , the ing secondary alcohol dehydrogenase is adh , adhB , EhAdh1, acetyl- CoA : acetoacetate - CoA transferase or acetate : ac- or homolog thereof. In some embodiments , the S - ADH is etoacetyl -CoA hydrolase is encoded by one or more nucleic predicted from homology and can be from Thermoanaero acid molecules obtained from a microorganism selected bacter mathranii, Micrococcus luteus, Nocardiopsis alba, from Clostridium sp . and E. coli . In another embodiment, 45 Mycobacterium hassiacum , Helicobacter suis , Candida the acetyl -CoA : acetoacetate - CoA transferase or acetate :ac- albicans, Candida parapsilosis, Candida orthopsilosis, etoacetyl - CoA hydrolase is encoded by one or more nucleic Candida metapsilosis, Grosmannia clavigera and Scheffer acid molecules obtained from E. coli . In some embodiments, somyces stipitis . In a further embodiment, the alcohol dehy the one or more nucleic acid molecules encoding the acetyl- drogenase comprises an amino acid sequence selected from CoA : acetoacetate - CoA transferase is atoA and / or atoD , or 50 the group consisting of SEQ ID NOs : 106 and 108. In yet homolog thereof. In another embodiment, the acetyl -CoA : another embodiment, the alcohol dehydrogenase is encoded acetoacetate - CoA transferase or acetate : acetoacetyl - CoA by a nucleic acid sequence selected from the group consist hydrolase is encoded by one or more nucleic acid molecules ing of SEQ ID NOs : 104 , 105 and 107 . obtained from Clostridium acetobutylicum . In some embodi- Enzyme Engineering ments, the one or more nucleic acid molecules encoding the 55 The enzymes in the recombinant microorganism can be acetate : acetoacetyl - CoA hydrolase is ctfA and / or ctfB , or engineered to improve one or more aspects of the substrate homolog thereof. In a further embodiment, the acetyl -CoA : to product conversion . Non - limiting examples of enzymes acetoacetate - CoA transferase or acetate : acetoacetyl -CoA that can be further engineered for use in methods of the hydrolase comprises an amino acid sequence selected from disclosure include an aldolase , an aldehyde reductase, an the group consisting of SEQ ID NOs : 43 , 46 , 97 , 99 , 101 and 60 acetoacetyl coenzyme A hydrolase, a xylose isomerase, a 103. In yet a further embodiment, the acetyl- CoA :acetoac- xylitol dehydrogenase and combinations thereof. These etate - CoA transferase or acetate : acetoacetyl -CoA hydrolase enzymes can be engineered for improved catalytic activity, is encoded by a nucleic acid sequence selected from the improved selectivity, improved stability, improved tolerance group consisting of SEQ ID NOs : 41 , 42 , 44 , 45 , 96 , 98 , 100 to various fermentation conditions ( temperature , pH , etc. ) , and 102 . 65 or improved tolerance to various metabolic substrates , prod In one embodiment of any aspect disclosed above , the ucts , by - products, intermediates, etc. The term “ improved acetoacetate decarboxylase is encoded by one or more catalytic activity ” as used herein with respect to a particular US 10,941,424 B2 93 94 enzymatic activity refers to a higher level of enzymatic As another example, the enzyme xylitol dehydrogenase activity than that measured relative to a comparable non- plays a role in the utilization of xylose along with xylose engineered enzyme. reductase. Xylose reductase ( XR ) reduces xylose to xylitol For example, engineering methods have been used to alter and then xylitol dehydrogenase ( XDH ) reoxidizes xylitol to the stability, substrate specificity and stereospecificity of 5 form xylulose . However, since XR prefers NADPH as aldolases to produce excellent enzymes for biocatalytic cosubstrate, while XDH exclusively uses NAD + as cosub strate , a cosubstrate recycling problem is encountered . One processes . The thermostability and solvent tolerance of solution is to engineer XDH such that its cosubstrate speci fructose - 1,6 - bisphosphate aldolase ( FBP - aldolase ) was ficity is altered from NAD + to NADP + ( Ehrensberger et al . increased using family DNA shuffling of the fda genes from 10 2006 Structure 14 : 567-575 ) . A crystal structure of the Escherichia coli and Edwardsiella ictaluri . A fourth genera Gluconobacter oxydans holoenzyme revealed that Asp38 is tion variant was identified which displayed an average largely responsible for the NAD + specificity of XDH . Asp38 280 - fold higher half - life at 53 ° C. than either parent. The interacts with the hydroxyls of the adenosine ribose , and same variant also displayed enhanced activity in various Met39 stacks under the purine ring and is also located near polar and non - polar organic solvents ( Hao and Berry 2004 15 the 2 ' hydroxyl. A double mutant ( D38S / M39R ) XDH was Protein Eng Des Sel 17 : 689-697 ) . constructed that exclusively used NADP + without loss of As another example , acetoacetyl coenzyme A hydrolase enzyme activity. can convert acetoacetyl - CoA to acetoacetate . However, the Metabolic Engineering Enzyme Overexpression or hydrolase is unspecific in that it also reacts with the same Enzyme Downregulation /Deletion for Increased Pathway magnitude of order with acetyl- CoA , which is the substrate 20 Flux required for acetoacetyl -CoA formation by the enzyme In various embodiments described herein , the exogenous thiolase . Thus, to create more efficient acetoacetyl - CoA and endogenous enzymes in the recombinant microorganism hydrolases, these enzymes have been engineered to have at participating in the biosynthesis pathways described herein least 10x higher activity for the acetoacetyl - CoA substrate may be overexpressed . than for acetyl - CoA substrate by replacing several glutamic 25 The terms " overexpressed ” or “ overexpression ” refers to acid residues in the enzyme beta subunit that is important for an elevated level ( e.g. , aberrant level ) of mRNAs encoding catalysis ( WO 2015/042588 ) . for a protein ( s ) , and / or to elevated levels of protein ( s ) in As another example, the E. coli YqhD enzyme is a broad cells as compared to similar corresponding unmodified cells substrate aldehyde reductase with NADPH - dependent expressing basal levels of mRNAs or having basal levels of reductase activity for more than 10 aldehyde substrates and 30 proteins. In particular embodiments, mRNA ( s) or protein ( s ) is a useful enzyme to produce biorenewable fuels and may be overexpressed by at least 2 - fold , 3 - fold , 4 - fold , chemicals ( Jarboe 2010 Applied Microbiology and Biotech- 5 - fold , 6 - fold , 8 - fold , 10 - fold , 12 - fold , 15 - fold or more in nology 89 : 249 ) . Though YqhD enzyme activity is beneficial microorganisms engineered exhibit increased gene through its scavenging of toxic aldehydes, the enzyme is mRNA , protein , and / or activity . also NADPH - dependent and contributes to NADPH deple- 35 In some embodiments, a recombinant microorganism of tion and growth inhibition of organisms. Error - prone PCR of the disclosure is generated from a host that contains the YqhD was performed in order to improve 1,3 - propanediol enzymatic capability to synthesize substrates such as D -xy production from 3 - hydroxypropionaldehyde ( 3 - HPA ). This lulose , D - ribulose , D - ribulose - 1 -phosphate , D -xylulose - 1 directed engineering yielded two mutants , D99QN147H and phosphate, D -xylonolactone , D -xylonate , 2 -keto - 3 -deoxy Q202A , with decreased Km and increased kcat for certain 40 xylonate , glycolaldehyde, DHAP, pyruvate, acetoacetyl aldehydes, particularly 3 -HPA ( Li et al . 2008 Prog. Nat . Sci . CoA or acetoacetate . In some embodiments, it can be useful 18 ( 12 ) : 1519-1524 ) . The improved catalytic activity of the to increase the synthesis or accumulation of, for example , D99QN147H mutant is consistent with what is known about D - xylulose , D - ribulose, D - ribulose - 1 - phosphate, D -xylu the structure of YqhD ( Sulzenbacher et al . 2004 J. Mol . Biol . lose - 1 -phosphate , D -xylonolactone , D -xylonate , 2 -keto -3 342 ( 2 ) : 489-502 ) , as residues Asp99 and Asn147 both inter- 45 deoxy - xylonate , glycolaldehyde, DHAP , pyruvate , act with NADPH . Use of the D99QN147H mutant increased acetoacetyl -CoA or acetoacetate, to increase the production 1,3 -propanediol production from 3 -HPA 2 - fold . Mutant of MEG and one or more three - carbon compounds. YqhD enzymes with increased catalytic efficiency increased In some embodiments , it may be useful to increase the Kcat /Km ) toward NADPH have also been described in WO expression of endogenous or exogenous enzymes involved 2011012697 A2 , which is herein incorporated in its entirety. 50 in the MEG and three - carbon compound biosynthesis path As another example, xylose isomerase is a metal -depen- ways to increase flux from , for example, D - xylulose , D - ribu dent enzyme that catalyzes the interconversion of aldose and lose , D - ribulose - 1 - phosphate , D - xylulose - 1 - phosphate , ketose sugars, primarily between xylose to xylulose and D - xylonolactone , D -xylonate , 2 -keto - 3 - deoxy -xylonate , glucose to fructose . It has lower affinity for lyxose , arabinose glycolaldehyde , DHAP , pyruvate , acetoacetyl - CoA or and mannose sugars . The hydroxyl groups of sugars may 55 acetoacetate , thereby resulting in increased synthesis or define the substrate preference of sugar isomerases . The accumulation of MEG and one or more three - carbon com aspartate at residue 256 of Thermus thermophilus xylose pounds . isomerase was replaced with arginine ( Patel et al . 2012 Increased synthesis or accumulation can be accomplished Protein Engineering, Design & Selection vol . 25 no . 7 pp . by, for example , overexpression of nucleic acids encoding 331-336 ) . This mutant xylose isomerase exhibited an 60 one or more of the above - described MEG and three - carbon increase in specificity for D -lyxose , L - arabinose and D -man- compound biosynthesis pathway enzymes . Overexpression nose . The catalytic efficiency of the D256R xylose of a MEG and three - carbon compound biosynthesis pathway isomerase mutant was also higher for these 3 substrates enzyme or enzymes can occur, for example, through compared to the wild type enzyme. It was hypothesized that increased expression of an endogenous gene or genes , or the arginine at residue 256 in the mutant enzyme may play 65 through the expression , or increased expression , of an exog a role in the catalytic reaction or influence changes in enous gene or genes . Therefore , naturally occurring organ substrate orientation . isms can be readily modified to generate non -natural , MEG US 10,941,424 B2 95 96 and three - carbon compound producing microorganisms Murray et al . ( 1989 ) Nucl. Acids Res . 17 : 477-508 ) can be through overexpression of one or more nucleic acid mol- prepared , for example, to increase the rate of translation or ecules encoding a MEG and three - carbon compound bio- to produce recombinant RNA transcripts having desirable synthesis pathway enzyme . In addition , a non - naturally properties, such as a longer half- life, as compared with occurring organism can be generated by mutagenesis of an 5 transcripts produced from a non - optimized sequence. Trans endogenous gene that results in an increase in activity of an lation stop codons can also be modified to reflect host enzyme in the MEG and three - carbon compound biosynthe- preference . For example , typical stop codons for S. cerevi sis pathways. siae and mammals are UAA and UGA , respectively . The Equipped with the present disclosure , the skilled artisan typical stop codon for monocotyledonous plants is UGA , will be able to readily construct the recombinant microor- 10 whereas insects and E. coli commonly use UAA as the stop ganisms described herein , as the recombinant microorgan- codon ( Dalphin et al . ( 1996 ) Nucl. Acids Res . 24 : 216-218 ) . isms of the disclosure can be constructed using methods well Those of skill in the art will recognize that, due to the known in the art as exemplified above to exogenously degenerate nature of the genetic code , a variety of nucleic express at least one nucleic acid encoding a MEG and acid sequences can be used to encode a given enzyme of the three -carbon compound biosynthesis pathway enzyme in 15 disclosure . The nucleic acid sequences encoding the biosyn sufficient amounts to produce MEG and one or more three- thetic enzymes are referenced herein merely to illustrate an carbon compounds. embodiment of the disclosure , and the disclosure includes Methods for constructing and testing the expression levels any nucleic acid sequences that encode the amino acid of a non - naturally occurring MEG and three - carbon com- sequences of the polypeptides and proteins of the enzymes pound -producing host can be performed , for example , by 20 of the present disclosure . In similar fashion , a polypeptide recombinant and detection methods well known in the art. can typically tolerate one or more amino acid substitutions, Such methods can be found described in , for example, deletions, and insertions in its amino acid sequence without Sambrook et al . , Molecular Cloning: A Laboratory Manual, loss or significant loss of a desired activity . The disclosure Third Ed . , Cold Spring Harbor Laboratory, New York includes such polypeptides with different amino acid ( 2001 ) ; Ausubo et al . , Current Protocols in Molecular 25 sequences than the specific proteins described herein so long Biology, John Wiley and Sons, Baltimore , Md . ( 1999 ) . as the modified or variant polypeptides have the enzymatic A variety of mechanisms known in the art can be used to anabolic or catabolic activity of the reference polypeptide . express , or overexpress, exogenous or endogenous genes . Furthermore , the amino acid sequences encoded by the For example , an expression vector or vectors can be con- nucleic acid sequences shown herein merely illustrate structed to harbor one or more MEG and three -carbon 30 embodiments of the disclosure. compound biosynthesis pathway enzymes encoding nucleic Expression control sequences are known in the art and acids as exemplified herein operably linked to expression include , for example, promoters , enhancers, polyadenylation control sequences functional in the host organism . Expres- signals, transcription terminato internal ribosome entry sion vectors applicable for use in the microbial host organ- sites ( IRES ) , and the like , that provide for the expression of isms of the invention include, for example, plasmids, phage 35 the polynucleotide sequence in a host cell . Expression vectors , viral vectors , episomes and artificial chromosomes, control sequences interact specifically with cellular proteins including vectors and selection sequences or markers oper- involved in transcription (Maniatis et al . , Science, 236 : able for stable integration into a host chromosome . Select- 1237-1245 ( 1987 ) ) . Exemplary expression control able marker genes also can be included that, for example, sequences are described in , for example, Goeddel, Gene provide resistance to antibiotics or toxins , complement 40 Expression Technology : Methods in Enzymology , Vol . 185 , auxotrophic deficiencies, or supply critical nutrients not in Academic Press, San Diego , Calif. ( 1990 ) . the culture media . Expression control sequences can include In various embodiments , an expression control sequence constitutive and inducible promoters, transcription enhanc- may be operably linked to a polynucleotide sequence . By ers , transcription terminators, and the like which are well “ operably linked ” is meant that a polynucleotide sequence known in the art . When two or more exogenous encoding 45 and an expression control sequence ( s) are connected in such nucleic acids are to be co -expressed , both nucleic acids can a way as to permit gene expression when the appropriate be inserted , for example , into a single expression vector or molecules ( e.g. , transcriptional activator proteins) are bound in separate expression vectors . For single vector expression , to the expression control sequence ( s ). Operably linked pro the encoding nucleic acids can be operationally linked to one moters are located upstream of the selected polynucleotide common expression control sequence or linked to different 50 sequence in terms of the direction of transcription and expression control sequences , such as one inducible pro- translation . Operably linked enhancers can be located moter and one constitutive promoter. The transformation of upstream , within , or downstream of the selected polynucle exogenous nucleic acid sequences involved in a metabolic or otide . synthetic pathway can be confirmed using methods well In some embodiments, the recombinant microorganism is known in the art . 55 manipulated to delete , disrupt, mutate, and / or reduce the As will be understood by those of skill in the art, it can be activity of one or more endogenous enzymes that catalyzes advantageous to modify a coding sequence to enhance its a reaction in a pathway that competes with the biosynthesis expression in a particular host . The genetic code is redun- pathway for the production of MEG and one or more dant with 64 possible codons , but most organisms typically three -carbon compounds. use a subset of these codons . The codons that are utilized 60 In some embodiments , the recombinant microorganism is most often in a species are called optimal codons , and those manipulated to delete , disrupt, mutate , and / or reduce the not utilized very often are classified as rare or low - usage activity of one or more endogenous enzymes that catalyzes codons . Codons can be substituted to reflect the preferred the conversion of D - xylulose to D -xylulose - 5 - phosphate . In codon usage of the host , a process sometimes called “ codon some such embodiments, the enzyme that catalyzes the optimization " or " controlling for species codon bias . " 65 conversion of D - xylulose to D - xylulose - 5 - phosphate is a Optimized coding sequences containing codons preferred D - xylulose - 5 - kinase . In some embodiments, the D - xylulose by a particular prokaryotic or eukaryotic host ( see also , 5 - kinase is from Escherichia coli . In some embodiments , the US 10,941,424 B2 97 98 D - xylulose - 5 - kinase is encoded by the xylB gene or the xylA gene or homologs thereof. In some embodiments, homologs thereof. In some embodiments , the manipulation the manipulation prevents conversion of D - xylose to D - xy prevents the conversion of D - xylulose to D - xylulose - 5- lulose and instead shunts the reaction toward the conversion phosphate and instead shunts the reaction toward conversion of D -xylose to D -xylonate . of D - xylulose to D - xylulose - 1 -phosphate . 5 In some embodiments, the recombinant microorganism is EXAMPLES manipulated to delete , disrupt, mutate , and / or reduce the activity of one or more endogenous enzymes that catalyzes Example la . Production of Ethylene Glycol in E. the conversion of glycolaldehyde to glycolic acid . In some coli such embodiments , the enzyme that catalyzes the conversion 10 of glycolaldehyde to glycolic acid is a glycolaldehyde The E. coli K12 strain MG1655 was used as host for the dehydrogenase. In some embodiments , the glycolaldehyde deletion of two genes that could divert the carbon flux from dehydrogenase is from Escherichia coli . In some embodi MEG + IPA pathway : aldA and xylB . The genes were suc ments, the glycolaldehyde dehydrogenase is encoded by the cessfully deleted and deletion confirmed by sequencing. A aldA gene or homologs thereof. In some embodiments, the 15 plasmid containing the dte gene , encoding the first enzyme manipulation prevents the production of glycolic acid from of the pathway ( D - tagatose 3 - epimerase , SEQ ID NO : 3 , glycolaldehyde and instead shunts the reaction toward con- encoded by nucleic acid sequence SEQ ID NO : 2 ) , was version of glycolaldehyde to MEG . expressed under the control of the proD promoter in a pUC In some embodiments, the recombinant microorganism is vector backbone. The plasmid was constructed using the manipulated to delete , disrupt, mutate, and / or reduce the 20 MoClo system and confirmed by sequencing. The confirmed activity of one or more endogenous enzymes that catalyzes plasmid was transformed in the deleted strain . Colonies the conversion of pyruvate to lactate . In some such embodi- from transformations were inoculated in 3 mL of LB media ments , the enzyme that catalyzes the conversion of pyruvate for pre - culture. After 16 hours of cultivation 10 % of the to lactate is a lactate dehydrogenase. In some embodiments, pre - culture was transferred to 50 mL of LB media containing the lactate dehydrogenase is from Escherichia coli. In some 25 15 g / L of xylose . The flasks were incubated at 37 ° C. , 250 embodiments, the lactate dehydrogenase is encoded by the rpm until complete consumption of xylose . The initial OD of IdhA gene or homologs thereof. In some embodiments , the the cultivation was 0.4 . Xylose was fully consumed after 96 manipulation prevents the production of lactate from pyru- hours of cultivation . Small amounts of MEG were detected vate and instead shunts the reaction toward production of a after 27 hours of cultivation . The highest MEG concentra three - carbon compound . 30 tion was measured after 100 hours of cultivation, reaching In some embodiments , the recombinant microorganism is 2.1 g / L . The overall yield of MEG production was 13.7 wt manipulated to delete , disrupt, mutate , and / or reduce the % ( FIG . 7 ) . activity of one or more endogenous enzymes that catalyzes the conversion of D - xylulose to D - xylulose - 5 -phosphate . In Example 1 b . Improved Production of Ethylene some such embodiments , the enzyme that catalyzes the 35 Glycol in E. coli conversion of D - xylulose to D - xylulose - 5 - phosphate is a D - xylulose - 5 - kinase . In some embodiments, the D - xylulose- The E. coli K12 strain MG1655 with aldA and xylB genes 5 - kinase is from Saccharomyces cerevisiae. In some deleted ( same strains as example 1a ) was used as host for the embodiments the D - xylulose - 5 - kinase is encoded by the implementation of a complete MEG pathway. An operon XKS1 gene or homologs thereof. In some embodiments , the 40 containing dte ( D - tagatose 3 -epimerase enzyme, SEQ ID D - xylulose - 5 -kinase is from Pichia stipitis. In some embodi- NO : 3 , encoded by nucleic acid sequence SEQ ID NO : 2 ) , ments the D - xylulose - 5 -kinase is encoded by the XYL3 fucA ( D -ribulose - 1 - phosphate aldolase enzyme, SEQ ID gene or homologs thereof. In some embodiments, the NO : 11 , encoded by nucleic acid sequence SEQ ID NO : 10 ) , manipulation prevents the conversion of D - xylulose to fuco ( aldehyde reductase enzyme , SEQ ID NO : 28 , encoded D - xylulose - 5 -phosphate and instead shunts the reaction 45 by nucleic acid sequence SEQ ID NO : 27 ) and fuck toward conversion of D - xylulose to D - xylulose - 1 -phos- ( D - ribulokinase enzyme , SEQ ID NO : 8 , encoded by nucleic phate. acid sequence SEQ ID NO : 7 ) genes under the control of the In some embodiments , the recombinant microorganism is proD promoter was constructed in a pET28a backbone . The manipulated to delete , disrupt, mutate , and / or reduce the plasmid was constructed using In - fusion commercial kit and activity of one or more endogenous enzymes that catalyzes 50 confirmed by sequencing. The confirmed plasmid was trans the conversion of D - xylulose - 5 -phosphate to D - xylulose . In formed in the MG1655 mutant strain . Colonies from trans some such embodiments, the enzyme that catalyzes the formation were inoculated in 3 mL of LB media for pre conversion of D - xylulose - 5 -phosphate to D -xylulose is an culture . After 16 hours of cultivation , the pre - culture was alkaline phosphatase. In some embodiments , the alkaline transferred to 50 mL of LB media containing 15 g / L of phosphatase is from S. cerevisiae . In some embodiments, the 55 xylose to an initial OD of 0.3 . The flasks were incubated at alkaline phosphatase is encoded by the PH013 gene or 37 ° C. , 250 rpm until complete consumption of xylose . After homologs thereof. In some embodiments , the manipulation 4 hours of cultivation , approximately 100 mg / L of MEG prevents the conversion of D - xylulose - 5 - phosphate to D - xy- could be detected . After 144 hours of cultivation , 4.3 g / L of lulose . MEG were produced and all xylose was consumed ( FIG . 8 ) . In some embodiments, the recombinant microorganism is 60 The overall yield and productivity were , respectively, 0.3 g / g manipulated to delete , disrupt, mutate , and / or reduce the and 0.03 g / L · h . activity of one or more endogenous enzymes that catalyzes the conversion of D - xylose to D - xylulose . In some such Example 2 : Co -Production of Ethylene Glycol and embodiments , the enzyme that catalyzes the conversion of Isopropanol in Saccharomyces cerevisiae D - xylose to D - xylulose is a D - xylose isomerase . In some 65 embodiments , the D - xylose isomerase is from E. coli . In The S. cerevisiae laboratory strain BY4730 , derived from some embodiments, the D - xylose isomerase is encoded by S288c , was used as host for the expression of MEG + IPA US 10,941,424 B2 99 100 pathways. The first step was the integration of the IPA ( D - ribulose - 1 - phosphate aldolase enzyme ), fuco ( aldehyde pathway into the genome of S. cerevisiae . One copy of each reductase enzyme) and fucK ( D - ribulokinase enzyme ). dte gene was integrated by homologous recombination under gene was codon optimized for E. coli ( Dte amino acid the control of the following promoters: ADH1 for thl gene sequence set forth in SEQ ID NO : 3 ) and synthesized . All ( thiolase, SEQ ID NO : 35 , encoded by nucleic acid sequence 5 other genes are native from E. coli and were PCR amplified SEQ ID NO : 34 ) ; TEF1 for ato A gene , PGK1 for ato D gene using the following primers: fucA and fuco ( Forward ( acetate : acetoacetyl -CoA transferase , SEQ ID NOs : 43 and Primer : CCTTTAATAAGGAGATATACCATG 46 , encoded by nucleic acid sequences SEQ ID NOs : 42 and GAACGAAATAAACTTGC ( SEQ ID NO : 111 ) and 45 , respectively ); TDH3 for adc gene ( acetoacetate decar Reverse Primer : GGTTATTCCTCCTTATT boxylase , SEQ ID NO : 49 , encoded by nucleic acid 10 TAGAGCTCTAAACGAATTCT sequence SEQ ID NO : 48 ) ; and TPI1 for adh gene ( second- TACCAGGCGGTATGGTAA A ( SEQ ID NO : 112 ) ) and ary alcohol dehydrogenase, SEQ ID NO : 106 , encoded by fuck ( Forward Primer : GAATTCGTT nucleic acid sequence SEQ ID NO : 105 ) . The integration TAGAGCTCTAAATAAGGAGGAATAACCATGAT was confirmed by PCR and sequencing . The second step was GAAACAAGAAGTTA T ( SEQ ID NO : 113 ) and Reverse the introduction of genes capable of consuming xylose in the 15 Primer: GAGCT CGGTACCCGGGGATC yeast genome. The pathway chosen for xylose consumption CAAAAAACCCCTCAAGACCC ( SEQ ID NO : 114 ) ) . An is composed of two genes : Xyll and Xy12 . Three copies of operon containing dte ( D - tagatose 3 - epimerase enzyme ), the Xyll gene ( SEQ ID NO : 84 , encoded by nucleic acid fucA ( D - ribulose - 1 - phosphate aldolase enzyme ), fuco (al sequence SEQ ID NO : 83 ) under control of TEF1 promoter dehyde reductase enzyme ), fucK ( D - ribulokinase enzyme ) and three copies of the Xy12 ( SEQ ID NO : 90 , encoded by 20 genes and T7 terminator under the control of proD promoter nucleic acid sequence SEQ ID NO : 89 ) gene also under ( constitutive promoter ) was constructed in a pET28a back control of TEF1 promoter were integrated into the yeast bone . For each gene a specific RBS sequence was utilized . genome through homologous recombination . The integra- The plasmid was constructed using In - fusion commercial kit tion was confirmed by PCR and sequencing. The third step and confirmed by sequencing. The entire operon under the was the integration of the MEG pathway. Two copies of the 25 control of proD promoter was subcloned in PZA31 and D - tagatose 3 - epimerase enzyme ( dte gene , SEQ ID NO : 3 , pZS * 13 backbones using restriction -ligation methodology . encoded by nucleic acid sequence SEQ ID NO : 2 ) under the Isopropanol pathway was also assembled in three differ control of TEF1 and TDH3 promoters, respectively, were ent vectors backbones: PZA31 , pZS * 13 and pET28a . Pro integrated into the genome along with the following genes : duction of isopropanol requires the expression of five genes : one copy of fuco gene ( glycolaldehyde reductase, SEQ ID 30 thl ( thiolase ), ato A / D ( acetate : acetoacetyl -CoA transferase ), NO : 28 , encoded by nucleic acid sequence SEQ ID NO : 27 ) adc ( acetoacetate decarboxylase ) and adh ( secondary alco under control of the PGK1 promoter; one copy of the fucA hol dehydrogenase ). Ato A / D gene is native from E. coli and gene ( D - ribulose - phosphate aldolase , SEQ ID NO : 11 , was PCR amplified (Forward Primer : CTGTTGTTATAT encoded by nucleic acid sequence SEQ ID NO : 10 ) using a TGTAATGATGTATGCAAGAGGGATAAA ( SEQ ID NO : PGK1 promoter and one copy of fuck gene ( D - ribulokinase , 35 115 ) and Reverse Primer: TATATCTCCTTCTTAAAGTT SEQ ID NO : 8 , encoded by nucleic acid sequence SEQ ID CATAAATCACCCCGTTGC ( SEQ ID NO : 116 ) ) . thl ( Thl NO : 7 ) under a PGK1 promoter. The final strain was amino acid sequence set forth in SEQ ID NO : 35 ) , adc ( Ade confirmed by PCR and sequencing. The strain was inocu- amino acid sequence set forth in SEQ ID NO : 49 ) , and adh lated in YPD media containing 20 g / L of glucose and (Adh amino acid sequence set forth in SEQ ID NO : 106 ) incubated at 30 ° C. , 200 rpm . After 16 hours of growth ,the 40 were codon optimized for E. coli and synthesized . An pre -culture was inoculated in YPDX media containing 30 operon containing thl ( thiolase ), adh ( secondary alcohol g / L of glucose and 10 g / L of xylose to an OD 2.0 . The flasks dehydrogenase ), adc ( acetoacetate decarboxylase ), ato A /D were incubated at 30 ° C. , 100 rpm . The typical behavior of ( acetate : acetoacetyl- CoA transferase ) genes and T1 termi C5 and C6 consumption in yeast was observed . 30 g / L of nator under the control of the inducible promoter PLLaco glucose was consumed in less than 60 hours , while 90 % of 45 was constructed in a PET28a backbone . For each gene a the initial xylose was consumed only after 200 hours. The specific RBS sequence was utilized . The plasmid was con OD reached a value of 55 after 200 hours of cultivation . structed in several steps using both In - fusion commercial kit Isopropanol was already being produced in the initial 60 and restriction - ligation methodology . The correct assemble hours of cultivation , while MEG was only detected after 160 was confirmed by sequencing. The entire operon under the hours of cultivation . The highest co - production was 50 control of the inducible promoter pLLaco was subcloned in obtained at 183 hours of cultivation , with 58 mg / L of MEG PZA31 and pZS * 13 backbones using restriction - ligation and 2.81 g / L of isopropanol. The overall yield for the methodology. co -production , from glucose and xylose , was 7.4 % ( FIG . 6 ) . Several co - transformations of MEG and IPA plasmids were performed in the strains with xylB and aldA deleted to Example 3. Co - Production of Ethylene Glycol 55 generate strains harboring all possible plasmid combina ( MEG ) , Acetone and Isopropanol ( IPA ) in E. coli tions . Table 2 describes the constructed strains . Using Ribulose - 1 -Phosphate Pathway TABLE 2 E. coli K12 strain MG1655 was used as host for the deletion of two genes that could divert the carbon flux from 60 Strain Plasmids and pathways MEG + IPA pathway: aldA and xylB . The genes were suc 1 MEG in pET28a and IPA in PZA31 cessfully deleted and the deletion was confirmed by 2 MEG in pET28a and IPA in pZS * 13 sequencing. Ribulose - 1 - phosphate pathway for MEG pro 3 MEG in PZA31 and IPA in pZS * 13 4 MEG in PZA31 and IPA in PET28a1 duction was assembled in three different vectors backbones : 5 MEG in pZS * 13 and IPA in PZA31 PZA31 , pZS * 13 and pET28a . Production of MEG through 65 6 ribulose - 1 -phosphate pathway requires the expression of MEG in pZS * 13 and IPA in PET28a four genes : dte ( D - tagatose 3 - epimerase enzyme ), fucA US 10,941,424 B2 101 102 Colonies from transformations were inoculated in 3 mL of E. coli and was PCR amplified ( Forward Primer: TB media for pre - culture . After 16 hours of cultivation , CTGTTGTTATATTGTAATGATGTATGCAAGAGGGA 100 % of the pre - culture was transferred to 100 mL of TB TAAA ( SEQ ID NO : 119 ) and Reverse Primer: media containing 15 g / L of xylose . The flasks were incu- TATATCTCCTTCTTAAAGTTCATAAAT bated at 37 ° C. , 250 rpm until complete consumption of 5 CACCCCGTTGC ( SEQ ID NO : 120 ) ) . thl ( Thl amino acid xylose . The initial OD of the cultivation was 0.3 . For the sequence set forth in SEQ ID NO : 35 ) , adc ( Adc amino acid induction of pLLacO promoter 1 mM of IPTG was added to sequence set forth in SEQ ID NO : 49 ) and adh ( Adh amino the culture after 2 hours ( OD = 1 ) . acid sequence set forth in SEQ ID NO : 106 ) were codon Xylose was fully consumed after 32 hours of cultivation optimized for E. coli and synthesized . for strains 1 to 4. Strain 5 only consumed xylose completely 10 An IPA integration cassette was composed of an operon after 55 hours and strain 6 was not able to consume all containing thl ( thiolase ), adh ( secondary alcohol dehydro xylose after 144 hours . genase ), adc ( acetoacetate decarboxylase ), atoA / D ( acetate : The overall yield of co - production was calculated con- acetoacetyl - CoA transferase ) genes and T1 terminator under sidering the amount of ethylene glycol , isopropanol and the control of a medium strength constitutive promoter acetone produced per gram of xylose consumed . The best 15 (modified from RecA ) flanked by regions homologous to yield was obtained after 48 hours of fermentation . The yield upstream and downstream of aldA gene . For each gene a ( g products / g xylose ) of all strains is depicted in FIG . 9 . specific RBS sequence was utilized . An antibiotic marker Strains 2 and 3 co -produced MEG , isopropanol and was included into the cassette for the selection of transfor acetone while strains 1 and 4 co - produced only MEG and mants . The cassette was constructed using In - fusion com acetone . Strains 5 and 6 produced only MEG . 20 mercial kit, confirmed by sequencing and transformed in E. Strain 2 showed the highest overall yield ( 0.28 g / g ) , as coli K12 MG1655 strain . The proper integration of an IPA well as the highest yield for ethylene glycol ( 0.25 g / g ) and pathway at aldA locus , yielding a deleted aldA strain with an isopropanol ( 0.03 g / g ) production. Strain 4 showed the IPA pathway integrated , was confirmed by sequencing. highest yield for acetone production ( 0.01 g / g ). The xylB aldA deleted strain with MEG and IPA pathways 25 integrated in the genome was inoculated in 3 mL of TB Example 4. Co - Production of Ethylene Glycol media for pre - culture . After 16 hours of cultivation , 100 % of ( MEG ) , Acetone and Isopropanol ( IPA ) in E. coli the pre - culture was transferred to 100 mL of TB media Using Xylulose - 1 - Phosphate Pathway containing 15 g / L of xylose . The flasks were incubated at 37 ° C. , 250 rpm until complete consumption of xylose . The E. coli K12 strain MG1655 was used as host for the 30 initial OD of the cultivation was 0.3 . expression of MEG + IPA pathways. Two genes that could Xylose was fully consumed after 30 hours of cultivation divert the carbon flux from MEG + IPA pathway were iden- ( FIG . 10 ) . Ethylene glycol , acetone and isopropanol reached tified as target for deletion : aldA and xylB genes . A MEG a maximum titer of 3.5 g / L , 70 mg / L and 400 mg / L pathway was integrated at xyl? locus , enabling a stable respectively integration concomitantly with xylB deletion . Production of 35 The overall yield of co - production was calculated con MEG through xylulose - 1 - phosphate pathway requires the sidering the amount of ethylene glycol , isopropanol and expression of three genes : khkC ( D -xylulose - 1 - kinase acetone produced per gram of xylose consumed . MEG is the enzyme ), aldoB ( D -xylulose - 1 -phosphate aldolase enzyme) product with the highest yield , 0.237 g / g, followed by and fuco ( aldehyde reductase enzyme ). khkC ( KhkC amino isopropanol , 0.029 g / g and acetone, 0.006 g / g ( FIG . 11 ) . The acid sequence set forth in SEQ ID NO : 55 ) and aldoB 40 best co - production yield , obtained after 48 hours of fermen ( AldoB amino acid sequence set forth in SEQ ID NO : 58 ) tation , was 0.27 g products / g xylose ( 44 % of maximum genes were codon optimized for E. coli and synthesized . theoretical yield ) . Fuco gene is native from E. coli and was PCR amplified ( Forward Primer : ATGGCTAACAGAATGATTCTG ( SEQ Example 5. Co -Production of Ethylene Glycol ID NO : 117 ) and Reverse Primer: 45 ( MEG ) , Acetone and Isopropanol ( IPA ) in E. coli TTACCAGGCGGTATGGTAAAGCT ( SEQ ID NO : 118 ) ) . Using Xylonate Pathway A MEG integration cassette was composed of an operon containing khkC ( D - xylulose - 1 - kinase enzyme ), aldoB E. coli K12 strain MG1655 was used as host for the ( D -xylulose - 1 -phosphate aldolase enzyme ), fuco ( aldehyde expression of MEG + IPA pathways. Two genes that could reductase enzyme) genes and rplM terminator under the 50 divert the carbon flux from MEG + IPA pathway were iden control of proD promoter ( constitutive promoter) flanked by tified as target for deletion : aldA and xylA genes . A MEG regions homologous to upstream and downstream of xy1B pathway was integrated at xylA locus , enabling a stable gene. For each gene a specific RBS sequence was utilized . integration concomitantly with xylA deletion . Production of An antibiotic marker was also added to the cassette for the MEG through a xylonate pathway requires the expression of selection of transformants . The cassette was constructed 55 two genes : xdh (Xdh amino acid sequence set forth in SEQ using In - fusion commercial kit , confirmed by sequencing ID NO : 61 ) from Caulobacter crescentus was codon opti and transformed in E. coli K12 MG1655 strain . The proper mized for E. coli and synthesized . Fuco gene is native from integration of a MEG pathway at xyl? locus , yielding a E. coli and was PCR amplified ( Forward Primer: deleted xylB strain with a MEG pathway integrated , was ATGGCTAACAGAATGATTCTG ( SEQ ID NO : 117 ) and confirmed by sequencing. 60 Reverse Primer : TTACCAGGCGGTATGGTAAAGCT The strain harboring a MEG pathway at xyl? locus was ( SEQ ID NO : 118 ) ) . Two other native enzymes could be used as host for integration of an IPA pathway at aldA locus , overexpressed to improve MEG production through a enabling a stable integration concomitantly with aldA dele- xylonate pathway : D - xylonate dehydratase ( yjhG , yagF , or tion . Production of isopropanol requires the expression of homologs thereof ) and aldolase ( yjhH , yagE , or homologs five genes: thl ( thiolase ), ato A / D ( acetate : acetoacetyl -COA 65 thereof ). transferase ), adc ( acetoacetate decarboxylase ) and adh ( sec- A MEG integration cassette was composed of an operon ondary alcohol dehydrogenase ). atoA / D gene is native from containing xdh ( D - xylose dehydrogenase ), fuco ( aldehyde US 10,941,424 B2 103 104 reductase enzyme) genes and rnpB terminator under the ration . Production of isopropanol requires the expression of control of proD promoter ( constitutive promoter) flanked by five genes : thl ( thiolase ), ato A / D ( acetate : acetoacetyl- CoA regions homologous to upstream and downstream of xylA transferase ), adc ( acetoacetate decarboxylase ) and adh ( sec gene . For each gene a specific RBS sequence was utilized . ondary alcohol dehydrogenase ) . ato A / D gene is native from An antibiotic marker was also added to the cassette for the 5 E. coli and was PCR amplified ( Forward Primer : selection of transformants. The cassette was constructed CTGTTGTTATATTGTAATGATGTATGCAAGAGGGA using In - fusion commercial kit , confirmed by sequencing TAAA ( SEQ ID NO : 119 ) and Reverse Primer : and transformed in E. coli K12 MG1655 strain . The proper TATATCTCCTTCTTAAAGTTCATAAAT integration of a MEG pathway at xylA locus , yielding a CACCCCGTTGC ( SEQ ID NO : 120 ) ) . thl ( Thl amino acid deleted xylA strain with a MEG pathway integrated , was 10 sequence set forth in SEQ ID NO : 35 ) , adc ( Adc amino acid confirmed by sequencing. sequence set forth in SEQ ID NO : 49 ) and adh ( Adh amino The strain harboring a MEG pathway at xylA locus was acid sequence set forth in SEQ ID NO : 106 ) were codon used as host for integration of an IPA pathway at aldA locus , optimized for E. coli and synthesized . An operon containing tionenabling. Production a stable ofintegration isopropanol concomitantly requires the with expression aldA dele of is thl ( thiolase ), adh (secondary alcohol dehydrogenase ), ade five genes : thl (thiolase ), ato A /D ( acetate : acetoacetyl - CoA ( acetoacetate decarboxylase ), ato A / D ( acetate : acetoacetyl transferase ), adc ( acetoacetate decarboxylase ) and adh ( sec CoA transferase ) genes and T1 terminator under the control ondary alcohol dehydrogenase ). Ato A / D gene is native from of the inducible promoter pLLaco was constructed in a E. coli and was PCR amplified ( Forward Primer : pZS * 13 backbone. The candidate selection was done using CTGTTGTTATATTGTAATGATGTATGCAAGAGGGA- 20 kanamycin and ampicillin in LB medium . The strain herein TAAA ( SEQ ID NO : 119 ) and Reverse Primer : was referred to as IPA + LinD . This combination of plasmids TATATCTCCTTCTTAAAGTTCATAAAT provides a strain capable of producing isopropanol from CACCCCGTTGC ( SEQ ID NO : 120 ) ) . thl ( Thl amino acid glu se and also expressing linalool isomerase dehydratase sequence set forth in SEQ ID NO : 35 ) , adc ( Adc amino acid enzyme. sequence set forth in SEQ ID NO : 49 ) and adh ( Adh amino 25 One single colony of IPA + Lind , pZs * 13_IPA and acid sequence set forth in SEQ ID NO : 106 ) were codon PET28a_LinD was inoculated in TB medium containing 10 optimized for E. coli and synthesized . g / L glycerol supplemented with kanamycin ( 50 ug / mL ) and An IPA integration cassette was composed of an operon ampicillin ( 100 ug /mL ) at 37 ° C. , 220 rpm . After 20 hours , containing thl ( thiolase ), adh ( secondary alcohol dehydro a new inoculation was done using optical density of 0.2 in genase ), adc ( acetoacetate decarboxylase ), atoA / D ( acetate: 30 TB medium containing 1.5 g / L glycerol supplemented with acetoacetyl - CoA transferase ) genes and T1 terminator under the control of a medium strength constitutive promoter appropriate antibiotics at 37 ° C. , 220 rpm . After 3 hours , the (modified from RecA ) flanked by regions homologous to OD achieved 1.0 at 600 nm and IPTG was added to a final upstream and downstream of aldA gene . For each gene a concentration of 1 mM . The flasks were incubated at 18 ° C. , specific RBS sequence was utilized . An antibiotic marker 35 220 rpm . was included into the cassette for the selection of transfor After 16 hours , the OD was measured and the cultures mants . The cassette was constructed using In - fusion com were concentrated to reach OD 20 using the following media mercial kit, confirmed by sequencing and transformed in E. as described for each assay : coli K12 MG1655 strain . The proper integration of an IPA ( a ) pZs * 13_IPA in TB 20 g / L glucose ( control for iso pathway at aldA locus, yielding a deleted aldA strain with an 40 propanol production ), IPA pathway integrated , was confirmed by sequencing . ( b ) IPA + LinD in TB 10 g / L glycerol and 3 g / L isopropa The xylA aldA deleted strain with MEG and IPA pathways nol ( control for propylene production ) , integrated in the genome was inoculated in 3 mL of TB ( c ) IPA + LinD in TB 20 g / L glucose and 3 g / L isopropanol media for pre - culture . After 16 hours of cultivation , 100 % of ( control for propylene production ), the pre - culture was transferred to 100 mL of TB media 45 ( d ) IPA + LinD in TB 20 g / L glucose ( candidate 1 for containing 15 g / L of xylose . The flasks were incubated at propylene production ), 37 ° C. , 250 rpm until complete consumption of xylose. The ( e ) IPA + LinD in TB 20 g / L glucose ( candidate 2 for initial OD of the cultivation was 0.3 . propylene production ) Xylose was fully consumed before 24 hours of cultivation One aliquot of all cultures were lysate for expression ( FIG . 12 ) . Ethylene glycol , acetone and isopropanol reached 50 analysis and the cells were collected by centrifugation at a maximum titer of 5 g / L , 170 mg / L and 420 mg / L respec- 5000 rpm for 20 min and 4 ° C. The pellet was kept in -80 ° tively . C. for 1 hour then it was thawed on ice and ressuspended in The overall yield of co - production was calculated con- 10 % of original volume in Tris -HCl 50 mM pH 7.5 . The sidering the amount of ethylene glycol, isopropanol and lysis was done by sonication ( 3-5 cycles , 10/10 minutes, acetone produced per gram of xylose consumed . MEG is the 55 25 % amplitude ) on ice after that to separate the soluble product with the highest yield , 0.339 g / g , followed by fraction it was centrifuged at 5000 rpm for 30 min at 4 ° C. isopropanol , 0.028 g / g and acetone , 0.008 g / g ( FIG . 13 ) . The The samples were heated at 95 ° C. for 10 minutes and best co - production yield , obtained after 48 hours of fermen- analyzed in SDS - PAGE ( FIG . 14 ) . tation , was 0.375 g products / g xylose ( 61 % of maximum 1.0 mL aliquots of each culture were placed in 2 mL theoretical yield ) . 60 headspace vials in triplicate and incubated at 37 ° C. , 225 rpm . At the end of 116 hours of incubation the vials were Example 6. Direct Production of Propylene from removed from the shaking incubator and the propylene and Glucose isopropanol concentration was analyzed in GC - MS . A con trol containing only TB medium 20 g / L glucose was done in Vectors pZs * 13 containing an IPA pathway in an operon 65 order to verify contamination in the end of incubation under plLaco promoter and pET28a containing LinD gene period. 1.0 mL of the headspace phase was injected in gas were co - transformed into BL21Star ( DE3 ) using electropo- chromatograph ( Focus GC - Thermo ) equipped with electron US 10,941,424 B2 105 106 impact mass spectrometer detector ( ISQ - Thermo ). Helium isopropanol standards and by comparison with a data base of was used as a carrier gas with a flow rate of 1.5 mL /min , the mass fragmentation. split rate used was 10 with a split flow of 15 mL /min . The The production of isopropanol in assays ( a ) , ( d ) and ( e ) volatile compounds were separated in a HP - Plot / Q column were 0.5 g / L and in ( b ) and ( c ) 3.0 g / L as expected . The ( Agilent) with initial temperature held at 90 ° C. for 1.0 min 5 production of 4 10-5 mM of propylene was observed in the followed by a first ramp at 13.3 ° C./min to 130 ° C. and a assay ( b ) positive control for propylene and a significant second one at 45 ° C./min to 200 ° C. held for 1 min . The production was observed in the assays ( d ) and ( e ) , candi retention time of propylene under these conditions was 1.51 dates with IPA + LinD co - transformed ( FIG . 15 ) . No amount min and of isopropanol was 4.3 min . The product reaction of propylene was observed in the control reaction that was identified both by comparison with propylene and contained only TB medium .

SEQUENCE LISTING SEQ ID NO : 1 Pseudomonas cichorii D - tagatose 3 - epimerase DTE NT sequence GTGAACAAAGTTGGCATGTTCTACACCTACTGGTCGACTGAGTGGATGGTCGACT TTCCGGCGACTGCGAAGCGCATTGCCGGGCTCGGCTTCGACTTAATGGAAATCTC GCTCGGCGAGTTTCACAATCTTTCCGACGCGAAGAAGCGTGAGCTAAAAGCCGTG GCTGATGATCTGGGGCTCACGGTGATGTGCTGTATCGGA?TGAAGTCTGAGTACG ACTTTGCCTCGCCGGACAAGAGCGTTCGTGATGCCGGCACGGAATATGTGAAGCG CTTGCTCGACGACTGTCACCTCCTCGGCGCGCCGGTCTTTGCTGGCCTTACGTTC TGCGCGTGGCCCCAATCTCCGCCGCTGGACATGAAGGATAAGCGCCCTTACGTCG ACCGTGCAATCGAAAGCGTTCGTCGTGTTATCAAGGTAGCTGAAGACTACGGGAT TATTTATGCACTGGAAGTGGTGAACCGATTCGAGCAGTGGCTTTGCAATGACGCC AAGGAAGCAATTGCGTTTGCCGACGCGGTTGACAGTCCGGCGTGCAAGGTCCAGC TCGACACATTCCACATGAATATCGAAGAGACTTCCTTCCGCGATGCAATCCTTGC CTGCAAGGGCAAGATGGGCCATTTCCATTTGGGCGAAGCGAACCGTCTGCCGCCG GGCGAGGGTCGCCTGCCGTGGGATGAAATATTCGGGGCGCTGAAGGAAATCGGAT ATGACGGCACCATCGTTATGGAACCGTTCATGCGCAAGGGCGGCTCGGTCAGCCG CGCGGTGGGCGTATGGCGGGATATGTCGAACGGTGCGACGGACGAAGAGATGGAC GAGCGCGCTCGCCGCTCGTTGCAGTTTGTTCGTGACAAGCTGGCCTGA SEO ID NO : 2 Pseudomonas cichorii D - tagatose 3 - epimerase DTE codon optimized NT sequence ATGAACAAAGTGGGTATGTTCTATACGTACTGGTCCACGGAATGGATGGTTGACT TTCCGGCAACCGCGAAACGTATTGCGGGCCTGGGCTTCGACCTGATGGAAATTTC TCTGGGCGAATTTCACAACCTGTCCGATGCGAAAAAGCGTGAACTGAAAGCCGTT GCCGACGATCTGGGTCTGACTGTGATGTGCTGTATCGGCCTGAAATCTGAATACG ATTTCGCGAGCCCGGATAAAAGCGTTCGCGACGCCGGTACTGAATATGTCAAACG TCTGCTGGATGACTGTCACCTGCTGGGCGCACCAGTGTTCGCGGGTCTGACCTTC TGTGCGTGGCCGCAGTCCCCACCGCTGGACATGAAGGATAAACGTCCGTACGTGG ACCGTGCCATCGAAAGCGTGCGCCGCGTAATCAAAGTCGCTGAAGATTATGGCAT TATTTACGCTCTGGAAGTTGTTAACCGTTTCGAACAGTGGCTGTGCAACGACGCG AAAGAGGCCATTGCCTTCGCTGACGCGGTGGATTCTCCGGCTTGCAAAGTTCAGC TGGACACTTTCCATATGAACATCGAGGAAACCTCCTTCCGTGACGCGATCCTGGC TTGCAAGGGTAAAATGGGCCATTTCCATCTGGGCGAAGCAAACCGCCTGCCGCCG GGCGAAGGTCGTCTGCCGTGGGACGAAATTTTTGGCGCTCTGAAGGAAATCGGCT ACGATGGCACGATTGTTATGGAGCCGTTCATGCGCAAAGGTGGCTCCGTTTCCCG TGCAGTTGGTGTTTGGCGTGATATGTCTAACGGTGCCACCGATGAAGAAATGGAC GAACGTGCACGTCGCTCCCTGCAATTCGTTCGCGATAAACTGGCGTAA SEQ ID NO : 3 Pseudomonas cichorii D - tagatose 3 - epimerase DTE AA sequence MNKVGMFYTYWSTEWMVDFPATAKRIAGLGFDLMEISLGEFHNLSDAKKRELKAV ADDLGL TVMCCIGLKSEYDFASPDKSVRDAGTEYVKRLLDDCHLLGAPVFAGLTF CAWPQSPPLDMKDKRPYVDRAIESVRRVIKVAEDYGIIYALEVVNRFEQWLCNDA KEAIAFADAVDSPACKVOLDTFHMNISETSFRDAILACKGKMGHFHLGEANRLPP GEGRLPWDEIFGALKEIGYDGTIVMEPFMRKGGSVSRAVGVWRDMSWGATDEEMD ERARRSLQEVRDKLA SEO ID NO : 4 Rhodobacter sphaeroides D - tagatose 3 - epimerase FJ851309.1 NT sequence GTGAAAAATCCTGTCGGCATCATCTCGATGCAGTTCATCCGGCCCTTCACCTCGG AGTCGCTGCATTTCCTGAAGAAGTCCCGGGCCCTGGGCTTCGATTTCATCGAGCT TCTCGTGCCCGAGCCCGAAGACGGGCTCGACGCGGCCGAGGTGCGGCGCATCTGC GAGGGCGAGGGGCTGGGCCTCGTTCTGGCCGCGCGCGTGAACCTCCAGCGCTCGA TCGCGAGCGAGGAGGCCGCGGCGCGGGCCGGCGGGCGCGACTATCTGAAATACTG CATCGAGGCCGCCGAGGCGCTCGGCGCGACCATCGTCGGCGGCCCGCTCTATGGC GAGCCGCTGGTCTTCGCCGGCCGCCCGCCCTTCCCCTGGACGGCCGAGCAGATCG CCACCCGCGCCGCCCGCACCGTCGAGGGGCTGGCCGAAGTGGCCCCGCTCGCCGC GAGCGCGGGCAAGGTCTTCGGGCTCGAGCCGCTGAACCGCTTCGAGACCGACATC GTGAACACGACCGCACAGGCCATCGAGGTGGTGGATGCGGTGGGCTCGCCCGGTC TCGGCGTCATGCTCGACACGTTCCACATGAACATGGAGGAACGCTCGATCCCCGA TGCGATCCGCGCCACAGGCGCGCGCCTCGTCCATTTTCAGGCCAACGAGAACCAC CGCGGCTTCCCCGGCACCGGCACCATGGACTGGACGGCCATCGCGCGGGCGCTGG GGCAGGCGGGCTACGCGGGTCCGGTCTCGCTCGAGCCTTTCCGGCGCGACGACGA GCGCGTGGCGCTGCCCATCGCCCACTGGCGCGCCCCGCACGAGGACGAGGACGAG AAGCTGCGCGCGGGGCTGGGTCTCATCCGCTCCGCGATCACCCTGGCGGAGGTGA CCCACTGA US 10,941,424 B2 107 108 - continued SEQUENCE LISTING SEQ ID NO : 5 Rhodobacter sphaeroides D - tagatose 3 - epimerase FJ851309.1 AA sequence MKNPVGIISMQFIRPFTSESLHFLKESRALGFDFIELLVPEPEDGLDAAEVRRIC EGEGLGLVLAARVNLQRSIASEEAAARAGGRDYLKYCIEAAEALGATIVGGPLYG EPLVFAGRPPFPWTAEQIATRAARTVEGLAEVAPLAASAGKVFGLEPLNRFETDI VNTTAQAI EVVDAVGSPGLGVMLDTFHMNMEERSIPDAIRATGARLVHFQANENH RGFPGTGTMDWTAIARALGQAGYAGPVSLEPFRRDDERVALPIAHWRAPHEDEDE KLRAGLGLIRSAITLAEVTH SEO ID NO : 6 Escherichia coli L - fuculokinase Fuck NT sequence ATGATGAAACAAGAAGTTATCCTGGTACTCGACTGTGGCGCGACCAATGTCAGGG CCATCGCGGTTAATCGGCAGGGCAAAATTGTTGCCCGCGCCTCAACGCCTAATGC CAGCGATATCGCGATGGAAAACAACACCTGGCACCAGTGGTCTTTAGACGCCATT TTGCAACGCTTTGCTGATTGCTGTCGGCAAATCAATAGTGAACTGACTGAATGCC ACATCCGCGGTATCGCCGTCACCACCTTTGGTGTGGATGGCGCTCTGGTAGATAA GCAAGGCAATCTGCTCTATCCGATTATTAGCTGGAAATGTCCGCGAACAGCAGCG GTTATGGACAATATTGAACGGTTAATCTCCGCACAGCGGTTGCAGGCTATTTCTG GCGTCGGAGCCTTTAGTTTCAATACGTTATATAAGTTGGTGTGGTTGAAAGAAAA TCATCCACAACTGCTGGAACGCGCGCACGCCTGGCTCTTTATTTCGTCGCTGATT AACCACCGTTTAACCGGCGAATTCACTACTGATATCACGATGGCCGGAACCAGCC AGATGCTGGATATCCAGCAACGCGATTTCAGTCCGCAAATTTTACAAGCCACCGG TATTCCACGCCGACTCTTCCCTCGTCTGGTGGAAGCGGGTGAACAGATTGGTACG CTACAGAACAGCGCCGCAGCAATGCTCGGCTTACCCGTTGGCATACCGGTGATTT CCGCAGGTCACGATACCCAGTTCGCCCTTTTTGGCGCTGGTGCTGAACAAAATGA ACCCGTGCTCTCTTCCGGTACATGGGAAATTTTAATGGTTCGCAGCGCCCAGGTT GATACTTCGCTGTTAAGT CAGTACGCCGGTTCCACCTGCGAACTGGATAGCCAGG CAGGGTTGTATAACCCAGGTATGCAATGGCTGGCATCCGGCGTGCTGGAATGGGT GAGAAAACTGTTCTGGACGGCTGAAACACCCTGGCAAATGTTGATTGAAGAAGCT CGTCTGATCGCGCCTGGCGCGGATGGCGTAAAAATGCAGTGTGATTTATTGTCGT GTCAGAACGCTGGCTGGCAAGGAGTGACGCTTAATACCACGCGGGGGCATTTCTA TCGCGCGGCGCTGGAAGGGTTAACTGCGCAATTACAGCGCAATCTACAGATGCTG GAAAAAATCGGGCACTTTAAGGCCTCTGAATTATTGTTAGTCGGTGGAGGAAGTC GCAACACATTGTGGAATCAGATTAAAGCCAATATGCTTGATATTCCGGTAAAAGT TCTCGACGACGCCGAAACGACCGTCGCAGGAGCTGCGCTGTTCGGTTGGTATGGC GTAGGGGAATTTAACAGCCCGGAAGAAGCCCGCGCACAGATTCATTATCAGTACC GTTATTTCTACCCGCAAACTGAACCTGAATTTATAGAGGAAGTGTGA

SEQ ID NO : 7 Escherichia coli L - fuculokinase Fuck codon optimized NT sequence ATGATGAAACAAGAAGTTATCCTGGTACTCGACTGTGGCGCGACCAATGTCAGGG CCATCGCGGTTAATCGGCAGGGCAAAATTGTTGCCCGCGCCTCAACGCCTAATGC CAGCGATATCGCGATGGAAAACAACACCTGGCACCAGTGGTCTTTAGACGCCATT TTGCAACGCTTTGCTGATTGCTGTCGGCAAATCAATAGTGAACTGACTGAATGCC ACATCCGCGGTATCGCCGTCACCACCTTTGGTGTGGATGGCGCTCTGGTAGATAA GCAAGGCAATCTGCTCTATCCGATTATTAGCTGGAAATGTCCGCGAACAGCAGCG GTTATGGACAATATTGAACGGTTAATCTCCGCACAGCGGTTGCAGGCTATTTCTG GCGTCGGAGCCTTTAGTTTCAATACGTTATATAAGTTGGTGTGGTTGAAAGAAAA TCATCCACAACTGCTGGAACGCGCGCACGCCTGGCTCTTTATTTCGTCGCTGATT AACCACCGTTTAACCGGCGAATTCACTACTGATATCACGATGGCCGGAACCAGCC AGATGCTGGATATCCAGCAACGCGATTTCAGTCCGCAAATTTTACAAGCCACCGG TATTCCACGCCGACTCTTCCCTCGTCTGGTGGAAGCGGGTGAACAGATTGGTACG CTACAGAACAGCGCCGCAGCAATGCTCGGCTTACCCGTTGGCATACCGGTGATTT CCGCAGGTCACGATACCCAGTTCGCCCTTTTTGGCGCTGGTGCTGAACAAAATGA ACCCGTGCTCTCTTCCGGTACATGGGAAATTTTAATGGTTCGCAGCGCCCAGGTT GATACTTCGCTGTTAAGTCAGTACGCCGGTTCCACCTGCGAACTGGATAGCCAGG CAGGGTTGTATAACCCAGGTATGCAATGGCTGGCATCCGGCGTGCTGGAATGGGT GAGAAAACTGTTCTGGACGGCTGAAACACCCTGGCAAATGTTGATTGAAGAAGCT CGTCTGATCGCGCCTGGCGCGGATGGCGTAAAAATGCAGTGTGATTTATTGTCGT GTCAGAACGCTGGCTGGCAAGGAGTGACGCTTAATACCACGCGGGGGCATTTCTA TCGCGCGGCGCTGGAAGGGTTAACTGCGCAATTACAGCGCAATCTACAGATGCTG GAAAAAATCGGGCACTTTAAGGCCTCTGAATTATTGTTAGTCGGTGGAGGAAGTC GCAACACATTGTGGAATCAGATTAAAGCCAATATGCTTGATATTCCGGTAAAAGT TCTCGACGACGCCGAAACGACCGTCGCAGGAGCTGCGCTGTTCGGTTGGTATGGC GTAGGGGAATTTAACAGCCCGGAAGAAGCCCGCGCACAGATTCATTATCAGTACC GTTATTTCTACCCGCAAACTGAACCTGAATTTATAGAGGAAGTGTGA

SEQ ID NO : 8 Escherichia coli L - fuculokinase fuck AA sequence MMKQEVILVLDCGATKVRAIAVNRQGKIVARASTPNASDIAMENNTWHQWSLDAI LQRFADCCRQINSELTECHIRGIAVTTFGVDGALVDKQGMLLYPIISWKCPRTAA VMDNIERLISAQRLQAISGVGAFSFNTLYKLVWLKENHPOLLERAHAWLFISSLI NHRLTGEFTTDI TMAGTSOMLDIQQRDFSPQILQATGIPRRLFPRLVEAGEQIGT LONSAAAMLGLPVGIPVISAGHDTQFALFGAGAEQNEPVLSSGTWEILMVRSAQV DTSLLSQYAGSTCELDSQAGLYNPGMQWLASGVLEWVRKLFWTAETPWQMLIEEA RLIAPGADGVKMQCDLLSCQNAGWQGVTLNTTRGHFYRAALEGLTAQLQRNLQML EKIGHFKASELLLVGGGSRNTLWNQIKANMLDIPVKVLDDAETTVAGAALFGWYG VGEFNSPEEARAQIHYQYRYFYPOTEPEFIEEV US 10,941,424 B2 109 110 - continued SEQUENCE LISTING SEQ ID NO : 9 Escherichia coli L - fuculose phosphate aldolase fucA NT sequence ATGGAACGAAATAAACTTGCTCGTCAGATTATTGACACTTGCCTGGAAATGACCC GCCTGGGA?TGAACCAGGGGACAGCGGGGAACGTCAGTGTACGTTATCAGGATGG GATGCTGATTACGCCTACAGGCATTCCATATGAAAAACTGACGGAGTCGCATATT GTCTTTATTGATGGCAACGGTAAACATGAGGAAGGAAAGCTCCCCTCAAGCGAAT GGCGTTTCCATATGGCAGCCTATCAAAGCAGACCGGATGCCAACGCGGTTGTTCA CAATCATGCCGTTCATTGCACGGCAGTTTCCATTCTTAACCGATCGATCCCCGCT ATTCACTACATGATTGCGGCGGCTGGCGGTAATTCTATTCCTTGCGCGCCTTATG CGACCTTTGGAACACGCGAACTTTCTGAACATGTTGCGCTGGCTCTCAAAAATCG TAAGGCAACTTTGTTACAACATCATGGGCTTATCGCTTGTGAGGTGAATCTGGAA AAAGCGTTATGGCTGGCGCATGAAGTTGAAGTGCTGGCGCAACTTTACCTGACGA CCCTGGCGATTACGGACCCGGTGCCAGTGCTGAGCGATGAAGAGATTGCCGTAGT GCTGGAGAAATTCAAAACCTATGGGTTACGAATTGAAGAGTAA SEQ ID NO : 10 Escherichia coli L - fuculose phosphate aldolase fucA codon optimized NT sequence ATGGAACGAAATAAACTTGCTCGTCAGATTATTGACACTTGCCTGGAAATGACCC GCCTGGGA?TGAACCAGGGGACAGCGGGGAACGTCAGTGTACGTTATCAGGATGG GATGCTGATTACGCCTACAGGCATTCCATATGAAAAACTGACGGAGTCGCATATT GTCTTTATTGATGGCAACGGTAAACATGAGGAAGGAAAGCTCCCCTCAAGCGAAT GGCGTTTCCATATGGCAGCCTATCAAAGCAGACCGGATGCCAACGCGGTTGTTCA CAATCATGCCGTTCATTGCACGGCAGTTTCCATTCTTAACCGATCGATCCCCGCT ATTCACTACATGATTGCGGCGGCTGGCGGTAATTCTATTCCTTGCGCGCCTTATG CGACCTTTGGAACACGCGAACTTTCTGAACATGTTGCGCTGGCTCTCAAAAATCG TAAGGCAACTTTGTTACAACATCATGGGCTTATCGCTTGTGAGGTGAATCTGGAA AAAGCGTTATGGCTGGCGCATGAAGTTGAAGTGCTGGCGCAACTTTACCTGACGA CCCTGGCGATTACGGACCCGGTGCCAGTGCTGAGCGATGAAGAGATTGCCGTAGT GCTGGAGAAATTCAAAACCTATGGGTTACGAATTGAAGAGTAA SEQ ID NO : 11 Escherichia coli L - fuculose phosphate aldolase fucA AA sequence MERNKIARQIIDTCLEMTRLGLNQGTAGNVSVRYQDGMLITPTGIPYEKLTESHI VFIDGNGKKEEGKLPSSEWRFHMAAYQSRPDANAVVHNHAVHCTAVSILNRSIPA IHYMIAAAGGNSIPCAPYATFGTRELSEHVALALKNRKATLLQHHGLIACEVNLE KALWLAHEVEVLAQLYLTTLAITDPVPVLSDEEIAVVLEKFKTYGLRIEE SEO ID NO : 12 Escherichia coli glycerol dehydrogenase gldA NT sequence ATGGACCGCATTATTCAATCACCGGGTAAATACATCCAGGGCGCTGATGTGATTA ATCGTCTGGGCGAATACCTGAAGCCGCTGGCAGAACGCTGGTTAGTGGTGGGTGA CAAATTTGTTTTAGGTTTTGCTCAATCCACTGTCGAGAAAAGCTTTAAAGATGCT GGACTGGTAGTAGAAATTGCGCCGTTTGGCGGTGAATGTTCGCAAAATGAGATCG ACCGTCTGCGTGGCATCGCGGAGACTGCGCAGTGTGGCGCAATTCTCGGTATCGG TGGCGGAAAAACCCTCGATACTGCCAAAGCACTGGCACATTTCATGGGTGTTCCG GTAGCGATCGCACCGACTATCGCCTCTACCGATGCACCGTGCAGCGCATTGTCTG TTATCTACACCGATGAGGGTGAGTTTGACCGCTATCTGCTGTTGCCAAATAACCC GAATATGGTCATTGTCGACACCAAAATCGTCGCTGGCGCACCTGCACGTCTGTTA GCGGCGGGTATCGGCGATGCGCTGGCAACCTGGTTTGAAGCGCGTGCCTGCTCTC GTAGCGGCGCGACCACCATGGCGGGCGGCAAGTGCACCCAGGCTGCGCTGGCACT GGCTGAACTGTGCTACAACACCCTGCTGGAAGAAGGCGAAAAAGCGATGCTTGCT GCCGAACAGCATGTAGTGACTCCGGCGCTGGAGCGCGTGATTGAAGCGAACACCT ATTTGAGCGGTGTTGGTTTTGAAAGTGGTGGTCTGGCTGCGGCGCACGCAGTGCA TAACGGCCTGACCGCTATCCCGGACGCGCATCACTATTATCACGGTGAAAAAGTG GCATTCGGTACGCTGACGCAGCTGGTTCTGGAAAATGCGCCGGTGGAGGAAATCG AAACCGTAGCTGCCCTTAGCCATGCGGTAGGTTTGCCAATAACTCTCGCTCAACT GGATATTAAAGAAGATGTCCCGGCGAAAATGCGAATTGTGGCAGAAGCGGCATGT GCAGAAGGTGAAACCATTCACAACATGCCTGGCGGCGCGACGCCAGATCAGGTTT ACGCCGCTCTGCTGGTAGCCGACCAGTACGGTCAGCGTTTCCTGCAAGAGTGGGA ATAA SEQ ID NO : 13 Escherichia coli glycerol dehydrogenase gldA AA sequence MDRIIQSPGKYIQGADVINRLGEYLKPLAERWLVVGDKFVLGFAQSTVEKSFKDA GLVVEIAPFGGECSQNEIDRLRGIAETAQCGAILGIGGGKTLDTAKALAHFMGVP VAIAPTIASTDAPCSALSVIYTDEGEFDRYLLLPNNPNMVIVDTKIVAGAPARLL AAGIGDALATWFEARACSRSGATTMAGGKCTQAALALAELCYNTLLEEGEKAMLA AEQHVVTPALERVIEANTYLSGVGFESGGLAAAHAVHNGL TAIPDAHHYYHGEKV AFGTLTOLVLENAPVEEIETVAALSHAVGLPITLAQLDIKEDVPAKMRIVAEAAC AEGETIHNMPGGATPDOVYAALLVADQYGQRFLQEWE SEQ ID NO : 14 Saccharomyces cerevisiae methylglyoxal reductase GRE2 NT sequence ATGTCAGTTTTCGTTTCAGGTGCTAACGGGTTCATTGCCCAACACATTGTCGATC TCCTGTTGAAGGAAGACTATAAGGTCATCGGTTCTGCCAGAAGTCAAGAAAAGGC CGAGAATTTAACGGAGGCCTTTGGTAACAACCCAAAATTCTCCATGGAAGTTGTC CCAGACATATCTAAGCTGGACGCATTTGACCATGTTTTCCAAAAGCACGGCAAGG US 10,941,424 B2 111 112 - continued SEQUENCE LISTING ATATCAAGATAGTTCTACATACGGCCTCTCCATTCTGCTTTGATATCACTGACAG TGAACGCGATTTATTAATTCCTGCTGTGAACGGTGTTAAGGGAATTCTCCACTCA ATTAAAAAATACGCCGCTGATTCTGTAGAACGTGTAGTTCTCACCTCTTCTTATG CAGCTGTGTTCGATATGGCAAAAGAAAACGATAAGTCTTTAACATTTAACGAAGA ATCCTGGAACCCAGCTACCTGGGAGAGTTGCCAAAGTGACCCAGTTAACGCCTAC TGTGGTTCTAAGAAGTTTGCTGAAAAAGCAGCTTGGGAATTTCTAGAGGAGAATA GAGACTCTGTAAAATTCGAATTAACTGCCGTTAACCCAGTTTACGTTTTTGGTCC GCAAATGTTTGACAAAGATGTGAAAAAACACTTGAACACATCTTGCGAACTCGTC AACAGCTTGATGCATTTATCACCAGAGGACAAGATACCGGAACTATTTGGTGGAT ACATTGATGTTCGTGATGTTGCAAAGGCTCATTTAGTTGCCTTCCAAAAGAGGGA AACAATTGGTCAAAGACTAATCGTATCGGAGGCCAGATTTACTATGCAGGATGTT CTCGATATCCTTAACGAAGACTTCCCTGTTCTAAAAGGCAATATTCCAGTGGGGA AACCAGGTTCTGGTGCTACCCATAACACCCTTGGTGCTACTCTTGATAATAAAAA GAGTAAGAAATTGTTAGGTTTCAAGTTCAGGAACTTGAAAGAGACCATTGACGAC ACTGCCTCCCAAATTTTAAAATTTGAGGGCAGAATATAA SEQ ID NO : 15 Saccharomyces cerevisiae methylglyoxal reductase GRE2 AA sequence MSVPISGANGFTAQHTVDLLLKEDYKVIGSARSCENAENLTEAFGNNPKFSMEW PDISRIDAFDHVFQKHGKDIKIVIHTASPECEDITDSERDLLIPAVNGVKGILHS IKKYAADSVERVULTSSYAAVFDMAKENDKSLTENEESWNPATWESCQSDPVNAY CGSKKFAEKAAWEFLEENRDSVKFELTAVNPVYVFGPQMFDKDVKKHLNTSCELV NSLMHLSPEDKTPELFGGYTDVRIWAKAHLVAFQKRETIGORLIVSEARFTMQDV LDILNEDFPVLKGNIPVGKPGSGATHNTLGATLDNKKSKKLLGFKFRNLKETIDD TASQILKFEGRI SEQ ID NO : 16 Saccharomyces cerevisiae aldose reductase GRE3 NT sequence ATGTCTTCACTGGTTACTCTTAATAACGGTCTGAAAATGCCCCTAGTCGGCTTAG GGTGCTGGAAAATTGACAAAAAAGTCTGTGCGAATCAAATTTATGAAGCTATCAA ATTAGGCTACCGTTTATTCGATGGTGCTTGCGACTACGGCAACGAAAAGGAAGTT GGTGAAGGTATCAGGAAAGCCATCTCCGAAGGTCTTGTTTCTAGAAAGGATATAT TTGTTGTTTCAAAGTTATGGAACAATTTTCACCATCCTGATCATGTAAAATTAGC TTTAAAGAAGACCTTAAGCGATATGGGACTTGATTATTTAGACCTGTATTATATT CACTTCCCAATCGCCTTCAAATATGTTCCATTTGAAGAGAAATACCCTCCAGGAT TCTATACGGGCGCAGATGACGAGAAGAAAGGTCACATCACCGAAGCACATGTACC AATCATAGATACGTACCGGGCTCTGGAAGAATGTGTTGATGAAGGCTTGATTAAG TCTATTGGTGTTTCCAACTTTCAGGGAAGCTTGATTCAAGATTTATTACGTGGTT GTAGAATCAAGCCCGTGGCTTTGCAAATTGAACACCATCCTTATTTGACTCAAGA ACACCTAGTTGAGTTTTGTAAATTAGACGATATCCAAGTAGTTGCTTACTCCTCC TTCGGTCCTCAATCATTCATTGAGATGGACTTACAGTTGGCAAAAACCACGCCAA CTCTGTTCGAGAATGATGTAATCAAGAAGGTCTCACAAAACCATCCAGGCAGTAC CACTTCCCAAGTATTGCTTAGATGGGCAACTCAGAGAGGCATTGCCGTCATTCCA AAATCTTCCAAGAAGGAAAGGTTACTTGGCAACCTAGAAATCGAAAAAAAGTTCA CTTTAACGGAGCAAGAATTGAAGGATATTTCTGCACTAAATGCCAACATCAGATT TAATGATCCATGGACCTGGTTGGATGGTAAATTCCCCACTTTTGCCTGA SEQ ID NO : 17 Saccharomyces cerevisiae aldose reductase GRE3 AA sequence MSSLVTLNNGLKMPLVGLGCWKIDKKVCANQIYEAI KLGYRLFDGACDYGNEKEV GEGIRKAISEGLVSRKDIFVVSKLWNMFHHPDHVKLALKKTLSDMGLDYLDLYYI HFPIAFKYVPFEEKYPPGFYTGADDEKKGHITEAHVPIIDTYPALEECVDEGLIK SIGVSNFQGSLIQDLLRGCRIKPVALQIEHHPYLTQEHLVEFCKLHDIQVVAYSS FGPOSFIEMDLQLAKTTPTLFENDVIKKVSQNKPGSTTSQVLLRWATORGIAVIP KSSKKERLLGNLEIEKKFTLTECELKDISALNANIRFNDPWTWLDGKFPTFA SEQ ID NO : 18 Escherichia coli alcohol dehydrogenase yqhD * NT sequence ATGAACAACTTTAATCTGCACACCCCAACCCGCATTCTGTTTGGTAAAGGCGCAA TCGCTGGTTTACGCGAACAAATTCCTCACGATGCTCGCGTATTGATTACCTACGG CGGCGGCAGCGTGAAAAAAACCGGCGTTCTCGATCAAGTTCTGGATGCCCTGAAA GGCATGGACGTGCTGGAATTTGGCGGTATTGAGCCAAACCCGGCTTATGAAACGC TGATGAACGCCGTGAAACTGGTTCGCGAACAGAAAGTGACTTTCCTGCTGGCGGT TGGCGGCGGTTCTGTACTGGACGGCACCAAATTTATCGCCGCAGCGGCTAACTAT CCGGAAAATATCGATCCGTGGCACATTCTGCAAACGGGCGGTAAAGAGATTAAAA GCGCCATCCCGATGGGCTGTGTGCTGACGCTGCCAGCAACCGGTTCAGAATCCAA CGCAGAAGCGGTGATCTCCCGTAAAACCACAGGCGACAAGCAGGCGTTCCATTCT GCCCATGTTCAGCCGGTATTTGCCGTGCTCGATCCGGTTTATACCTACACCCTGC CGCCGCGTCAGGTGGCTAACGGCGTAGTGGACGCCTTTGTACACACCGTGGAACA GTATGTTACCAAACCGGTTGATGCCAAAATTCAGGACCGTTTCGCAGAAGGCATT TTGCTGACGCTAATCGAAGATGGTCCGAAAGCCCTGAAAGAGCCAGAAAACTACG ATGTGCGCGCCAACGTCATGTGGGCGGCGACTCAGGCGCTGAACGGTTTGATTGG CGCTGGCGTACCGCAGGACTGGGCAACGCATATGCTGGGCCACGAACTGACTGCG ATGCACGGTCTGGATCACGCGCAAACACTGGCTATCGTCCTGCCTGCACTGTGGA ATGAAAAACGCGATACCAAGCGCGCTAAGCTGCTGCAATATGCTGAACGCGTCTG GAACATCACTGAAGGTTCCGATGATGAGCGTATTGACGCCGCGATTGCCGCAACC CGCAATTTCTTTGAGCAATTAGGCGTGCCGACCCACCTCTCCGACTACGGTCTGG US 10,941,424 B2 113 114 - continued SEQUENCE LISTING ACGGCAGCTCCATCCCGGCTTTGCTGAAAAAACTGGAAGAGCACGGCATGACCCA ACTGGGCGAAAATCATGACATTACGTTGGATGTCAGCCGCCGTATATACGAAGCC GCCCGCTAA SEQ ID NO : 19 Escherichia coli alcohol dehydrogenase yqhD * codon optimized NT sequence ATGAACAATTTTAATTTGCATACTCCAACTAGAATATTATTTGGAAAAGGTGCAA TTGCAGGTTTAAGGGAACAAATACCACATGATGCAAGGGTATTAATCACATACGG TGGTGGTTCTGTCAAGAAAACTGGTGTATTGGATCAAGTATTGGATGCTTTAAAG GGTATGGATGTCTTGGAATTTGGAGGAATCGAACCAAACCCTGCTTACGAGACTT TAATGAATGCTGTCAAATTGGTCAGAGAACAAAAGGTAACATTCTTATTGGCTGT TGGAGGTGGATCAGTATTAGATGGTACAAAGTTCATTGCTGCTGCAGCAAATTAT CCAGAAAACATTGATCCATGGCATATATTGCAAACTGGTGGTAAGGAAATAAAGT CAGCTATCCCAATGGGATGTGTTTTGACATTGCCTGCAACAGGATCAGAATCAAA CGCTGAAGCAGTCATCTCAAGAAAGACTACAGGTGACAAACAGGCATTCCATTCT GCCCATGTCCAACCTGTATTTGCTGTTTTAGACCCTGTATACACTTACACATTAC CACCAAGGCAAGTCGCAAATGGAGTTGTCGATGCCTTTGTTCACACTGTAGAACA GTACGTCACCAAACCAGTCGATGCAAAGATCCAGGACAGGTTTGCAGAAGGTATT TTATTGACATTAATCGAAGATGGACCAAAAGCATTGAAAGAGCCAGAGAACTATG ACGTTAGGGCAAATGTTATGTGGGCTGCTACCCAGGCATTGAACGGTTTAATTGG TGCAGGAGTTCCACAAGATTGGGCTACACACATGTTGGGTCACGAGTTGACCGCC ATGCACGGTTTGGACCATGCACAGACTTTAGCCATTGTTTTGCCTGCCTTATGGA ACGAGAAAAGAGATACTAAGAGGGCTAAGTTATTACAATACGCTGAAAGGGTTTG GAATATCACCGAGGGATCTGATGATGAAAGGATTGATGCCGCTATTGCAGCCACT AGAAACTTCTTTGAACAATTAGGTGTTCCAACTCACTTGTCTGACTATGGTTTAG ATGGATCATCTATTCCAGCTTTGTTGAAGAAATTGGAAGAGCACGGTATGACCCA GTTGGGTGAGAATCATGATATAACCTTAGATGTATCTAGGAGAATCTACGAGGCT GCTAGATAATGA SEQ ID NO : 20 Escherichia coli alcohol dehydrogenase yqhD * AA sequence MNNFNLHTPTRILFGKGAIAGLREQIPHDARVLITYGGGSVKKTGVLDQVLDALK GMDVLEFGGIEPNPAYETLMNAVKLVREQKVTFLLAVGGGSVLDGTKFIAAAANY PENIDPWHILQTGGKEIKSAIPMGCVLTLPATGSESNAEAVISRKTTGDKQAFHS AHVQPVFAVLDPVYTYTLPPROVANGVVDAFVHTVEQYVTKPVDAKIQDRFAEGI LLTLI EDGPKALKEPENYDVRANVMWAATQALNGLIGAGVPQDWATHMLGHELTA MHGLDHAQTLAIVLPALWNEKRDTKRAKLLQYAERVWNITEGSDDERIDAAIAAT RNFFEQLGVPTHLSDYGLDGSSIPALLKKLEEHGMTQLGENHDITLDVSRRIYEA AR SEQ ID NO : 21 Escherichia coli alcohol dehydrogenase yqhD NT sequence ATGAACAACTTTAATCTGCACACCCCAACCCGCATTCTGTTTGGTAAAGGCGCAA TCGCTGGTTTACGCGAACAAATTCCTCACGATGCTCGCGTATTGATTACCTACGG CGGCGGCAGCGTGAAAAAAACCGGCGTTCTCGATCAAGTTCTGGATGCCCTGAAA GGCATGGACGTGCTGGAATTTGGCGGTATTGAGCCAAACCCGGCTTATGAAACGC TGATGAACGCCGTGAAACTGGTTCGCGAACAGAAAGTGACTTTCCTGCTGGCGGT TGGCGGCGGTTCTGTACTGGACGGCACCAAATTTATCGCCGCAGCGGCTAACTAT CCGGAAAATATCGATCCGTGGCACATTCTGCAAACGGGCGGTAAAGAGATTAAAA GCGCCATCCCGATGGGCTGTGTGCTGACGCTGCCAGCAACCGGTTCAGAATCCAA CGCAGGCGCGGTGATCTCCCGTAAAACCACAGGCGACAAGCAGGCGTTCCATTCT GCCCATGTTCAGCCGGTATTTGCCGTGCTCGATCCGGTTTATACCTACACCCTGC CGCCGCGTCAGGTGGCTAACGGCGTAGTGGACGCCTTTGTACACACCGTGGAACA GTATGTTACCAAACCGGTTGATGCCAAAATTCAGGACCGTTTCGCAGAAGGCATT TTGCTGACGCTAATCGAAGATGGTCCGAAAGCCCTGAAAGAGCCAGAAAACTACG ATGTGCGCGCCAACGTCATGTGGGCGGCGACTCAGGCGCTGAACGGTTTGATTGG CGCTGGCGTACCGCAGGACTGGGCAACGCATATGCTGGGCCACGAACTGACTGCG ATGCACGGTCTGGATCACGCGCAAACACTGGCTATCGTCCTGCCTGCACTGTGGA ATGAAAAACGCGATACCAAGCGCGCTAAGCTGCTGCAATATGCTGAACGCGTCTG GAACATCACTGAAGGTTCCGATGATGAGCGTATTGACGCCGCGATTGCCGCAACC CGCAATTTCTTTGAGCAATTAGGCGTGCCGACCCACCTCTCCGACTACGGTCTGG ACGGCAGCTCCATCCCGGCTTTGCTGAAAAAACTGGAAGAGCACGGCATGACCCA ACTGGGCGAAAATCATGACATTACGTTGGATGTCAGCCGCCGTATATACGAAGCC GCCCGCTAA SEQ ID NO : 22 Escherichia coli alcohol dehydrogenase yqhD codon optimized NT sequence ATGAACAACTTTAATCTGCACACCCCAACCCGCATTCTGTTTGGTAAAGGCGCAA TCGCTGGTTTACGCGAACAAATTCCTCACGATGCTCGCGTATTGATTACCTACGG CGGCGGCAGCGTGAAAAAAACCGGCGTTCTCGATCAAGTTCTGGATGCCCTGAAA GGCATGGACGTGCTGGAATTTGGCGGTATTGAGCCAAACCCGGCTTATGAAACGC TGATGAACGCCGTGAAACTGGTTCGCGAACAGAAAGTGACTTTCCTGCTGGCGGT TGGCGGCGGTTCTGTACTGGACGGCACCAAATTTATCGCCGCAGCGGCTAACTAT CCGGAAAATATCGATCCGTGGCACATTCTGCAAACGGGCGGTAAAGAGATTAAAA GCGCCATCCCGATGGGCTGTGTGCTGACGCTGCCAGCAACCGGTTCAGAATCCAA CGCAGGCGCGGTGATCTCCCGTAAAACCACAGGCGACAAGCAGGCGTTCCATTCT GCCCATGTTCAGCCGGTATTTGCCGTGCTCGATCCGGTTTATACCTACACCCTGC CGCCGCGTCAGGTGGCTAACGGCGTAGTGGACGCCTTTGTACACACCGTGGAACA US 10,941,424 B2 115 116 - continued SEQUENCE LISTING GTATGTTACCAAACCGGTTGATGCCAAAATTCAGGACCGTTTCGCAGAAGGCATT TTGCTGACGCTAATCGAAGATGGTCCGAAAGCCCTGAAAGAGCCAGAAAACTACG ATGTGCGCGCCAACGTCATGTGGGCGGCGACTCAGGCGCTGAACGGTTTGATTGG CGCTGGCGTACCGCAGGACTGGGCAACGCATATGCTGGGCCACGAACTGACTGCG ATGCACGGTCTGGATCACGCGCAAACACTGGCTATCGTCCTGCCTGCACTGTGGA ATGAAAAACGCGATACCAAGCGCGCTAAGCTGCTGCAATATGCTGAACGCGTCTG GAACATCACTGAAGGTTCCGATGATGAGCGTATTGACGCCGCGATTGCCGCAACC CGCAATTTCTTTGAGCAATTAGGCGTGCCGACCCACCTCTCCGACTACGGTCTGG ACGGCAGCTCCATCCCGGCTTTGCTGAAAAAACTGGAAGAGCACGGCATGACCCA ACTGGGCGAAAATCATGACATTACGTTGGATGTCAGCCGCCGTATATACGAAGCC GCCCGCTAA SEQ ID NO : 23 Escherichia coli alcohol dehydrogenase yqhD AA sequence MNNFNLHTPTRILFGKGAIAGLREQIPHDARVLITYGGGSVKKTGVLDOVLDALK GMDVLEFGGIEPNPAYETLMNAVKLVREQKVTFLLAVGGGSVLDGTKFIAAAANY PENIDPWHILQTGGKEIKSAI PMGCVLTLPATGSESNAGAVISRKTTGDKQAFHS AHVQPVFAVLDPVYTYTLPPROVANGVVDAFVHTVEQYVTKPVDAKIQDRFAEGI LLTLIEDGPKALKEPENYDVRANVMWAATQALNGLIGAGVPQDWATHMLGHELTA MHGLDHAQTLAIVLPALWNEKRDTKRAKLLQYAERVWNITEGSDDERIDAAIAAT RNFFEQLGVPTHLSDYGLDGSSIPALLKKLEEHGMTQLGENHDITLDVSRRIYEA AR SEQ ID NO : 24 Escherichia coli methylglyoxal reductase ydjG NT sequence ATGAAAAAGATACCTTTAGGCACAACGGATATTACGCTTTCGCGAATGGGGTTGG GGACATGGGCCATTGGCGGCGGTCCTGCATGGAATGGCGATCTCGATCGGCAAAT ATGTATTGATACGATTCTTGAAGCCCATCGTTGTGGCATTAATCTGATTGATACT GCGCCAGGATATAACTTTGGCAATAGTGAAGTTATCGTCGGTCAGGCGTTAAAAA AACTGCCCCGTGAACAGGTTGTAGTAGAAACCAAATGCGGCATTGTCTGGGAACG AAAAGGAAGTTTATTCAACAAAGTTGGCGATCGGCAGTTGTATAAAAACCTTTCC CCGGAATCTATCCGCGAAGAGGTAGCAGCGAGCTTGCAACGTCTGGGTATTGATT ACATCGATATCTACATGACGCACTGGCAGTCGGTGCCGCCATTTTTTACGCCGAT CGCTGAAACTGTCGCAGTGCTTAATGAGTTAAAGTCTGAAGGGAAAATTCGCGCT ATAGGCGCTGCTAACGTCGATGCTGACCATATCCGCGAGTATCTGCAATATGGTG AACTGGATATTATTCAGGCGAAATACAGTATCCTCGACCGGGCAATGGAAAACGA ACTGCTGCCACTATGTCGTGATAATGGCATTGTGGTTCAGGTTTATTCCCCGCTA GAGCAGGGATTGTTGACCGGCACCATCACTCGTGATTACGTTCCGGGCGGCGCTC GGGCAAATAAAGTCTGGTTCCAGCGTGAAAACATGCTGAAAGTGATTGATATGCT TGAACAGTGGCAGCCACTTTGTGCTCGTTATCAGTGCACAATTCCCACTCTGGCA CTGGCGTGGATATTAAAACAGAGTGATTTAATCTCCATTCTTAGTGGGGCTACTG CACCGGAACAGGTACGCGAAAATGTCGCGGCACTGAATATCAACTTATCGGATGC AGACGCAACATTGATGAGGGAAATGGCAGAGGCCCTGGAGCGTTAA SEQ ID NO : 25 Escherichia coli methylglyoxal reductase ydjG AA sequence MKKIPLGTTDITLSRMGLGTWAIGGGPAWNGDLDRQICIDTILEAHRCGINLIDT APGYNFGNSEVIVGQALKKLPREQVVVETKCGIVWERKGSLFNKVGDROLYKNLS PESIREEVAASLQRLGIDYIDIYMTHWQSVPPFFTPIAETVAVLNELKSEGKIRA IGAANVDADHIREYLQYGELDIIQAKYSILDRAMENELLPLCRDNGIVVQVYSPL EQGLLTGTITRDYVPGGARANKVWFQRENMLKVIDMLEQWQPLCARYQCTIPTLA LAWILKQSDLISILSGATAPEQVRENVAALNINLSDADATLMREMAEALER SEQ ID NO : 26 Escherichia coli lactaldehyde reductase fuco NT sequence ATGGCTAACAGAATGATTCTGAACGAAACGGCATGGTTTGGTCGGGGTGCTGTTG GGGCTTTAACCGATGAGGTGAAACGCCGTGGTTATCAGAAGGCGCTGATCGTCAC CGATAAAACGCTGGTGCAATGCGGCGTGGTGGCGAAAGTGACCGATAAGATGGAT GCTGCAGGGCTGGCATGGGCGATTTACGACGGCGTAGTGCCCAACCCAACAATTA CTGTCGTCAAAGAAGGGCTCGGTGTATTCCAGAATAGCGGCGCGGATTACCTGAT CGCTATTGGTGGTGGTTCTCCACAGGATACTTGTAAAGCGATTGGCATTATCAGC AACAACCCGGAGTTTGCCGATGTGCGTAGCCTGGAAGGGCTTTCCCCGACCAATA AACCCAGTGTACCGATTCTGGCAATTCCTACCACAGCAGGTACTGCGGCAGAAGT GACCATTAACTACGTGATCACTGACGAAGAGAAACGGCGCAAGTTTGTTTGCGTT GATCCGCATGATATCCCGCAGGTGGCGTTTATTGACGCTGACATGATGGATGGTA TGCCTCCAGCGCTGAAAGCTGCGACGGGTGTCGATGCGCTCACTCATGCTATTGA GGGGTATATTACCCGTGGCGCGTGGGCGCTAACCGATGCACTGCACATTAAAGCG ATTGAAATCATTGCTGGGGCGCTGCGAGGATCGGTTGCTGGTGATAAGGATGCCG GAGAAGAAATGGCGCTCGGGCAGTATGTTGCGGGTATGGGCTTCTCGAATGTTGG GTTAGGGTTGGTGCATGGTATGGCGCATCCACTGGGCGCGTTTTATAACACTCCA CACGGTGTTGCGAACGCCATCCTGTTACCGCATGTCATGCGTTATAACGCTGACT TTACCGGTGAGAAGTACCGCGATATCGCGCGCGTTATGGGCGTGAAAGTGGAAGG TATGAGCCTGGAAGAGGCGCGTAATGCCGCTGTTGAAGCGGTGTTTGCTCTCAAC CGTGATGTCGGTATTCCGCCACATTTGCGTGATGTTGGTGTACGCAAGGAAGACA TTCCGGCACTGGCGCAGGCGGCACTGGATGATGTTTGTACCGGTGGCAACCCGCG TGAAGCAACGCTTGAGGATATTGTAGAGCTTTACCATACCGCCTGGTAA US 10,941,424 B2 117 118 - continued SEQUENCE LISTING SEQ ID NO : 27 Escherichia coli lactaldehyde reductase fuco codon optimized NT sequence ATGGCTAACAGAATGATTCTGAACGAAACGGCATGGTTTGGTCGGGGTGCTGTTG GGGCTTTAACCGATGAGGTGAAACGCCGTGGTTATCAGAAGGCGCTGATCGTCAC CGATAAAACGCTGGTGCAATGCGGCGTGGTGGCGAAAGTGACCGATAAGATGGAT GCTGCAGGGCTGGCATGGGCGATTTACGACGGCGTAGTGCCCAACCCAACAATTA CTGTCGTCAAAGAAGGGCTCGGTGTATTCCAGAATAGCGGCGCGGATTACCTGAT CGCTATTGGTGGTGGTTCTCCACAGGATACTTGTAAAGCGATTGGCATTATCAGC AACAACCCGGAGTTTGCCGATGTGCGTAGCCTGGAAGGGCTTTCCCCGACCAATA AACCCAGTGTACCGATTCTGGCAATTCCTACCACAGCAGGTACTGCGGCAGAAGT GACCATTAACTACGTGATCACTGACGAAGAGAAACGGCGCAAGTTTGTTTGCGTT GATCCGCATGATATCCCGCAGGTGGCGTTTATTGACGCTGACATGATGGATGGTA TGCCTCCAGCGCTGAAAGCTGCGACGGGTGTCGATGCGCTCACTCATGCTATTGA GGGGTATATTACCCGTGGCGCGTGGGCGCTAACCGATGCACTGCACATTAAAGCG ATTGAAATCATTGCTGGGGCGCTGCGAGGATCGGTTGCTGGTGATAAGGATGCCG GAGAAGAAATGGCGCTCGGGCAGTATGTTGCGGGTATGGGCTTCTCGAATGTTGG GTTAGGGTTGGTGCATGGTATGGCGCATCCACTGGGCGCGTTTTATAACACTCCA CACGGTGTTGCGAACGCCATCCTGTTACCGCATGTCATGCGTTATAACGCTGACT TTACCGGTGAGAAGTACCGCGATATCGCGCGCGTTATGGGCGTGAAAGTGGAAGG TATGAGCCTGGAAGAGGCGCGTAATGCCGCTGTTGAAGCGGTGTTTGCTCTCAAC CGTGATGTCGGTATTCCGCCACATTTGCGTGATGTTGGTGTACGCAAGGAAGACA TTCCGGCACTGGCGCAGGCGGCACTGGATGATGTTTGTACCGGTGGCAACCCGCG TGAAGCAACGCTTGAGGATATTGTAGAGCTTTACCATACCGCCTGGTAA SEQ ID NO : 28 Escherichia coli lactaldehyde reductase fuco AA sequence MANRMILNETAWFGRGAVGALTDEVKRRGYQKALIVTDKTLVQCGVVAKVTDKMD AAGLAWAI YDGVVPNPTITVVKEGLGVFQNSGADYLIAIGGGSPODTCKAIGIIS NNPEFADVRSLEGLSPTNKPSVPILAIPTTAGTAAEVTINYVITDEEKRRKFVCV DPHDIPQVAFIDADMMDGMPPALKAATGVDALTHAI EGYI TRGAWALTDALHIKA IEIIAGALRGSVAGDKDAGEEMALGQYVAGMGFSNVGLGLVHGMAHPLGAFYNTP HGVANAILLPHVMRYNADFTGEKYRDIARVMGVKVEGMSLEEARNAAVEAVFALN RDVGIPPHLRDVGVRKEDIPALAQAALDDVCTGGNPREATLEDIVELYHTAW SEQ ID NO : 29 Escherichia coli methylglyoxal reductase yafB ( dkgB ) [ multifunctional ] NT sequence ATGGCTATCCCTGCATTTGGTTTAGGTACTTTCCGTCTGAAAGACGACGTTGTTA TTTCATCTGTGATAACGGCGCTTGAACTTGGTTATCGCGCAATTGATACCGCACA AATCTATGATAACGAAGCCGCAGTAGGTCAGGCGATTGCAGAAAGTGGCGTGCCA CGTCATGAACTCTACATCACCACTAAAATCTGGATTGAAAATCTCAGCAAAGACA AATTGATCCCAAGTCTGAAAGAGAGCCTGCAAAAATTGCGTACCGATTATGTTGA TCTGACGCTAATCCACTGGCCGTCACCAAACGATGAAGTCTCTGTTGAAGAGTTT ATGCAGGCGCTGCTGGAAGCCAAAAAACAAGGGCTGACGCGTGAGATCGGTATTT CCAACTTCACGATCCCGTTGATGGAAAAAGCGATTGCTGCTGTTGGTGCTGAAAA CATCGCTACTAACCAGATTGAACTCTCTCCTTATCTGCAAAACCGTAAAGTGGTT GCCTGGGCTAAACAGCACGGCATCCATATTACTTCCTATATGACGCTGGCGTATG GTAAGGCCCTGAAAGATGAGGTTATTGCTCGTATCGCAGCTAAACACAATGCGAC TCCGGCACAAGTGATTCTGGCGTGGGCTATGGGGGAAGGTTACTCAGTAATTCCT TCTTCTACTAAACGTAAAAACCTGGAAAGTAATCTTAAGGCACAAAATTTACAGC TTGATGCCGAAGATAAAAAAGCGATCGCCGCACTGGATTGCAACGACCGCCTGGT TAGCCCGGAAGGTCTGGCTCCTGAATGGGATTAA SEQ ID NO : 30 Escherichia coli methylglyoxal reductase yafB ( dkgB ) [ multifunctional ] AA sequence MAIPAFGLGTFRLKDDVVISSVITALELGYRAIDTAQIYDNEAAVGQAIAESGVP RHELYITTKIWIENLSKDKLIPSLKESLQKLRTDYVDLTLIHWPSPNDEVSVEEF MQALLEAKKQGL TREIGISNFTIPLMEKAIAAVGAENIATNQIELSPYLONRKVV AWAKQHGIHITSYMTLAYGKALKDEVIARIAAKHNATPAQVILAWAMGEGYSVIP SSTKRKNLESNLKAQNLQLDAEDKKAIAALDCNDRLVSPEGLAPEWD SEO ID NO : 31 Escherichia coli 2,5 - diketo - D - gluconic acid reductase A yqhE ( dkgA ) NT sequence ATGGCTAATCCAACCGTTATTAAGCTACAGGATGGCAATGTCATGCCCCAGCTGG GACTGGGCGTCTGGCAAGCAAGTAATGAGGAAGTAATCACCGCCATTCAAAAAGC GTTAGAAGTGGGTTATCGCTCGATTGATACCGCCGCGGCCTACAAGAACGAAGAA GGTGTCGGCAAAGCCCTGAAAAATGCCTCAGTCAACAGAGAAGAACTGTTCATCA CCACTAAGCTGTGGAACGACGACCACAAGCGCCCCCGCGAAGCCCTGCTCGACAG CCTGAAAAAACTCCAGCTTGATTATATCGACCTCTACTTAATGCACTGGCCCGTT CCCGCTATCGACCATTATGTCGAAGCATGGAAAGGCATGATCGAATTGCAAAAAG AGGGATTAATCAAAAGCATCGGCGTGTGCAACTTCCAGATCCATCACCTGCAACG CCTGATTGATGAAACTGGCGTGACGCCTGTGATAAACCAGATCGAACTTCATCCG CTGATGCAACAACGCCAGCTACACGCCTGGAACGCGACACACAAAATCCAGACCG AATCCTGGAGCCCATTAGCGCAAGGAGGGAAAGGCGTTTTCGATCAGAAAGTCAT TCGCGATCTGGCAGATAAATACGGCAAAACCCCGGCGCAGATTGTTATCCGCTGG CATCTGGATAGCGGCCTGGTGGTGATCCCGAAATCGGTCACACCTTCACGTATTG CCGAAAACTTTGATGTCTGGGATTTCCGTCTCGACAAAGACGAACTCGGCGAAAT US 10,941,424 B2 119 120 - continued SEQUENCE LISTING TGCAAAACTCGATCAGGGCAAGCGTCTCGGTCCCGATCCTGACCAGTTCGGCGGC TAA SEQ ID NO : 32 Escherichia coli 2,5 - diketo - D - gluconic acid reductase A yqhE ( dkgA ) AA sequence MANPTVIKLQDGNVMPOLGLGVWQASNEEVITAIQKALEVGYRSIDTAAAYKNEE GVGKALKNASVNREELFITTKLWNDDHKRPREALLDSLKKLQLDYIDLYLMHWPV PAIDHYVEAWKGMI ELQKEGLIKSIGVCNFQIHHLQRLIDETGVTPVINQIELHP LMQQRQLHAWNATHKIQTESWSPLAQGGKGVFDOKVIRDLADKYGKTPAQIVIRW HLDSGLWIPKSVTPSRIAENFDVWDFRLDKDELGEIAKLDOGKRLGPDPDQFGG SEQ ID NO : 33 Clostridium acetobutylicum acetyl coenzyme A acetyltransferase thlA NT sequence ATGAAAGAAGTTGTAATAGCTAGTGCAGTAAGAACAGCGATTGGATCTTATGGAA AGTCTCTTAAGGATGTACCAGCAGTAGATTTAGGAGCTACAGCTATAAAGGAAGC AGTTAAAAAAGCAGGAATAAAACCAGAGGATGTTAATGAAGTCATTTTAGGAAAT GTTCTTCAAGCAGGTTTAGGACAGAATCCAGCAAGACAGGCATCTTTTAAAGCAG GATTACCAGTTGAAATTCCAGCTATGACTATTAATAAGGTTTGTGGTTCAGGACT TAGAACAGTTAGCTTAGCAGCACAAATTATAAAAGCAGGAGATGCTGACGTAATA ATAGCAGGTGGTATGGAAAATATGTCTAGAGCTCCTTACTTAGCGAATAACGCTA GATGGGGATATAGAATGGGAAACGCTAAATTTGTTGATGAAATGATCACTGACGG ATTGTGGGATGCATTTAATGATTACCACATGGGAATAACAGCAGAAAACATAGCT GAGAGATGGAACATTTCAAGAGAAGAACAAGATGAGTTTGCTCTTGCATCACAAA AAAAAGCTGAAGAAGCTATAAAATCAGGTCAATTTAAAGATGAAATAGTTCCTGT AGTAATTAAAGGCAGAAAGGGAGAAACTGTAGTTGATACAGATGAGCACCCTAGA TTTGGATCAACTATAGAAGGACTTGCAAAATTAAAACCTGCCTTCAAAAAAGATG GAACAGTTACAGCTGGTAATGCATCAGGATTAAATGACTGTGCAGCAGTACTTGT AATCATGAGTGCAGAAAAAGCTAAAGAGCTTGGAGTAAAACCACTTGCTAAGATA GTTTCTTATGGTTCAGCAGGAGTTGACCCAGCAATAATGGGATATGGACCTTTCT ATGCAACAAAAGCAGCTATTGAAAAAGCAGGTTGGACAGTTGATGAATTAGATTT AATAGAATCAAATGAAGCTTTTGCAGCTCAAAGTTTAGCAGTAGCAAAAGATTTA AAATTTGATATGAATAAAGTAAATGTAAATGGAGGAGCTATTGCCCTTGGTCATC CAATTGGAGCATCAGGTGCAAGAATACTCGTTACTCTTGTACACGCAATGCAAAA AAGAGATGCAAAAAAAGGCTTAGCAACTTTATGTATAGGTGGCGGACAAGGAACA GCAATATTGCTAGAAAAGTGCTAG SEO ID NO : 34 Clostridium acetobutylicum acetyl coenzyme A acetyltransferase thlA codon optimized NT sequence ATGAAAGAAGTTGTTATTGCGAGCGCGGTTCGTACCGCGATTGGCAGCTATGGCA AGAGCCTGAAGGATGTTCCGGCGGTGGACCTGGGTGCGACCGCGATCAAAGAGGC GGTTAAGAAAGCGGGCATTAAACCGGAGGATGTGAACGAAGTTATCCTGGGTAAC GTGCTGCAAGCGGGTCTGGGCCAAAACCCGGCGCGTCAGGCGAGCTTCAAGGCGG GCCTGCCGGTTGAAATCCCGGCGATGACCATTAACAAAGTTTGCGGTAGCGGCCT GCGTACCGTGAGCCTGGCGGCGCAAATCATTAAGGCGGGTGACGCGGATGTTATC ATTGCGGGTGGCATGGAGAACATGAGCCGTGCGCCGTACCTGGCGAACAACGCGC GTTGGGGTTATCGTATGGGCAACGCGAAATTCGTGGACGAAATGATTACCGACGG TCTGTGGGATGCGTTTAACGACTACCACATGGGCATCACCGCGGAGAACATTGCG GAACGTTGGAACATTAGCCGTGAGGAACAAGATGAGTTCGCGCTGGCGAGCCAGA AGAAAGCGGAGGAAGCGATCAAGAGCGGCCAGTTTAAAGACGAAATCGTTCCGGT GGTTATTAAGGGTCGTAAGGGTGAAACCGTGGTGGACACCGATGAACACCCGCGT TTCGGTAGCACCATTGAGGGCCTGGCGAAGCTGAAACCGGCGTTTAAGAAAGATG GCACCGTGACCGCGGGTAACGCGAGCGGCCTGAACGACTGCGCGGCGGTGCTGGT TATCATGAGCGCGGAGAAGGCGAAAGAACTGGGTGTGAAGCCGCTGGCGAAAATT GTTAGCTACGGTAGCGCGGGTGTGGACCCGGCGATCATGGGTTACGGCCCGTTTT ATGCGACCAAGGCGGCGATTGAGAAAGCGGGTTGGACCGTGGACGAACTGGATCT GATCGAGAGCAACGAAGCGTTCGCGGCGCAAAGCCTGGCGGTGGCGAAGGATCTG AAATTTGACATGAACAAGGTGAACGTGAACGGTGGTGCGATTGCGCTGGGTCACC CGATTGGTGCGAGCGGCGCGCGTATCCTGGTGACCCTGGTTCACGCGATGCAGAA ACGTGACGCGAAGAAAGGTCTGGCGACCCTGTGCATTGGTGGTGGTCAAGGCACC GCGATTCTGCTGGAAAAGTGCTAA SEQ ID NO : 35 Clostridium acetobutylicum acetyl coenzyme A acetyltransferase thiA AA sequence MKEVIASAVRTAIGSYGKSLKDVPAVDLGATAI KEAVKKAGIKPEDVNEVILGN VLQAGLGQNPARQASFKAGLPVEIPAMTINKVCGSGLRTVSLAAQIIKAGDADVI IAGGMENMSRAPYLANNARWGYRMGNAKFVDEMI TDGLWDAFNDYHMGITAENIA ERWNISREEQDEFALASQKKAEEAI KSGQFKDEIVPVVIKGRKGETVVDTDEHPR FGSTIEGLAKLKPAFKKDGTVTAGNASGLNDCAAVLVIMSAEKAKELGVKPLAKI VSYGSAGVDPAIMGYGPFYATKAAIEKAGWTVDELDLIESNEAFAAQSLAVAKDL KFDMNKVNVNGGAIALGHPIGASGARILVTLVHAMQKRDAKKGLATLCIGGGQGT AILLEKC SEQ ID NO : 36 Escherichia coli acetyl coenzyme A acetyltransferase atoB NT sequence ATGAAAAATTGTGTCATCGTCAGTGCGGTACGTACTGCTATCGGTAGTTTTAACG GTTCACTCGCTTCCACCAGCGCCATCGACCTGGGGGCGACAGTAATTAAAGCCGC CATTGAACGTGCAAAAATCGATTCACAACACGTTGATGAAGTGATTATGGGTAAC US 10,941,424 B2 121 122 - continued SEQUENCE LISTING GTGTTACAAGCCGGGCTGGGGCAAAATCCGGCGCGTCAGGCACTGTTAAAAAGCG GGCTGGCAGAAACGGTGTGCGGATTCACGGTCAATAAAGTATGTGGTTCGGGTCT TAAAAGTGTGGCGCTTGCCGCCCAGGCCATTCAGGCAGGTCAGGCGCAGAGCATT GTGGCGGGGGGTATGGAAAATATGAGTTTAGCCCCCTACTTACTCGATGCAAAAG CACGCTCTGGTTATCGTCTTGGAGACGGACAGGTTTATGACGTAATCCTGCGCGA TGGCCTGATGTGCGCCACCCATGGTTATCATATGGGGATTACCGCCGAAAACGTG GCTAAAGAGTACGGAATTACCCGTGAAATGCAGGATGAACTGGCGCTACATTCAC AGCGTAAAGCGGCAGCCGCAATTGAGTCCGGTGCTTTTACAGCCGAAATCGTCCC GGTAAATGTTGTCACTCGAAAGAAAACCTTCGTCTTCAGTCAAGACGAATTCCCG AAAGCGAATTCAACGGCTGAAGCGTTAGGTGCATTGCGCCCGGCCTTCGATAAAG CAGGAACAGTCACCGCTGGGAACGCGTCTGGTATTAACGACGGTGCTGCCGCTCT GGTGATTATGGAAGAATCTGCGGCGCTGGCAGCAGGCCTTACCCCCCTGGCTCGC ATTAAAAGTTATGCCAGCGGTGGCGTGCCCCCCGCATTGATGGGTATGGGGCCAG TACCTGCCACGCAAAAAGCGTTACAACTGGCGGGGCTGCAACTGGCGGATATTGA TCTCATTGAGGCTAATGAAGCATTTGCTGCACAGTTCCTTGCCGTTGGGAAAAAC CTGGGCTTTGATTCTGAGAAAGTGAATGTCAACGGCGGGGCCATCGCGCTCGGGC ATCCTATCGGTGCCAGTGGTGCTCGTATTCTGGTCACACTATTACATGCCATGCA GGCACGCGATAAAACGCTGGGGCTGGCAACACTGTGCATTGGCGGCGGTCAGGGA ATTGCGATGGTGATTGAACGGTTGAATTAA SEQ ID NO : 37 Escherichia coli acetyl coenzyme A acetyltransferase atoB AA sequence MKNCVIVSAVRTAIGSFNGSLASTSAIDLGATVIKAAI ERAKIDSQHVDEVIMGN VLQAGLGONPARQALLKSGLAETVCGFTVNKVCGSGLKSVALAAQAI QAGQAQSI VAGGMENMSLAPYLLDAKARSGYRLGDGQVYDVILRDGLMCATHGYHMGITAENV AKEYGITREMODELALHSQRKAAAAIESGAFTAEIVPVNVVTRKKTFVFSQDEFP KANSTAEALGALRPAFDKAGTVTAGNASGINDGAAALVIMEESAALAALTPLAR IKSYASGGVPPALMGMGPVPATOKALQLAGLQLADIDLIEANEAFAAQFLAVGKN LGFDSEKVNVNGGAIALGHPIGASGARILVTLLHAMQARDKTLGLATLCIGGGQG IAMVIERLN SEO ID NO : 38 Saccharomyces cerevisiae acetyl coenzyme A acetyltransferase ERG10 NT sequence ATGTCTCAGAACGTTTACATTGTATCGACTGCCAGAACCCCAATTGGTTCATTCC AGGGTTCTCTATCCTCCAAGACAGCAGTGGAATTGGGTGCTGTTGCTTTAAAAGG CGCCTTGGCTAAGGTTCCAGAATTGGATGCATCCAAGGATTTTGACGAAATTATT TTTGGTAACGTTCTTTCTGCCAATTTGGGCCAAGCTCCGGCCAGACAAGTTGCTT TGGCTGCCGGTTTGAGTAATCATATCGTTGCAAGCACAGTTAACAAGGTCTGTGC ATCCGCTATGAAGGCAATCATTTTGGGTGCTCAATCCATCAAATGTGGTAATGCT GATGTTGTCGTAGCTGGTGGTTGTGAATCTATGACTAACGCACCATACTACATGC CAGCAGCCCGTGCGGGTGCCAAATTTGGCCAAACTGTTCTTGTTGATGGTGTCGA AAGAGATGGGTTGAACGATGCGTACGATGGTCTAGCCATGGGTGTACACGCAGAA AAGTGTGCCCGTGATTGGGATATTACTAGAGAACAACAAGACAATTTTGCCATCG AATCCTACCAAAAATCTCAAAAATCTCAAAAGGAAGGTAAATTCGACAATGAAAT TGTACCTGTTACCATTAAGGGATTTAGAGGTAAGCCTGATACTCAAGTCACGAAG GACGAGGAACCTGCTAGATTACACGTTGAAAAATTGAGATCTGCAAGGACTGTTT TCCAAAAAGAAAACGGTACTGTTACTGCCGCTAACGCTTCTCCAATCAACGATGG TGCTGCAGCCGTCATCTTGGTTTCCGAAAAAGTTTTGAAGGAAAAGAATTTGAAG CCTTTGGCTATTATCAAAGGTTGGGGTGAGGCCGCTCATCAACCAGCTGATTTTA CATGGGCTCCATCTCTTGCAGTTCCAAAGGCTTTGAAACATGCTGGCATCGAAGA CATCAATTCTGTTGATTACTTTGAATTCAATGAAGCCTTTTCGGTTGTCGGTTTG GTGAACACTAAGATTTTGAAGCTAGACCCATCTAAGGTTAATGTATATGGTGGTG CTGTTGCTCTAGGTCACCCATTGGGTTGTTCTGGTGCTAGAGTGGTTGTTACACT GCTATCCATCTTACAGCAAGAAGGAGGTAAGATCGGTGTTGCCGCCATTTGTAAT GGTGGTGGTGGTGCTTCCTCTATTGTCATTGAAAAGATATGA SEQ ID NO : 39 Saccharomyces cerevisiae acetyl coenzyme A acetyltransferase ERG10 codon optimized NT sequence ATGTCTCAGAACGTTTACATTGTATCGACTGCCAGAACCCCAATTGGTTCATTCC AGGGTTCTCTATCCTCCAAGACAGCAGTGGAATTGGGTGCTGTTGCTTTAAAAGG CGCCTTGGCTAAGGTTCCAGAATTGGATGCATCCAAGGATTTTGACGAAATTATT TTTGGTAACGTTCTTTCTGCCAATTTGGGCCAAGCTCCGGCCAGACAAGTTGCTT TGGCTGCCGGTTTGAGTAATCATATCGTTGCAAGCACAGTTAACAAGGTCTGTGC ATCCGCTATGAAGGCAATCATTTTGGGTGCTCAATCCATCAAATGTGGTAATGCT GATGTTGTCGTAGCTGGTGGTTGTGAATCTATGACTAACGCACCATACTACATGC CAGCAGCCCGTGCGGGTGCCAAATTTGGCCAAACTGTTCTTGTTGATGGTGTCGA AAGAGATGGGTTGAACGATGCGTACGATGGTCTAGCCATGGGTGTACACGCAGAA AAGTGTGCCCGTGATTGGGATATTACTAGAGAACAACAAGACAATTTTGCCATCG AATCCTACCAAAAATCTCAAAAATCTCAAAAGGAAGGTAAATTCGACAATGAAAT TGTACCTGTTACCATTAAGGGATTTAGAGGTAAGCCTGATACTCAAGTCACGAAG GACGAGGAACCTGCTAGATTACACGTTGAAAAATTGAGATCTGCAAGGACTGTTT TCCAAAAAGAAAACGGTACTGTTACTGCCGCTAACGCTTCTCCAATCAACGATGG TGCTGCAGCCGTCATCTTGGTTTCCGAAAAAGTTTTGAAGGAAAAGAATTTGAAG CCTTTGGCTATTATCAAAGGTTGGGGTGAGGCCGCTCATCAACCAGCTGATTTTA CATGGGCTCCATCTCTTGCAGTTCCAAAGGCTTTGAAACATGCTGGCATCGAAGA CATCAATTCTGTTGATTACTTTGAATTCAATGAAGCCTTTTCGGTTGTCGGTTTG GTGAACACTAAGATTTTGAAGCTAGACCCATCTAAGGTTAATGTATATGGTGGTG US 10,941,424 B2 123 124 - continued SEQUENCE LISTING CTGTTGCTCTAGGTCACCCATTGGGTTGTTCTGGTGCTAGAGTGGTTGTTACACT GCTATCCATCTTACAGCAAGAAGGAGGTAAGATCGGTGTTGCCGCCATTTGTAAT GGTGGTGGTGGTGCTTCCTCTATTGTCATTGAAAAGATATGA SEQ ID NO : 40 Saccharomyces cerevisiae acetyl coenzyme A acetyltransferase ERG10 AA sequence MSQNVYIVSTARTPIGSFQGSLSSKTAVELGAVALKGALAKVPELDASKDFDEII FGNVLSANLGQAPAROVALAAGLSNHIVASTVNKVCASAMKAIILGAQSIKCGNA DVVVAGGCESMTNAPYYMPAARAGAKFGQTVLVDGVERDGLNDAYDGLAMGVHAE KCARDWDITREQQDNFAIESYQKSQKSQKEGKFDNEIVPVTIKGFRGKPDTQVTK DEEPARLHVEKLRSARTVFQKENGTVTAANASPINDGAAAVILVSEKVLKEKNLK PLAIIKGWGEAAHQPADFTWAPSLAVPKALKHAGIEDINSVDYFEFNEAFSVVGL VNTKILKLDPSKVNVYGGAVALGHPLGCSGARVVVTLLSILQQEGGKIGVAAICN GGGGASSIVIEKI SEQ ID NO : 41 Escherichia coli Acetyl - CoA : acetoacetate - COA transferase subunit atoA NT sequence ATGGATGCGAAACAACGTATTGCGCGCCGTGTGGCGCAAGAGCTTCGTGATGGTG ACATCGTTAACTTAGGGATCGGTTTACCCACAATGGTCGCCAATTATTTACCGGA GGGTATTCATATCACTCTGCAATCGGAAAACGGCTTCCTCGGTTTAGGCCCGGTC ACGACAGCGCATCCAGATCTGGTGAACGCTGGCGGGCAACCGTGCGGTGTTTTAC CCGGTGCAGCCATGTTTGATAGCGCCATGTCATTTGCGCTAATCCGTGGCGGTCA TATTGATGCCTGCGTGCTCGGCGGTTTGCAAGTAGACGAAGAAGCAAACCTCGCG AACTGGGTAGTGCCTGGGAAAATGGTGCCCGGTATGGGTGGCGCGATGGATCTGG TGACCGGGTCGCGCAAAGTGATCATCGCCATGGAACATTGCGCCAAAGATGGTTC AGCAAAAATTTTGCGCCGCTGCACCATGCCACTCACTGCGCAACATGCGGTGCAT ATGCTGGTTACTGAACTGGCTGTCTTTCGTTTTATTGACGGCAAAATGTGGCTCA CCGAAATTGCCGACGGGTGTGATTTAGCCACCGTGCGTGCCAAAACAGAAGCTCG GTTTGAAGTCGCCGCCGATCTGAATACGCAACGGGGTGATTTATGA SEQ ID NO : 42 Escherichia coli Acetyl - COA : acetoacetate - COA transferase subunit atoA codon optimized NT sequence ATGGATGCGAAACAACGTATTGCGCGCCGTGTGGCGCAAGAGCTTCGTGATGGTG ACATCGTTAACTTAGGGATCGGTTTACCCACAATGGTCGCCAATTATTTACCGGA GGGTATTCATATCACTCTGCAATCGGAAAACGGCTTCCTCGGTTTAGGCCCGGTC ACGACAGCGCATCCAGATCTGGTGAACGCTGGCGGGCAACCGTGCGGTGTTTTAC CCGGTGCAGCCATGTTTGATAGCGCCATGTCATTTGCGCTAATCCGTGGCGGTCA TATTGATGCCTGCGTGCTCGGCGGTTTGCAAGTAGACGAAGAAGCAAACCTCGCG AACTGGGTAGTGCCTGGGAAAATGGTGCCCGGTATGGGTGGCGCGATGGATCTGG TGACCGGGTCGCGCAAAGTGATCATCGCCATGGAACATTGCGCCAAAGATGGTTC AGCAAAAATTTTGCGCCGCTGCACCATGCCACTCACTGCGCAACATGCGGTGCAT ATGCTGGTTACTGAACTGGCTGTCTTTCGTTTTATTGACGGCAAAATGTGGCTCA CCGAAATTGCCGACGGGTGTGATTTAGCCACCGTGCGTGCCAAAACAGAAGCTCG GTTTGAAGTCGCCGCCGATCTGAATACGCAACGGGGTGATTTATGA SEQ ID NO : 43 Escherichia coli Acetyl - CoA : acetoacetate - COA transferase subunit atoA AA sequence MDAKORIARRVAQELRDGDIVNLGIGLPTMVANYLPEGIHITLQSENGFLGLGPV TTAHPDLVNAGGQPCGVLPGAAMFDSAMSFALIRGGHIDACVLGGLOVDEEANLA NWVVPGKMVPGMGGAMDLVTGSRKVIIAMEHCAKDGSAKILRRCTMPLTAQHAVH MLVTELAVFRFIDGKMWLTEIADGCDLATVRAKTEARFEVAADLNTQRGDL SEQ ID NO : 44 Escherichia coli Acetyl - CoA : acetoacetate - COA transferase subunit atoD NT sequence ATGAAAACAAAATTGATGACATTACAAGACGCCACCGGCTTCTTTCGTGACGGCA TGACCATCATGGTGGGCGGATTTATGGGGATTGGCACTCCATCCCGCCTGGTTGA AGCATTACTGGAATCTGGTGTTCGCGACCTGACATTGATAGCCAATGATACCGCG TTTGTTGATACCGGCATCGGTCCGCTCATCGTCAATGGTCGAGTCCGCAAAGTGA TTGCTTCACATATCGGCACCAACCCGGAAACAGGTCGGCGCATGATATCTGGTGA GATGGACGTCGTTCTGGTGCCGCAAGGTACGCTAATCGAGCAAATTCGCTGTGGT GGAGCTGGACTTGGTGGTTTTCTCACCCCAACGGGTGTCGGCACCGTCGTAGAGG AAGGCAAACAGACACTGACACTCGACGGTAAAACCTGGCTGCTCGAACGCCCACT GCGCGCCGACCTGGCGCTAATTCGCGCTCATCGTTGCGACACACTTGGCAACCTG ACCTATCAACTTAGCGCCCGCAACTTTAACCCCCTGATAGCCCTTGCGGCTGATA TCACGCTGGTAGAGCCAGATGAACTGGTCGAAACCGGCGAGCTGCAACCTGACCA TATTGTCACCCCTGGTGCCGTTATCGACCACATCATCGTTTCACAGGAGAGCAAA TAA SEQ ID NO : 45 Escherichia coli Acetyl - CoA : acetoacetate - COA transferase subunit atod codon optimized NT sequence ATGAAAACAAAATTGATGACATTACAAGACGCCACCGGCTTCTTTCGTGACGGCA TGACCATCATGGTGGGCGGATTTATGGGGATTGGCACTCCATCCCGCCTGGTTGA AGCATTACTGGAATCTGGTGTTCGCGACCTGACATTGATAGCCAATGATACCGCG TTTGTTGATACCGGCATCGGTCCGCTCATCGTCAATGGTCGAGTCCGCAAAGTGA TTGCTTCACATATCGGCACCAACCCGGAAACAGGTCGGCGCATGATATCTGGTGA GATGGACGTCGTTCTGGTGCCGCAAGGTACGCTAATCGAGCAAATTCGCTGTGGT GGAGCTGGACTTGGTGGTTTTCTCACCCCAACGGGTGTCGGCACCGTCGTAGAGG US 10,941,424 B2 125 126 - continued SEQUENCE LISTING AAGGCAAACAGACACTGACACTCGACGGTAAAACCTGGCTGCTCGAACGCCCACT GCGCGCCGACCTGGCGCTAATTCGCGCTCATCGTTGCGACACACTTGGCAACCTG ACCTATCAACTTAGCGCCCGCAACTTTAACCCCCTGATACCCTTGCGGCTGATA TCACGCTGGTAGAGCCAGATGAACTGGTCGAAACCGGCGAGCTGCAACCTGACCA TATTGTCACCCCTGGTGCCGTTATCGACCACATCATCGTTTCACAGGAGAGCAAA TAA SEQ ID NO : 46 Escherichia coli Acetyl - COA : acetoacetate - COA transferase subunit atoD AA sequence MKTKLMTLQDATGFFRDGMTIMVGGFMGIGTPSRLVEALLESGVRDLTLIANDTA FVDTGIGPLIVNGRVRKVIASHIGTNPETGRRMISGEMDVVLVPQGTLIEQIRCG GAGLGGFLTPTGVGTVVEEGKQTLTLDGKTWLLERPLRADLALIRAHRCDTLGNL TYQLSARNFNPLIALAADITLVEPDELVETGELQPDHIVTPGAVIDHIIVSQESK SEQ ID NO : 47 Clostridium acetobutylicum acetoacetate decarboxylase adc NT sequence ATGTTAAAGGATGAAGTAATTAAACAAATTAGCACGCCATTAACTTCGCCTGCAT TTCCTAGAGGACCCTATAAATTTCATAATCGTGAGTATTTTAACATTGTATATCG TACAGATATGGATGCACTTCGTAAAGTTGTGCCAGAGCCTTTAGAAATTGATGAG CCCTTAGTCAGGTTTGAAATTATGGCAATGCATGATACGAGTGGACTTGGTTGTT ATACAGAAAGCGGACAGGCTATTCCCGTAAGCTTTAATGGAGTTAAGGGAGATTA TCTTCATATGATGTATTTAGATAATGAGCCTGCAATTGCAGTAGGAAGGGAATTA AGTGCATATCCTAAAAAGCTCGGGTATCCAAAGCTTTTTGTGGATTCAGATACTT TAGTAGGAACTTTAGACTATGGAAAACTTAGAGTTGCGACAGCTACAATGGGGTA CAAACATAAAGCCTTAGATGCTAATGAAGCAAAGGATCAAATTTGTCGCCCTAAT TATATGTTGAAAATAATACCCAATTATGATGGAAGCCCTAGAATATGTGAGCTTA TAAATGCGAAAATCACAGATGTTACCGTACATGAAGCTTGGACAGGACCAACTCG ACTGCAGTTATTTGATCACGCTATGGCGCCACTTAATGATTTGCCAGTAAAAGAG ATTGTTTCTAGCTCTCACATTCTTGCAGATATAATATTGCCTAGAGCTGAAGTTA TATATGATTATCTTAAGTAA SEO ID NO : 48 Clostridium acetobutylicum acetoacetate decarboxylase adc codon optimized NT sequence ATGCTGAAGGACGAGGTTATTAAGCAGATTAGCACCCCGCTGACCAGCCCGGCGT TCCCGCGTGGTCCGTACAAGTTCCATAATCGCGAATACTTCAACATTGTGTATCG TACCGACATGGATGCGCTGCGTAAGGTGGTTCCGGAGCCGCTGGAAATTGACGAG CCGCTGGTTCGTTTCGAAATCATGGCGATGCACGATACCAGCGGTCTGGGCTGCT ACACCGAGAGCGGTCAGGCGATTCCGGTGAGCTTTAACGGTGTTAAAGGCGACTA CCTGCACATGATGTATCTGGATAACGAACCGGCGATTGCGGTGGGTCGTGAGCTG AGCGCGTACCCGAAGAAACTGGGCTATCCGAAGCTGTTCGTGGACAGCGATACCC TGGTGGGCACCCTGGACTACGGCAAACTGCGTGTTGCGACCGCGACCATGGGCTA TAAGCACAAAGCGCTGGACGCGAACGAAGCGAAGGATCAGATTTGCCGTCCGAAC TACATGCTGAAAATCATTCCGAACTATGACGGTAGCCCGCGTATCTGCGAACTGA TTAACGCGAAGATCACCGATGTTACCGTTCATGAGGCGTGGACCGGCCCGACCCG TCTGCAACTGTTTGACCACGCGATGGCGCCGCTGAACGATCTGCCGGTGAAAGAG ATCGTTAGCAGCAGCCACATCCTGGCGGACATCATCCTGCCGCGTGCGGAAGTTA TCTACGATTACCTGAAGTAA SEQ ID NO : 49 C. stridium acetobutylicum acetoacetate decarboxylase adc AA sequence MLKDEVIKQISTPLTSPAFPRGPYKFHNREYFNIVYRTDMDALRKVVPEPLEIDE PLVRFEIMAMHDTSGLGCYTESGQAIPVSFNGVKGDYLHMMYLDNEPAIAVGREL SAYPKKLGYPKLFVDSDTLVGTLDYGKLRVATATMGYKHKALDANEAKDQICRPN YMLKIIPNYDGS PRICELINAKITDVTVHEAWTGPTRLQLFDHAMAPLNDLPVKE IVSSSHILADIILPRAEVIYDYLK SEQ ID NO : 50 Clostridium beijerinckii acetoacetate decarboxylase adc NT sequence ATGTTAGAAAGTGAAGTATCTAAACAAATTACAACTCCACTTGCTGCTCCAGCGT TTCCTAGAGGACCATATAGGTTTCACAATAGAGAATATCTAAACATTATTTATCG AACTGATTTAGATGCTCTTCGAAAAATAGTACCAGAGCCACTTGAATTAGATAGA GCATATGTTAGATTTGAAATGATGGCTATGCCTGATACAACCGGACTAGGCTCAT ATACAGAATGTGGTCAAGCTATTCCAGTAAAATATAATGGTGTTAAGGGTGACTA CTTGCATATGATGTATCTAGATAATGAACCTGCTATTGCTGTTGGAAGAGAAAGT AGCGCTTATCCAAAAAAGCTTGGCTATCCAAAGCTATTTGTTGATTCAGATACTT TAGTTGGGACACTTAAATATGGTACATTACCAGTAGCTACTGCAACAATGGGATA TAAGCACGAGCCTCTAGATCTTAAAGAAGCCTATGCTCAAATTGCAAGACCCAAT TTTATGCTAAAAATCATTCAAGGTTACGATGGTAAGCCAAGAATTTGTGAACTAA TATGTGCAGAAAATACTGATATAACTATTCACGGTGCTTGGACTGGAAGTGCACG TCTACAATTATTTAGCCATGCACTAGCTCCTCTTGCTGATTTACCTGTATTAGAG ATTGTATCAGCATCTCATATCCTCACAGATTTAACTCTTGGAACACCTAAGGTTG TACATGATTATCTTTCAGTAAAATAA SEO ID NO : 51 Clostridium beijerinckii acetoacetate decarboxylase adc codon optimized NT sequence ATGCTGGAGAGCGAAGTTAGCAAACAAATCACCACCCCGCTGGCGGCGCCGGCGT TCCCGCGTGGCCCGTACCGTTTTCATAACCGTGAGTACCTGAACATCATTTATCG US 10,941,424 B2 127 128 - continued SEQUENCE LISTING TACCGACCTGGATGCGCTGCGTAAGATTGTGCCGGAGCCGCTGGAACTGGACCGT GCGTACGTTCGTTTCGAGATGATGGCGATGCCGGATACCACCGGTCTGGGCAGCT ACACCGAATGCGGTCAGGCGATCCCGGTGAAGTATAACGGTGTTAAAGGCGACTA CCTGCACATGATGTATCTGGATAACGAGCCGGCGATTGCGGTGGGTCGTGAAAGC AGCGCGTACCCGAAGAAACTGGGCTATCCGAAGCTGTTTGTGGACAGCGATACCC TGGTGGGCACCCTGAAATATGGCACCCTGCCGGTTGCGACCGCGACCATGGGCTA CAAGCACGAGCCGCTGGACCTGAAAGAAGCGTATGCGCAGATTGCGCGTCCGAAC TTCATGCTGAAGATCATTCAAGGTTATGACGGCAAACCGCGTATCTGCGAGCTGA TTTGCGCGGAAAACACCGATATCACCATCCATGGTGCGTGGACCGGCAGCGCGCG TCTGCAACTGTTTAGCCATGCGCTGGCGCCGCTGGCGGATCTGCCGGTGCTGGAA ATCGTTAGCGCGAGCCACATTCTGACCGATCTGACCCTGGGCACCCCGAAGGTTG TGCATGACTATCTGAGCGTGAAGTAA SEQ ID NO : 52 Clostridium beijerinckii acetoacetate decarboxylase adc AA sequence MLESEVSKQITTPLAAPAFPRGPYRFHNREYLNIIYRTDLDALRKIVPEPLELDR AYVRFEMMAMPDTTGLGSYTECGQAIPVKYNGVKGDYLHMMYLDNEPAIAVGRES SAYPKKLGYPKLFVDSDTLVGTLKYGTLPVATATMGYKHEPLDLKEAYAQIARPN FMLKIIQGYDGKPRICELICAENTDITIHGAWTGSARLQLFSHALAPLADLPVLE IVSASHILTDLTLGTPKVVHDYLSVK SEQ ID NO : 53 Homo sapiens ketohexokinase c khk - C cDNA sequence ATGGAAGAGAAGCAGATCCTGTGCGTGGGGCTAGTGGTGCTGGACGTCATCAGCC TGGTGGACAAGTACCCTAAGGAGGACTCGGAGATAAGGTGTTTGTCCCAGAGATG GCAGCGCGGAGGCAACGCGTCCAACTCCTGCACCGTTCTCTCCCTGCTCGGAGCC CCCTGTGCCTTCATGGGCTCAATGGCTCCTGGCCATGTTGCTGATTTTGTCCTGG ATGACCTCCGCCGCTATTCTGTGGACCTACGCTACACAGTCTTTCAGACCACAGG CTCCGTCCCCATCGCCACGGTCATCATCAACGAGGCCAGTGGTAGCCGCACCATC CTATACTATGACAGGAGCCTGCCAGATGTGTCTGCTACAGACTTTGAGAAGGTTG ATCTGACCCAGTTCAAGTGGATCCACATTGAGGGCCGGAACGCATCGGAGCAGGT GAAGATGCTGCAGCGGATAGACGCACACAACACCAGGCAGCCTCCAGAGCAGAAG ATCCGGGTGTCCGTGGAGGTGGAGAAGCCACGAGAGGAGCTCTTCCAGCTGTTTG GCTACGGAGACGTGGTGTTTGTCAGCAAAGATGTGGCCAAGCACTTGGGGTTCCA GTCAGCAGAGGAAGCCTTGAGGGGCTTGTATGGTCGTGTGAGGAAAGGGGCTGTG CTTGTCTGTGCCTGGGCTGAGGAGGGCGCCGACGCCCTGGGCCCTGATGGCAAAT TGCTCCACTCGGATGCTTTCCCGCCACCCCGCGTGGTGGATACACTGGGAGCTGG AGACACCTTCAATGCCTCCGTCATCTTCAGCCTCTCCCAGGGGAGGAGCGTGCAG GAAGCACTGAGATTCGGGTGCCAGGTGGCCGGCAAGAAGTGTGGCCTGCAGGGCT TTGATGGCATCGTTTAA SEO ID NO : 54 Homo sapiens ketohexokinase c khk - C codon optimized cDNA sequence ATGGAGGAAAAGCAAATTCTGTGCGTTGGTCTGGTGGTTCTGGACGTGATTAGCC TGGTTGATAAGTACCCGAAAGAGGATAGCGAAATCCGTTGCCTGAGCCAGCGTTG GCAACGTGGTGGCAACGCGAGCAATAGCTGCACCGTTCTGAGCCTGCTGGGTGCG CCGTGCGCGTTCATGGGTAGCATGGCGCCGGGTCATGTTGCGGACTTCCTGGTGG CGGATTTTCGTCGTCGTGGTGTGGACGTTAGCCAGGTTGCGTGGCAAAGCAAGGG CGATACCCCGAGCTCCTGCTGCATCATTAACAACAGCAACGGTAACCGTACCATT GTGCTGCACGACACCAGCCTGCCGGATGTTAGCGCGACCGACTTCGAGAAGGTGG ATCTGACCCAGTTTAAATGGATTCACATTGAGGGCCGTAACGCGAGCGAACAGGT TAAAATGCTGCAACGTATTGATGCGCACAACACCCGTCAGCCGCCGGAACAAAAG ATTCGTGTGAGCGTTGAGGTGGAAAAACCGCGTGAGGAACTGTTCCAACTGTTTG GTTACGGCGACGTGGTTTTCGTTAGCAAGGATGTGGCGAAACACCTGGGTTTTCA AAGCGCGGAGGAAGCGCTGCGTGGTCTGTATGGCCGTGTGCGTAAAGGCGCGGTT CTGGTGTGCGCGTGGGCGGAGGAAGGCGCGGATGCGCTGGGTCCGGATGGCAAAC TGCTGCACAGCGATGCGTTCCCGCCGCCGCGTGTGGTTGACACCCTGGGTGCGGG CGATACCTTCAACGCGAGCGTTATCTTTAGCCTGAGCCAGGGCCGTAGCGTGCAA GAGGCGCTGCGTTTCGGCTGCCAAGTTGCGGGTAAAAAATGCGGTCTGCAAGGCT TTGACGGTATCGTGTAA SEQ ID NO : 55 Homo sapiens ketohexokinase C khk - C AA sequence MEEKQILCVGLVVLDVISLVDKYPKEDSEIRCLSQRWQRGGNASNSCTVLSLLGA PCAFMGSMAPGHVADFLVADFRRRGVDVSQVAWQSKGDTPSSCCIINNSNGNRTI VLHDTSLPDVSATDFEKVDLTQFKWIHIEGRNASEQVKMLQRIDAHNTROPPEQK IRVSVEVEKPREELFQLFGYGDVVFVSKDVAKHLGFQSAEEALRGLYGRVRKGAV LVCAWAEEGADALGPDGKLLHSDAFPPPRVVDTLGAGDTFNASVIFSLSQGRSVQ EALRFGCOVAGKKCGLQGFDGIV SEQ ID NO : 56 Homo sapiens Fructose - bisphosphate aldolase B aldoB cDNA sequence ATGGCCCACCGATTTCCAGCCCTCACCCAGGAGCAGAAGAAGGAGCTCTCAGAAA TTGCCCAGAGCATTGTTGCCAATGGAAAGGGGATCCTGGCTGCAGATGAATCTGT AGGTACCATGGGGAACCGCCTGCAGAGGATCAAGGTGGAAAACACTGAAGAGAAC CGCCGGCAGTTCCGAGAAATCCTCTTCTCTGTGGACAGTTCCATCAACCAGAGCA TCGGGGGTGTGATCCTTTTCCACGAGACCCTCTACCAGAAGGACAGCCAGGGAAA GCTGTTCAGAAACATCCTCAAGGAAAAGGGGATCGTGGTGGGAATCAAGTTAGAC CAAGGAGGTGCTCCTCTTGCAGGAACAAACAAAGAAACCACCATTCAAGGGCTTG US 10,941,424 B2 129 130 - continued SEQUENCE LISTING ATGGCCTCTCAGAGCGCTGTGCTCAGTACAAGAAAGATGGTGTTGACTTTGGGAA GTGGCGTGCTGTGCTGAGGATTGCCGACCAGTGTCCATCCAGCCTCGCTATCCAG GAAAACGCCAACGCCCTGGCTCGCTACGCCAGCATCTGTCAGCAGAATGGACTGG TACCTATTGTTGAACCAGAGGTAATTCCTGATGGAGACCATGACCTGGAACACTG CCAGTATGTTACTGAGAAGGTCCTGGCTGCTGTCTACAAGGCCCTGAATGACCAT CATGTTTACCTGGAGGGCACCCTGCTAAAGCCCAACATGGTGACTGCTGGACATG CCTGCACCAAGAAGTATACTCCAGAACAAGTAGCTATGGCCACCGTAACAGCTCT CCACCGTACTGTTCCTGCAGCTGTTCCTGGCATCTGCTTTTTGTCTGGTGGCATG AGTGAAGAGGATGCCACTCTCAACCTCAATGCTATCAACCTTTGCCCTCTACCAA AGCCCTGGAAACTAAGTTTCTCTTATGGACGGGCCCTGCAGGCCAGTGCACTGGC TGCCTGGGGTGGCAAGGCTGCAAACAAGGAGGCAACCCAGGAGGCTTTTATGAAG CGGGCCATGGCTAACTGCCAGGCGGCCAAAGGACAGTATGTTCACACGGGTTCTT CTGGGGCTGCTTCCACCCAGTCGCTCTTCACAGCCTGCTATACCTACTAG SEQ ID NO : 57 Homo sapiens Fructose - bisphosphate aldolase B aldos codon optimized cDNA sequence ATGGCGCACCGTTTTCCGGCGCTGACCCAAGAGCAGAAGAAGGAGCTGAGCGAGA TTGCGCAGAGCATCGTGGCGAATGGTAAAGGTATTCTGGCGGCGGATGAGAGCGT TGGTACCATGGGCAACCGTCTGCAGCGTATTAAGGTGGAGAACACCGAGGAAAAC CGTCGTCAATTCCGTGAAATCCTGTTTAGCGTTGATAGCAGCATCAACCAGAGCA TTGGTGGCGTGATCCTGTTCCACGAAACCCTGTACCAGAAGGACAGCCAAGGTAA ACTGTTTCGTAACATTCTGAAGGAAAAAGGTATTGTGGTTGGCATCAAGCTGGAT CAAGGTGGCGCGCCGCTGGCGGGCACCAACAAGGAAACCACCATCCAGGGTCTGG ACGGCCTGAGCGAACGTTGCGCGCAATATAAGAAAGATGGTGTTGACTTCGGCAA GTGGCGTGCGGTGCTGCGTATTGCGGACCAGTGCCCGAGCAGCCTGGCGATCCAA GAAAACGCGAACGCGCTGGCGCGTTACGCGAGCATCTGCCAGCAAAACGGTCTGG TGCCGATTGTTGAGCCGGAAGTTATCCCGGACGGCGATCACGACCTGGAGCACTG CCAGTATGTGACCGAAAAGGTTCTGGCGGCGGTGTACAAAGCGCTGAACGATCAC CACGTTTATCTGGAGGGTACCCTGCTGAAACCGAACATGGTGACCGCGGGCCATG CGTGCACCAAGAAATACACCCCGGAACAGGTGGCGATGGCGACCGTGACCGCGCT GCACCGTACCGTTCCGGCGGCGGTGCCGGGTATTTGCTTTCTGAGCGGTGGCATG AGCGAAGAGGACGCGACCCTGAACCTGAACGCGATCAACCTGTGCCCGCTGCCGA AGCCGTGGAAACTUAGCTTCAGCTACGGCCGTGCGCTGCAGGCGAGCGCGCTGGC GGCGTGGGGTGGCAAGGCGGCGAACAAAGAGGCGACCCAAGAAGCGTTTATGAAG CGTGCGATGGCGAACTGCCAGGCGGCGAAAGGTCAATATGTGCATACCGGCAGCA GCGGTGCGGCGAGCACCCAGAGCCTGTTTACCGCGTGCTATACCTATTAA SEO ID NO : 58 Homo sapiens Fructose - bisphosphate aldolase B aldoB AA sequence MAHRFPALTQEQKKELSEIAQSIVANGKGILAADESVGTMGNRLQRIKVENTEEN RRQFREILFSVDSSINQSIGGVILFHETLYQKDSQGKLFRNILKEKGIVVGIKLD QGGAPLAGTNKETTIQGLDGLSERCAQYKKDGVDFGKWRAVLRIADQCPSSLAIQ ENANALARYASICQQNGLVPIVEPEVIPDGDHDLEHCQYVTEKVLAAVYKALNDH HVYLEGTLLKPNMVTAGHACTKKYTPEQVAMATVTALHRTVPAAVPGICFLSGGM SEEDATLNLNAINLCPLPKPWKLSFSYGRALQASALAAWGGKAANKEATQEAFMK RAMANCQAAKGQYVHTGSSGAASTOSLFTACYTY SEQ ID NO : 59 Caulobacter crescentus D - xylose 1 - dehydrogenase xylB NT sequence ATGTCCTCAGCCATCTATCCCAGCCTGAAGGGCAAGCGCGTCGTCATCACCGGCG GCGGCTCGGGCATCGGGGCCGGCCTCACCGCCGGCTTCGCCCGTCAGGGCGCGGA GGTGATCTTCCTCGACATCGCCGACGAGGACTCCAGGGCTCTTGAGGCCGAGCTG GCCGGCTCGCCGATCCCGCCGGTCTACAAGCGCTGCGACCTGATGAACCTCGAGG CGATCAAGGCGGTCTTCGCCGAGATCGGCGACGTCGACGTGCTGGTCAACAACGC CGGCAATGACGACCGCCACAAGCTGGCCGACGTGACCGGCGCCTATTGGGACGAG CGGATCAACGTCAACCTGCGCCACATGCTGTTCTGCACCCAGGCCGTCGCGCCGG GCATGAAGAAGCGTGGCGGCGGGGCGGTGATCAACTTCGGTTCGATCAGCTGGCA CCTGGGGCTTGAGGACCTCGTCCTCTACGAAACCGCCAAGGCCGGCATCGAAGGC ATGACCCGCGCGCTGGCCCGGGAGCTGGGTCCCGACGACATCCGCGTCACCTGCG TGGTGCCGGGCAACGTCAAGACCAAGCGCCAGGAGAAGTGGTACACGCCCGAAGG CGAGGCCCAGATCGTGGCGGCCCAATGCCTGAAGGGCCGCATCGTCCCGGAGAAC GTCGCCGCGCTGGTGCTGTTCCTGGCCTCGGATGACGCGTCGCTCTGCACCGGCC ACGAATACTGGATCGACGCCGGCTGGCGTTGA SEQ ID NO : 60 Caulobacter crescentus D - xylose 1 - dehydrogenase xylB codon optimized NT sequence ATGAGCAGCGCGATCTACCCGAGCCTGAAAGGTAAACGTGTGGTGATTACCGGCG GCGGCAGCGGCATTGGTGCGGGCCTGACCGCGGGCTTCGCGCGTCAGGGTGCGGA AGTGATCTTTCTGGACATTGCGGACGAAGATAGCCGTGCGCTGGAGGCGGAACTG GCGGGCAGCCCGATCCCGCCGGTGTACAAGCGTTGCGATCTGATGAACCTGGAGG CGATCAAAGCGGTTTTCGCGGAAATTGGCGACGTGGATGTTCTGGTGAACAACGC GGGTAACGACGACCGTCACAAGCTGGCGGATGTGACCGGTGCGTATTGGGATGAG CGTATTAACGTTAACCTGCGTCACATGCTGTTCTGCACCCAGGCGGTGGCGCCGG GTATGAAGAAACGTGGTGGCGGTGCGGTTATCAACTTTGGCAGCATTAGCTGGCA CCTGGGTCTGGAGGACCTGGTGCTGTACGAAACCGCGAAAGCGGGCATCGAGGGT ATGACCCGTGCGCTGGCGCGTGAACTGGGTCCGGACGATATTCGTGTGACCTGCG TGGTTCCGGGTAACGTTAAGACCAAACGTCAAGAGAAGTGGTATACCCCGGAGGG US 10,941,424 B2 131 132 - continued SEQUENCE LISTING TGAAGCGCAGATTGTTGCGGCGCAATGCCTGAAAGGTCGTATTGTTCCGGAAAAC GTGGCGGCGCTGGTTCTGTTTCTGGCGAGCGATGATGCGAGCCTGTGCACCGGCC ATGAGTATTGGATTGATGCGGGCTGGCGTTAA SEQ ID NO : 61 Caulobacter crescentus D - xylose 1 - dehydrogenase xylB AA sequence MSSAIYPSLKGKRVVITGGGSGIGAGLTAGFARQGAEVIFLDIADEDSRALEAEL AGSPIPPVYKRCDLMNLEAIKAVFAEIGDVDVLVNNAGNDDRHKLADVTGAYWDE RINVNLRHMLFCTQAVAPGMKKRGGGAVINFGSISWHLGLEDLVLYETAKAGIEG MTRALARELGPDDIRVTCVVPGNVKTKRQEKWYTPEGEAQIVAAQCLKGRIVPEN VAALVLFLASDDASLCTGHEYWIDAGWR SEQ ID NO : 62 Haloferax volcanii D - xylose 1 - dehydrogenase xdhi , HVO B0028 NT sequence ATGAGCCCCGCCCCCACCGACATCGTCGAGGAGTTCACGCGCCGCGACTGGCAGG GAGACGACGTGACGGGCACCGTGCGGGTCGCCATGATCGGCCTCGGCTGGTGGAC CCGCGACGAGGCGATTCCCGCGGTCGAGGCGTCCGAGTTCTGCGAGACGACGGTC GTCGTCAGCAGTTCGAAGGAGAAAGCCGAGGGCGCGACGGCGTTGACCGAGTCGA TAACCCACGGCCTCACCTACGACGAGTTCCACGAGGGGGTCGCCGCCGACGCCTA CGACGCGGTGTACGTCGTCACGCCGAACGGTCTGCATCTCCCGTACGTCGAGACC GCCGCCGAGTTGGGGAAGGCGGTCCTCTGCGAGAAACCGCTGGAAGCGTCGGTCG AGCGGGCCGAAAAGCTCGTCGCCGCCTGCGACCGCGCCGACGTGCCCCTGATGGT CGCCTATCGGATGCAGACCGAGCCGGCCGTCCGGCGCGCCCGCGAACTCGTCGAG GCCGGCGTCATCGGCGAGCCGGTGTTCGTCCACGGCCACATGTCCCAGCGCCTGC TCGACGAGGTCGTCCCCGACCCCGACCAGTGGCGGCTCGACCCCGAACTCTCCGG CGGCGCGACCGTCATGGACATCGGGCTCTACCCGCTGAACACCGCCCGGTTCGTC CTCGACGCCGACCCCGTCCGCGTCAGGGCGACCGCCCGCGTCGACGACGAGGCGT TCGAGGCCGTCGGCGACGAGCACGTCAGTTTCGGCGTCGACTTCGACGACGGCAC GCTCGCGGTCTGCACCGCCAGCCAGTCGGCTTACCAGTTGAGCCACCTCCGGGTG ACCGGCACCGAGGGCGAACTCGAAATCGAGCCCGCGTTCTACAACCGCCAAAAGC GGGGATTCCGACTGTCGTGGGGGGACCAGTCCGCCGACTACGACTTCGAGCAGGT AAACCAGATGACGGAGGAGTTCGACTACTTCGCGTCCCGGCTCCTGTCGGATTCC GACCCCGCGCCCGACGGCGACCACGCGCTCGTGGACATGCGCGCGATGGACGCGA TTTACGCCGCGGCGGAGCGCGGGACCGATGTCGCCGTCGACGCCGCCGACTCCGA TTCCGCCGACTCCGATTCCGCCGACGCTGCCGCCGCCAACCACGACGCCGACCCC GATTCCGACGGGACGTAG SEO ID NO : 63 Haloferax volcanii D - xylose 1 - dehydrogenase xdhi , HVO B0028 AA sequence MSPAPTDIVEEFTRRDWQGDDVTGTVRVAMIGLGWWTRDEAIPAVEASEFCETTV VVSSSKEKAEGATALTESITHGL TYDEFHEGVAADAYDAVYVVTPNGLHLPYVET AAELGKAVLCEKPLEASVERAEKLVAACDRADVPLMVAYRMQTEPAVRRARELVE AGVIGEPVFVHGHMSQRLLDEVVPDPDQWRLDPELSGGATVMDIGLYPLNTARFV LDADPVRVRATARVDDEAFEAVGDEHVSFGVDFDDGTLAVCTASQSAYQLSHLRV TGTEGELEIEPAFYNROKRGFRLSWGDQSADYDFEQVNQMTEEFDYFASRLLSDS DPAPDGDHALVDMRAMDAIYAAAERGTDVAVDAADSDSADSDSADAAAANHDADP DSDGT SEQ ID NO : 64 Trichoderma reesei D - xylose 1 - dehydrogenase xydi NT sequence ATGGCGTCTGGAAACCCTTACACCCTGAAATGGGGCATCATGGCCACCGGCGGAA TCGCAGAGACCTTCTGCAAGGATCTCCTGTGCAACCCCGCGATTCGAGGCGCCGA TGATGTGCGCCACGAGATTGTGGCCGTGGCCTCTTCCAGCAGCAGCAAGAGAGCA GAGGAGTTCCTCCAGAGAATCGACGGTGCCTTTGACGCCAAGACGTACGGATCAT ACCCGGAACTTGTGGCAGACCCCAACGTCGACATCGTCTATGTGGCAACTCCCCA CAGCCACCACTTCCAGAACACCATGCTGGCGCTGGAAGCCGGCAAGAACGTCTTG TGCGAAAAGGCTTTCACCGTGACGGCCGCGCAGGCCCGAAAGCTGGTTGAGACGG CCAAGGCCAAGAAGCTCTTCCTGATGGAAGCTGTGTGGACACGGTACTTTCCGCT GAGTATCAAGATTCGAGAGCTCATTGCCGCCGGCGAGATTGGCACTGTCTTTCGA ACAATCGCCGACTTGTCCATCAACGCAAACTCAGAGCAGGGTCAAGCCCTGAAAT TCGCAGACTCACATCGAATGGTCAACCCGGACCTCGCAGGCGGTGCCACCTTGGA TCTCGGAGTCTATCCCTTGACCTGGGTGTTCCAGACCCTGTATCATTTGCAACCG GAGGAAGACAAGGAGGCTCCCACCGTGGTTGCTTCCAGCAACAAGTACACCACTG GCGCAGACGAGAATACCGCCATCATCTGCAGCTTCCCTCGCCACAACAGCATTGG AATTGCTTCGACGACGATGAGGGCGGACACCGACCCCGAGAAGGACACCATTCCG GCGGTCCGAATTCAAGGATCCAAGGGAGAAATCCAAGTCTTCTTCCCGACCTACC GACCGCTCAAGTACAAGGTGGTGAAGACGAACGGCGAGGCGCAACGGTTGACTG CCCCATCCCCGGAGACCCCGCGCGCAAGGGCTCGGGCCACGGAATGTTCTGGGAG GCGGACGAGTGTGCTCGATGCCTTCGCGATGGCAAGTTGGAGAGTGCCACGTTGC CATGGAAGGAGAGCATTGTCATTATGGAAACGATGGAGGAGGCGCTGAGGCAGGG TGGCGTCACGTATCCGGAGCTGATTACCACGGATGTCTATGATCCCAAGAGCCCT CTCAACACGGGGAATCAGTAG SEO ID NO : 65 Trichoderma reesei D - xylose 1 - dehydrogenase xydi AA sequence MASGNPYTLKWGIMATGGIAETFCKDLLCNPAIRGADDVRHEIVAVASSSSSKRA EEFLORIDGAFDAKTYGSYPELVADPNVDIVYVATPHSHHFQNTMLALEAGKNVL US 10,941,424 B2 133 134 - continued SEQUENCE LISTING CEKAFTVTAAQARKLVETAKAKKLFLMEAVWTRYFPLSIKIRELIAAGEIGTVFR TIADLSINANSEQGQALKFADSHRMVNPDLAGGATLDLGVYPLTWVFQTLYHLQP EEDKEAPTVVASSNKYTTGADENTAIICSFPRHNSIGIASTTMRADTDPEKDTIP AVRIQGSKGEIQVFFPTYRPLKYKVVKTNGEAQTVDCPIPGDPARKGSGHGMFWE ADECARCLRDGKLESATLPWKESIVIMETMEEALRQGGVTYPELITTDVYDPKSP LNTGNQ SEQ ID NO : 66 Caulobacter crescentus Xylonolactonase xyic NT sequence ATGACCGCTCAAGTCACTTGCGTATGGGATCTGAAGGCCACGTTGGGCGAAGGCC CGATCTGGCATGGCGACACCCTGTGGTTCGTCGACATCAAGCAGCGTAAAATCCA CAACTACCACCCCGCCACCGGCGAGCGCTTCAGCTTCGACGCGCCGGAT CAGGTG ACCTTCCTCGCGCCGATCGTCGGCGCGACCGGCTTTGTCGTCGGTCTGAAGACCG GGATTCACCGCTTCCACCCGGCCACGGGCTTCAGCCTGCTGCTCGAGGTCGAGGA CGCGGCGCTGAACAACCGCCCCAACGACGCCACGGTCGACGCGCAAGGCCGTCTG TGGTTCGGCACCATGCACGACGGGGAAGAGAACAATAGCGGCTCGCTCTATCGGA TGGACCTCACCGGCGTCGCCCGGATGGACCGCGACATCTGCATCACCAACGGCCC GTGCGTCTCGCCCGACGGCAAGACCTTCTACCACACCGACACCCTGGAAAAGACG ATCTACGCCTTCGACCTGGCCGAGGACGGCCTGCTGTCGAACAAGCGCGTCTTCG TGCAGTTCGCCCTGGGCGACGATGTCTATCCGGACGGTTCGGTCGTCGATTCCGA AGGCTATCTGTGGACCGCCCTGTGGGGCGGTTTCGGCGCGGTCCGCTTCTCGCCG CAAGGCGACGCCGTGACGCGCATCGAACTGCCCGCCCCCAACGTCACCAAGCCCT GCTTCGGCGGGCCTGACCTGAAGACCCTCTATTTCACCACCGCCCGCAAGGGCCT GAGCGACGAGACCCTGGCCCAGTACCCGCTGGCCGGCGGTGTGTTCGCCGTTCCG GTCGATGTGGCCGGCCAACCCCAGCATGAGGTCCGCCTTGTCTAA SEQ ID NO : 67 Caulobacter crescentus Xylonolactonase xylC AA sequence MTAQVTCVWDLKATLGEGPIWHGDTLWFVDI KORKIHNYHPATGERFSFDAPDOV TFLAPIVGATGFWVGLKTGIHRFHPATGFSLLLEVEDAALNNRPNDATVDAQGRL WFGTMHDGEENNSGSLYRMDL TGVARMDRDICITNGPCVSPDGKTFYHTDTLEKT IYAFDLAEDGLLSNKRVFVQFALGDDVYPDGSVVDSEGYLWTALWGGFGAVRFSP QGDAVTRIELPAPNVTKPCFGGPDLKTLYFTTARKGLSDETLAQYPLAGGVFAVP VDVAGQPQHEVRLV SEQ ID NO : 68 Caulobacter crescentus xylonate dehydratase xyld NT sequence TTGTCTAACCGCACGCCCCGCCGGTTCCGGTCCCGCGATTGGTTCGATAACCCCG ACCATATCGACATGACCGCGCTCTATCTGGAGCGCTTCATGAACTACGGGATCAC GCCGGAGGAGCTGCGCAGCGGCAAGCCGATCATCGGCATCGCCCAGACCGGCAGC GACATCTCGCCCTGCAACCGCATCCACCTGGACCTGGTCCAGCGGGTGCGGGACG GGATCCGCGACGCCGGGGGCATCCCCATGGAGTTCCCGGTCCATCCGATCTTCGA GAACTGCCGTCGCCCGACGGCGGCGCTGGACCGGAACCTCTCGTACCTGGGTCTC GTCGAGACCCTGCACGGCTATCCGATCGACGCCGTGGTTCTGACCACCGGCTGCG ACAAGACCACCCCGGCCGGGATCATGGCCGCCACCACGGTCAATATCCCGGCCAT CGTGCTGTCGGGCGGCCCGATGCTGGACGGCTGGCACGAGAACGAGCTCGTGGGC TCGGGCACCGTGATCTGGCGCTCGCGCCGCAAGCTGGCGGCCGGCGAGATCACCG AGGAAGAGTTCATCGACCGCGCCGCCAGCTCGGCGCCGTCGGCGGGCCACTGCAA CACCATGGGCACGGCCTCGACCATGAACGCCGTGGCCGAGGCGCTGGGCCTGTCG CTGACCGGCTGCGCGGCCATCCCCGCCCCCTACCGCGAGCGCGGCCAGATGGCCT ACAAGACCGGCCAGCGCATCGTCGATCTGGCCTATGACGACGTCAAACCGCTCGA CATCCTGACCAAGCAAGCCTTCGAGAACGCCATCGCCCTGGTGGCGGCGGCCGGC GGCTCGACCAACGCCCAGCCGCACATCGTGGCCATGGCCCGTCACGCCGGCGTCG AGATCACCGCCGACGACTGGCGCGCGGCCTATGACATCCCGCTGATCGTCAACAT GCAGCCGGCCGGCAAGTATCTGGGCGAGCGCTTCCACCGAGCCGGCGGCGCGCCG GCGGTGCTGTGGGAGCTGTTGCAGCAAGGCCGCCTGCACGGCGACGTGCTGACCG TCACCGGCAAGACGATGAGCGAGAACCTGCAAGGCCGCGAAACCAGCGACCGCGA GGTGATCTTCCCGTACCACGAGCCGCTGGCCGAGAAGGCCGGGTTCCTGGTTCTC AAGGGCAACCTCTTCGACTTCGCGATCATGAAGTCCAGCGTGATCGGCGAGGAGT TCCGCAAGCGCTACCTGTCGCAGCCCGGCCAGGAAGGCGTGTTCGAAGCCCGCGC CATCGTGTTCGACGGCTCGGACGACTATCACAAGCGGATCAACGATCCGGCCCTG GAGATCGACGAGCGCTGCATCCTGGTGATCCGCGGCGCGGGTCCGATCGGCTGGC CCGGCTCGGCCGAGGTCGTCAACATGCAGCCGCCGGATCACCTTCTGAAGAAGGG GATCATGAGCCTGCCCACCCTGGGCGATGGCCGTCAGTCGGGCACCGCCGACAGC CCCTCGATCCTGAACGCCTCGCCCGAAAGCGCGATCGGCGGCGGCCTGTCGTGGC TGCGCACCGGCGACACCATCCGCATCGACCTCAACACCGGCCGCTGCGACGCCCT GGTCGACGAGGCGACGATCGCCGCGCGCAAGCAGGACGGCATCCCGGCGGTTCCC GCCACCATGACGCCCTGGCAGGAAATCTACCGCGCCCACGCCAGTCAGCTCGACA CCGGCGGCGTGCTGGAGTTCGCGGTCAAGTACCAGGACCTGGCGGCCAAGCTGCC CCGCCACAACCACTGA SEO ID NO : 69 Caulobacter crescentus xylonate dehydratase xylD AA sequence MSNRTPRRFRSRDWFDNPDHIDMTALYLERFMNYGITPEELRSGKPIIGIAQTGS DISPCNRIHLDLVORVRDGIRDAGGIPMEFPVHPIFENCRRPTAALDRNLSYLGL VETLHGYPIDAVVLTTGCDKTTPAGIMAATTVNIPAIVLSGGPMLDGWHENELVG SGTVIWRSRRKLAAGEITEEEFIDRAASSAPSAGHCNTMGTASTMNAVAEALGLS LTGCAAIPAPYRERGQMAYKTGQRIVDLAYDDVKPLDILTKQAFENAIALVAAAG GSTNAQPHIVAMARHAGVEITADDWRAAYDIPLIVNMQPAGKYLGERFHRAGGAP US 10,941,424 B2 135 136 - continued SEQUENCE LISTING AVLWELLQQGRLHGDVLTVTGKTMS ENLQGRETSDREVIFPYHEPLAEKAGFLVL KGNLFDFAIMKSSVIGEEFRKRYLSQPGQEGVFEARAIVFDGSDDYHKRINDPAL EIDERCILVIRGAGPIGWPGSAEVVNMQPPDHLLKKGIMSLPTLGDGRQSGTADS PSILNASPESAIGGGLSWLRTGDTIRIDLNTGRCDALVDEATIAARKQDGIPAVP ATMTPWQEIYRAHASQLDTGGVLEFAVKYQDLAAKLPRHNH SEQ ID NO : 70 Escherichia coli xylonate dehydratase yjhG NT sequence ATGTCTGTTCGCAATATTTTTGCTGACGAGAGCCACGATATTTACACCGTCAGAA CGCACGCCGATGGCCCGGACGGCGAACTCCCATTAACCGCAGAGATGCTTATCAA CCGCCCGAGCGGGGATCTGTTCGGTATGACCATGAATGCCGGAATGGGTTGGTCT CCGGACGAGCTGGATCGGGACGGTATTTTACTGCTCAGTACACTCGGTGGCTTAC GCGGCGCAGACGGTAAACCCGTGGCGCTGGCGTTGCACCAGGGGCATTACGAACT GGACATCCAGATGAAAGCGGCGGCCGAGGTTATTAAAGCCAACCATGCCCTGCCC TATGCCGTGTACGTCTCCGATCCTTGTGACGGGCGTACTCAGGGTACAACGGGGA TGTTTGATTCGCTACCATACCGAAATGACGCATCGATGGTAATGCGCCGCCTTAT TCGCTCTCTGCCCGACGCGAAAGCAGTTATTGGTGTGGCGAGTTGCGATAAGGGG CTTCCGGCCACCATGATGGCACTCGCCGCGCAGCACAACATCGCAACCGTGCTGG TCCCCGGCGGCGCGACGCTGCCCGCAAAGGATGGAGAAGACAACGGCAAGGTGCA AACCATTGGCGCACGCTTCGCCAATGGCGAATTATCTCTACAGGACGCACGCCGT GCGGGCTGTAAAGCCTGTGCCTCTTCCGGCGGCGGCTGTCAATTTTTGGGCACTG CCGGGACATCTCAGGTGGTGGCCGAAGGATTGGGACTGGCAATCCCACATTCAGC CCTGGCCCCTTCCGGTGAGCCTGTGTGGCGGGAGATCGCCAGAGCTTCCGCGCGA GCTGCGCTGAACCTGAGTCAAAAAGGCATCACCACCCGGGAAATTCTCACCGATA AAGCGATAGAGAATGCGATGACGGTCCATGCCGCGTTCGGTGGTTCAACAAACCT GCTGTTACACATCCCGGCAATTGCTCACCAGGCAGGTTGCCATATCCCGACCGTT GATGACTGGATCCGCATCAACAAGCGCGTGCCCCGACTGGTGAGCGTACTGCCTA ATGGCCCGGTTTATCATCCAACGGTCAATGCCTTTATGGCAGGTGGTGTGCCGGA AGTCATGTTGCATCTGCGCAGCCTCGGATTGTTGCATGAAGACGTTATGACGGTT ACCGGCAGCACGCTGAAAGAAAACCTCGACTGGTGGGAGCACTCCGAACGGCGTC AGCGGTTCAAGCAACTCCTGCTCGATCAGGAACAAATCAACGCTGACGAAGTGAT CATGTCTCCGCAGCAAGCAAAAGCGCGCGGATTAACCTCAACTATCACCTTCCCG GTGGGCAATATTGCGCCAGAAGGTTCGGTGATCAAATCCACCGCCATTGACCCCT CGATGATTGATGAGCAAGGTATCTATTACCATAAAGGTGTGGCGAAGGTTTATCT GTCCGAGAAAAGTGCGATTTACGATATCAAACATGACAAGATCAAGGCGGGCGAT ATTCTGGTCATTATTGGCGTTGGACCTTCAGGTACAGGGATGGAAGAAACCTACC AGGTTACCAGTGCCCTGAAGCATCTGTCATACGGTAAGCATGTTTCGTTAATCAC CGATGCACGTTTCTCGGGCGTTTCTACTGGCGCGTGCATCGGCCATGTGGGGCCA GAAGCGCTGGCCGGAGGCCCCATCGGTAAATTACGCACCGGGGATTTAATTGAAA TTAAAATTGATTGTCGCGAGCTTCACGGCGAAGTCAATTTCCTCGGAACCCGTAG CGATGAACAATTACCTTCACAGGAGGAGGCAACTGCAATATTAAATGCCAGACCC AGCCATCAGGATTTACTTCCCGATCCTGAATTGCCAGATGATACCCGGCTATGGG CAATGCTTCAGGCCGTGAGTGGTGGGACATGGACCGGTTGTATTTATGATGTAAA CAAAATTGGCGCGGCTTTGCGCGATTTTATGAATAAAAACTGA SEQ ID NO : 71 Escherichia coli xylonate dehydratase yjhg codon optimized NT sequence ATGTCTGTTCGCAATATTTTTGCTGACGAGAGCCACGATATTTACACCGTCAGAA CGCACGCCGATGGCCCGGACGGCGAACTCCCATTAACCGCAGAGATGCTTATCAA CCGCCCGAGCGGGGATCTGTTCGGTATGACCATGAATGCCGGAATGGGTTGGTCT CCGGACGAGCTGGATCGGGACGGTATTTTACTGCTCAGTACACTCGGTGGCTTAC GCGGCGCAGACGGTAAACCCGTGGCGCTGGCGTTGCACCAGGGGCATTACGAACT GGACATCCAGATGAAAGCGGCGGCCGAGGTTATTAAAGCCAACCATGCCCTGCCC TATGCCGTGTACGTCTCCGATCCTTGTGACGGGCGTACTCAGGGTACAACGGGGA TGTTTGATTCGCTACCATACCGAAATGACGCATCGATGGTAATGCGCCGCCTTAT TCGCTCTCTGCCCGACGCGAAAGCAGTTATTGGTGTGGCGAGTTGCGATAAGGGG CTTCCGGCCACCATGATGGCACTCGCCGCGCAGCACAACATCGCAACCGTGCTGG TCCCCGGCGGCGCGACGCTGCCCGCAAAGGATGGAGAAGACAACGGCAAGGTGCA AACCATTGGCGCACGCTTCGCCAATGGCGAATTATCTCTACAGGACGCACGCCGT GCGGGCTGTAAAGCCTGTGCCTCTTCCGGCGGCGGCTGTCAATTTTTGGGCACTG CCGGGACATCTCAGGTGGTGGCCGAAGGATTGGGACTGGCAATCCCACATTCAGC CCTGGCCCCTTCCGGTGAGCCTGTGTGGCGGGAGATCGCCAGAGCTTCCGCGCGA GCTGCGCTGAACCTGAGTCAAAAAGGCATCACCACCCGGGAAATTCTCACCGATA AAGCGATAGAGAATGCGATGACGGTCCATGCCGCGTTCGGTGGTTCAACAAACCT GCTGTTACACATCCCGGCAATTGCTCACCAGGCAGGTTGCCATATCCCGACCGTT GATGACTGGATCCGCATCAACAAGCGCGTGCCCCGACTGGTGAGCGTACTGCCTA ATGGCCCGGTTTATCATCCAACGGTCAATGCCTTTATGGCAGGTGGTGTGCCGGA AGTCATGTTGCATCTGCGCAGCCTCGGATTGTTGCATGAAGACGTTATGACGGTT ACCGGCAGCACGCTGAAAGAAAACCTCGACTGGTGGGAGCACTCCGAACGGCGTC AGCGGTTCAAGCAACTCCTGCTCGATCAGGAACAAATCAACGCTGACGAAGTGAT CATGTCTCCGCAGCAAGCAAAAGCGCGCGGATTAACCTCAACTATCACCTTCCCG GTGGGCAATATTGCGCCAGAAGGTTCGGTGATCAAATCCACCGCCATTGACCCCT CGATGATTGATGAGCAAGGTATCTATTACCATAAAGGTGTGGCGAAGGTTTATCT GTCCGAGAAAAGTGCGATTTACGATATCAAACATGACAAGATCAAGGCGGGCGAT ATTCTGGTCATTATTGGCGTTGGACCTTCAGGTACAGGGATGGAAGAAACCTACC AGGTTACCAGTGCCCTGAAGCATCTGTCATACGGTAAGCATGTTTCGTTAATCAC CGATGCACGTTTCTCGGGCGTTTCTACTGGCGCGTGCATCGGCCATGTGGGGCCA GAAGCGCTGGCCGGAGGCCCCATCGGTAAATTACGCACCGGGGATTTAATTGAAA US 10,941,424 B2 137 138 - continued SEQUENCE LISTING TTAAAATTGATTGTCGCGAGCTTCACGGCGAAGTCAATTTCCTCGGAACCCGTAG CGATGAACAATTACCTTCACAGGAGGAGGCAACTGCAATATTAAATGCCAGACCC AGCCATCAGGATTTACTTCCCGATCCTGAATTGCCAGATGATACCCGGCTATGGG CAATGCTTCAGGCCGTGAGTGGTGGGACATGGACCGGTTGTATTTATGATGTAAA CAAAATTGGCGCGGCTTTGCGCGATTTTATGAATAAAAACTGA SEQ ID NO : 72 Escherichia coli xylonate dehydratase yjhG AA sequence MSVRNIFADESHDIYTVRTHADGPDGELPLTAEMLINRPSGDLFGMTMNAGMGWS PDELDRDGILLLSTLGGLRGADGKPVALALHQGHYELDIQMKAAAEVIKANHALP YAVYVSDPCDGRTQGTTGMFDSLPYRNDASMVMRRLIRSLPDAKAVIGVASCDKG LPATMMALAAQHNIATVLVPGGATLPAKDGEDNGKVOTIGARFANGELSLQDARR AGCKACASSGGGCOFLGTAGTSQVVAEGLGLAIPHSALAPSGEPVWREIARASAR AALNLSQKGITTREILTDKAI ENAMTVHAAFGGSTNLLLHIPAIAHQAGCHIPTV DDWIRINKRVPRLVSVLPNGPVYHP TVNAFMAGGVPEVMLHLRSLGLLHEDVMTV TGSTLKENLDWWEHSERRORFKQLLLDQEQINADEVIMSPQQAKARGLTSTITFP VGNIAPEGSVIKSTAIDPSMIDEQGIYYHKGVAKVYLSEKSAIYDIKHDKI KAGD ILVIIGVGPSGTGMEETYQVTSALKHLSYGKHVSLITDARFSGVSTGACIGHVGP EALAGGPIGKLRTGDLIEIKIDCRELHGEVNFLGTRSDEQLPSQEEATAILNARP SHQDLLPDPELPDDTRLWAMLQAVSGGTWTGCIYDVNKIGAALRDFMNKN SEQ ID NO : 73 Escherichia coli xylonate dehydratase yagF NT sequence ATGACCATTGAGAAAATTTTCACCCCGCAGGACGACGCGTTTTATGCGGTGATCA CCCACGCGGCGGGGCCGCAGGGCGCTCTGCCGCTGACCCCGCAGATGCTGATGGA ATCTCCCAGCGGCAACCTGTTCGGCATGACGCAGAACGCCGGGATGGGCTGGGAC GCCAACAAGCTCACCGGCAAAGAGGTGCTGATTATCGGCACTCAGGGCGGCATCC GCGCCGGAGACGGACGCCCAATCGCGCTGGGCTACCACACCGGGCATTGGGAGAT CGGCATGCAGATGCAGGCGGCGGCGAAGGAGATCACCCGCAATGGCGGGATCCCG TTCGCGGCCTTCGTCAGCGATCCGTGCGACGGGCGCTCGCAGGGCACGCACGGTA TGTTCGATTCCCTGCCGTACCGCAACGACGCGGCGATCGTGTTTCGCCGCCTGAT CCGCTCCCTGCCGACGCGGCGGGCGGTGATCGGCGTAGCGACCTGCGATAAAGGG CTGCCCGCCACCATGATTGCGCTGGCCGCGATGCACGACCTGCCGACTATTCTGG TGCCGGGCGGGGCGACGCTGCCGCCGACCGTCGGGGAAGACGCGGGCAAGGTGCA GACCATCGGCGCGCGTTTCGCCAACCACGAACTCTCCCTGCAGGAGGCCGCCGAA CTGGGCTGTCGCGCCTGCGCCTCGCCGGGCGGCGGGTGTCAGTTCCTCGGCACGG CGGGCACCTCGCAGGTGGTCGCGGAGGCGCTGGGTCTGGCGCTGCCGCACTCCGC GCTGGCGCCGTCCGGGCAGGCGGTGTGGCTGGAGATCGCCCGCCAGTCGGCGCGC GCGGTCAGCGAGCTGGATAGCCGCGGCATCACCACGCGGGATATCCTCTCCGATA AAGCCATCGAAAACGCGATGGTGATCCACGCGGCGTTCGGCGGCTCCACCAATTT ACTGCTGCACATTCCGGCCATCGCCCACGCGGCGGGCTGCACGATCCCGGACGTT GAGCACTGGACGCGCATCAACCGTAAAGTGCCGCGTCTGGTGAGCGTGCTGCCCA ACGGCCCGGACTATCACCCGACCGTGCGCGCCTTCCTCGCGGGCGGCGTGCCGGA GGTGATGCTCCACCTGCGCGACCTCGGCCTGCTGCATCTGGACGCCATGACCGTG ACCGGCCAGACGGTGGGCGAGAACCTTGAATGGTGGCAGGCGTCCGAGCGCCGGG CGCGCTTCCGCCAGTGCCTGCGCGAGCAGGACGGCGTAGAGCCGGATGACGTGAT CCTGCCGCCGGAGAAGGCAAAAGCGAAAGGGCTGACCTCGACGGTCTGCTTCCCG ACGGGCAACATCGCTCCGGAAGGTTCGGTGATCAAGGCCACGGCGATCGACCCGT CGGTGGTGGGCGAAGATGGCGTATACCACCACACCGGCCGGGTGCGGGTGTTTGT CTCGGAAGCGCAGGCGATCAAGGCGATCAAGCGGGAAGAGATTGTGCAGGGCGAT ATCATGGTGGTGATCGGCGGCGGGCCGTCCGGCACCGGCATGGAAGAGACCTACC AGCTCACCTCCGCGCTAAAGCATATCTCGTGGGGCAAGACGGTGTCGCTCATCAC CGATGCGCGCTTCTCGGGCGTGTCGACGGGCGCCTGCTTCGGCCACGTGTCGCCG GAGGCGCTGGCGGGCGGGCCGATTGGCAAGCTGCGCGATAACGACATCATCGAGA TTGCCGTGGATCGTCTGACGTTAACTGGCAGCGTGAACTTCATCGGCACCGCGGA CAACCCGCTGACGCCGGAAGAGGGCGCGCGCGAGCTGGCGCGGCGGCAGACGCAC CCGGACCTGCACGCCCACGACTTTTTGCCGGACGACACCCGGCTGTGGGCGGCAC TGCAGTCGGTGAGCGGCGGCACCTGGAAAGGCTGTATTTATGACACCGATAAAAT TATCGAGGTAATTAACGCCGGTAAAAAAGCGCTCGGAATTTAA SEQ ID NO : 74 Escherichia coli xylonate dehydratase yagF codon optimized NT sequence ATGACCATTGAGAAAATTTTCACCCCGCAGGACGACGCGTTTTATGCGGTGATCA CCCACGCGGCGGGGCCGCAGGGCGCTCTGCCGCTGACCCCGCAGATGCTGATGGA ATCTCCCAGCGGCAACCTGTTCGGCATGACGCAGAACGCCGGGATGGGCTGGGAC GCCAACAAGCTCACCGGCAAAGAGGTGCTGATTATCGGCACTCAGGGCGGCATCC GCGCCGGAGACGGACGCCCAATCGCGCTGGGCTACCACACCGGGCATTGGGAGAT CGGCATGCAGATGCAGGCGGCGGCGAAGGAGATCACCCGCAATGGCGGGATCCCG TTCGCGGCCTTCGTCAGCGATCCGTGCGACGGGCGCTCGCAGGGCACGCACGGTA TGTTCGATTCCCTGCCGTACCGCAACGACGCGGCGATCGTGTTTCGCCGCCTGAT CCGCTCCCTGCCGACGCGGCGGGCGGTGATCGGCGTAGCGACCTGCGATAAAGGG CTGCCCGCCACCATGATTGCGCTGGCCGCGATGCACGACCTGCCGACTATTCTGG TGCCGGGCGGGGCGACGCTGCCGCCGACCGTCGGGGAAGACGCGGGCAAGGTGCA GACCATCGGCGCGCGTTTCGCCAACCACGAACTCTCCCTGCAGGAGGCCGCCGAA CTGGGCTGTCGCGCCTGCGCCTCGCCGGGCGGCGGGTGTCAGTTCCTCGGCACGG CGGGCACCTCGCAGGTGGTCGCGGAGGCGCTGGGTCTGGCGCTGCCGCACTCCGC GCTGGCGCCGTCCGGGCAGGCGGTGTGGCTGGAGATCGCCCGCCAGTCGGCGCGC GCGGTCAGCGAGCTGGATAGCCGCGGCATCACCACGCGGGATATCCTCTCCGATA AAGCCATCGAAAACGCGATGGTGATCCACGCGGCGTTCGGCGGCTCCACCAATTT US 10,941,424 B2 139 140 - continued SEQUENCE LISTING ACTGCTGCACATTCCGGCCATCGCCCACGCGGCGGGCTGCACGATCCCGGACGTT GAGCACTGGACGCGCATCAACCGTAAAGTGCCGCGTCTGGTGAGCGTGCTGCCCA ACGGCCCGGACTATCACCCGACCGTGCGCGCCTTCCTCGCGGGCGGCGTGCCGGA GGTGATGCTCCACCTGCGCGACCTCGGCCTGCTGCATCTGGACGCCATGACCGTG ACCGGCCAGACGGTGGGCGAGAACCTTGAATGGTGGCAGGCGTCCGAGCGCCGGG CGCGCTTCCGCCAGTGCCTGCGCGAGCAGGACGGCGTAGAGCCGGATGACGTGAT CCTGCCGCCGGAGAAGGCAAAAGCGAAAGGGCTGACCTCGACGGTCTGCTTCCCG ACGGGCAACATCGCTCCGGAAGGTTCGGTGATCAAGGCCACGGCGATCGACCCGT CGGTGGTGGGCGAAGATGGCGTATACCACCACACCGGCCGGGTGCGGGTGTTTGT CTCGGAAGCGCAGGCGATCAAGGCGATCAAGCGGGAAGAGATTGTGCAGGGCGAT ATCATGGTGGTGATCGGCGGCGGGCCGTCCGGCACCGGCATGGAAGAGACCTACC AGCTCACCTCCGCGCTAAAGCATATCTCGTGGGGCAAGACGGTGTCGCTCATCAC CGATGCGCGCTTCTCGGGCGTGTCGACGGGCGCCTGCTTCGGCCACGTGTCGCCG GAGGCGCTGGCGGGCGGGCCGATTGGCAAGCTGCGCGATAACGACATCATCGAGA TTGCCGTGGATCGTCTGACGTTAACTGGCAGCGTGAACTTCATCGGCACCGCGGA CAACCCGCTGACGCCGGAAGAGGGCGCGCGCGAGCTGGCGCGGCGGCAGACGCAC CCGGACCTGCACGCCCACGACTTTTTGCCGGACGACACCCGGCTGTGGGCGGCAC TGCAGTCGGTGAGCGGCGGCACCTGGAAAGGCTGTATTTATGACACCGATAAAAT TATCGAGGTAATTAACGCCGGTAAAAAAGCGCTCGGAATTTAA SEQ ID NO : 75 Escherichia coli xylonate dehydratase yagF AA sequence MTIEKIFTPODDAFYAVI THAAGPQGALPLTPQMLMESPSGNLFGMTQNAGMGWD ANKLTGKEVLIIGTQGGIRAGDGRPIALGYHTGHWEIGMQMQAAAKEITRNGGIP FAAFVSDPCDGRSQGTHGMFDSLPYRNDAAIVFRRLIRSLPTRRAVIGVATCDKG LPATMIALAAMHDLPTILVPGGATLPPTVGEDAGKVQTIGARFANHELSLQEAAE LGCRACASPGGGCOFLGTAGTSQVVAEALGLALPHSALAPSGQAVWLEIARQSAR AVSELDSRGITTRDILSDKAI ENAMVIHAAFGGSTNLLLHIPAIAHAAGCTIPDV EHWTRINRKVPRLVSVLPNGPDYHPTVRAFLAGGVPEVMLHLRDLGLLHLDAMTV TGQTVGENLEWWQASERRARFRQCLREQDGVEPDDVILPPEKAKAKGLTSTVCFP TGNIAPEGSVIKATAIDPSVVGEDGVYHHTGRVRVFVSEAQAIKAIKREEIVQGD IMVVIGGGPSGTGMEETYQLTSALKHI SWGKTVSLITDARFSGVSTGACFGHVSP EALAGGPIGKLRDNDIIEIAVDRLTLTGSVNFIGTADNPLTPEEGARELARROTH PDLHAHDFLPDDTRLWAALQSVSGGTWKGCIYDTDKIIEVINAGKKALGI SEQ ID NO : 76 Escherichia coli Uncharacterized lyase yjhH NT sequence ATGAAAAAATTCAGCGGCATTATTCCACCGGTATCCAGCACGTTTCATCGTGACG GAACCCTTGATAAAAAGGCAATGCGCGAAGTTGCCGACTTCCTGATTAATAAAGG GGTCGACGGGCTGTTTTATCTGGGTACCGGTGGTGAATTTAGCCAAATGAATACA GCCCAGCGCATGGCACTCGCCGAAGAAGCTGTAACCATTGTCGACGGGCGAGTGC CGGTATTGATTGGCGTCGGTTCCCCTTCCACTGACGAAGCGGTCAAACTGGCGCA GCATGCGCAAGCCTACGGCGCTGATGGTATCGTCGCCATCAACCCCTACTACTGG AAAGTCGCACCACGAAATCTTGACGACTATTACCAGCAGATCGCCCGTAGCGTCA CCCTACCGGTGATCCTGTACAACTTTCCGGATCTGACGGGTCAGGACTTAACCCC GGAAACCGTGACGCGTCTGGCTCTGCAAAACGAGAATATCGTTGGCATCAAAGAC ACCATCGACAGCGTTGGTCACTTGCGTACGATGATCAACACAGTTAAGTCGGTAC GCCCGTCGTTTTCGGTATTCTGCGGTTACGATGATCATTTGCTGAATACGATGCT GCTGGGCGGCGACGGTGCGATAACCGCCAGCGCTAACTTTGCTCCGGAACTCTCC GTCGGCATCTACCGCGCCTGGCGTGAAGGCGATCTGGCGACCGCTGCGACGCTGA ATAAAAAACTACTACAACTGCCCGCTATTTACGCCCTCGAAACACCGTTTGTCTC ACTGATCAAATACAGCATGCAGTGTGTCGGGCTGCCTGTAGAGACATATTGCTTA CCACCGATTCTTGAAGCATCTGAAGAAGCAAAAGATAAAGTCCACGTGCTGCTTA CCGCGCAGGGCATTTTACCAGTCTGA SEQ ID NO : 77 Escherichia coli Uncharacterized lyase yjhH codon optimized NT sequence ATGAAAAAATTCAGCGGCATTATTCCACCGGTATCCAGCACGTTTCATCGTGACG GAACCCTTGATAAAAAGGCAATGCGCGAAGTTGCCGACTTCCTGATTAATAAAGG GGTCGACGGGCTGTTTTATCTGGGTACCGGTGGTGAATTTAGCCAAATGAATACA GCCCAGCGCATGGCACTCGCCGAAGAAGCTGTAACCATTGTCGACGGGCGAGTGC CGGTATTGATTGGCGTCGGTTCCCCTTCCACTGACGAAGCGGTCAAACTGGCGCA GCATGCGCAAGCCTACGGCGCTGATGGTATCGTCGCCATCAACCCCTACTACTGG AAAGTCGCACCACGAAATCTTGACGACTATTACCAGCAGATCGCCCGTAGCGTCA CCCTACCGGTGATCCTGTACAACTTTCCGGATCTGACGGGTCAGGACTTAACCCC GGAAACCGTGACGCGTCTGGCTCTGCAAAACGAGAATATCGTTGGCATCAAAGAC ACCATCGACAGCGTTGGTCACTTGCGTACGATGATCAACACAGTTAAGTCGGTAC GCCCGTCGTTTTCGGTATTCTGCGGTTACGATGATCATTTGCTGAATACGATGCT GCTGGGCGGCGACGGTGCGATAACCGCCAGCGCTAACTTTGCTCCGGAACTCTCC GTCGGCATCTACCGCGCCTGGCGTGAAGGCGATCTGGCGACCGCTGCGACGCTGA ATAAAAAACTACTACAACTGCCCGCTATTTACGCCCTCGAAACACCGTTTGTCTC ACTGATCAAATACAGCATGCAGTGTGTCGGGCTGCCTGTAGAGACATATTGCTTA CCACCGATTCTTGAAGCATCTGAAGAAGCAAAAGATAAAGTCCACGTGCTGCTTA CCGCGCAGGGCATTTTACCAGTCTGA SEO ID NO : 78 Escherichia coli Uncharacterized lyase yjhH AA sequence MKKFSGIIPPVSSTFHRDGTLDKKAMREVADFLINKGVDGLFYLGTGGEFSQMNT AQRMALAEEAVTIVDGRVPVLIGVGSPSTDEAVKLAQHAQAYGADGIVAINPYYW KVAPRNLDDYYQQIARSVTLPVILYNFPDLTGODLTPETVTRLALONENIVGIKD US 10,941,424 B2 141 142 - continued SEQUENCE LISTING TIDSVGHLRTMINTVKSVRPSFSVFCGYDDHLLNTMLLGGDGAITASANFAPELS VGI YRAWREGDLATAATLNKKLLQLPAIYALETPFVSLIKYSMQCVGLPVETYCL PPILEASEEAKDKVHVLLTAQGILPV SEQ ID NO : 79 Escherichia coli Probable 2 - keto - 3 - deoxy - galactonate aldolase yage NT sequence ATGCCGCAGTCCGCGTTGTTCACGGGAATCATTCCCCCTGTCTCCACCATTTTTA CCGCCGACGGCCAGCTCGATAAGCCGGGCACCGCCGCGCTGATCGACGATCTGAT CAAAGCAGGCGTTGACGGCCTGTTCTTCCTGGGCAGCGGTGGCGAGTTCTCCCAG CTCGGCGCCGAAGAGCGTAAAGCCATTGCCCGCTTTGCTATCGATCATGTCGATC GTCGCGTGCCGGTGCTGATCGGCACCGGCGGCACCAACGCCCGGGAAACCATCGA ACTCAGCCAGCACGCGCAGCAGGCGGGCGCGGACGGCATCGTGGTGATCAACCCC TACTACTGGAAAGTGTCGGAAGCGAACCTGATCCGCTATTTCGAGCAGGTGGCCG ACAGCGTCACGCTGCCGGTGATGCTCTATAACTTCCCGGCGCTGACCGGGCAGGA TCTGACTCCGGCGCTGGTGAAAACCCTCGCCGACTCGCGCAGCAATATTATCGGC ATCAAAGACACCATCGACTCCGTCGCCCACCTGCGCAGCATGATCCATACCGTCA AAGGTGCCCATCCGCACTTCACCGTGCTCTGCGGCTACGACGATCATCTGTTCAA TACCCTGCTGCTCGGCGGCGACGGGGCGATATCGGCGAGCGGCAACTTTGCCCCG CAGGTGTCGGTGAATCTTCTGAAAGCCTGGCGCGACGGGGACGTGGCGAAAGCGG CCGGGTATCATCAGACCTTGCTGCAAATTCCGCAGATGTATCAGCTGGATACGCC GTTTGTGAACGTGATTAAAGAGGCGATCGTGCTCTGCGGTCGTCCTGTCTCCACG CACGTGCTGCCGCCCGCCTCGCCGCTGGACGAGCCGCGCAAGGCGCAGCTGAAAA CCCTGCTGCAACAGCTCAAGCTTTGCTGA SEO ID NO : 80 Escherichia coli Probable 2 - keto - 3 - deoxy - galactonate aldolase yage codon optimized NT sequence ATGCCGCAGTCCGCGTTGTTCACGGGAATCATTCCCCCTGTCTCCACCATTTTTA CCGCCGACGGCCAGCTCGATAAGCCGGGCACCGCCGCGCTGATCGACGATC TGAT CAAAGCAGGCGTTGACGGCCTGTTCTTCCTGGGCAGCGGTGGCGAGTTCTCCCAG CTCGGCGCCGAAGAGCGTAAAGCCATTGCCCGCTTTGCTATCGATCATGTCGATC GTCGCGTGCCGGTGCTGATCGGCACCGGCGGCACCAACGCCCGGGAAACCATCGA ACTCAGCCAGCACGCGCAGCAGGCGGGCGCGGACGGCATCGTGGTGATCAACCCC TACTACTGGAAAGTGTCGGAAGCGAACCTGATCCGCTATTTCGAGCAGGTGGCCG ACAGCGTCACGCTGCCGGTGATGCTCTATAACTTCCCGGCGCTGACCGGGCAGGA TCTGACTCCGGCGCTGGTGAAAACCCTCGCCGACTCGCGCAGCAATATTATCGGC ATCAAAGACACCATCGACTCCGTCGCCCACCTGCGCAGCATGATCCATACCGTCA AAGGTGCCCATCCGCACTTCACCGTGCTCTGCGGCTACGACGATCATCTGTTCAA TACCCTGCTGCTCGGCGGCGACGGGGCGATATCGGCGAGCGGCAACTTTGCCCCG CAGGTGTCGGTGAATCTTCTGAAAGCCTGGCGCGACGGGGACGTGGCGAAAGCGG CCGGGTATCATCAGACCTTGCTGCAAATTCCGCAGATGTATCAGCTGGATACGCC GTTTGTGAACGTGATTAAAGAGGCGATCGTGCTCTGCGGTCGTCCTGTCTCCACG CACGTGCTGCCGCCCGCCTCGCCGCTGGACGAGCCGCGCAAGGCGCAGCTGAAAA CCCTGCTGCAACAGCTCAAGCTTTGCTGA

SEQ ID NO : 81 Escherichia coli Probable 2 - keto - 3 - deoxy - galactonate aldolase yagE AA sequence MPQSALFTGIIPPVSTIFTADGQLDKPGTAALIDDLIKAGVDGLFFLGSGGEFSQ LGAEERKAIARFAIDHVDRRVPVLIGTGGTNARETIELSQHAQQAGADGIVVINP YYWKVSEANLIRYFEQVADSVTLPVMLYNFPALTGQDLTPALVKTLADSRSNIIG IKDTIDSVAHLRSMIHTV KGAHPHFTVLCGYDDHLFNTLLLGGDGAISASGNFAP QVSVNLLKAWRDGDVAKAAGYHQTLLQIPOMYOLDTPFVNVIKEAIVLCGRPVST HVLPPASPLDEPRKAQLKTLLQQLKLC SEQ ID NO : 82 Scheffersomyces stipitis D - xylose reductase xyli NT sequence ATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTCGGTTTCGGCT GTTGGAAAGTCGACGTCGACACCTGTTCTGAACAGATCTACCGTGCTATCAAGAC CGGTTACAGATTGTTCGACGGTGCCGAAGATTACGCCAACGAAAAGTTAGTTGGT GCCGGTGTCAAGAAGGCCATTGACGAAGGTATCGTCAAGCGTGAAGATTTGTTCC TTACCTCCAAGTTGTGGAACAACTACCACCACCCAGACAACGTCGAAAAGGCCTT GAACAGAACCCTTTCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCAC TTCCCAGTCACCTTCAAGTTCGTTCCATTAGAAGAAAAGTACCCACCAGGATTCT ACTGTGGTAAGGGTGACAACTTCGACTACGAAGATGTTCCAATTTTAGAGACTTG GAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGATCTATCGGTGTTTCT AACTTCCCAGGTGCTTTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCAT CTGTCTTGCAAGTTGAACACCACCCATACTTGCAACAACCAAGATTGATCGAATT CGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCGTTCGGTCCTCAATCT TTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTCCATTGTTCGAGAACG AAACTATCAAGGCTATCGCTGCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTGTT GAGATGGTCATCCCAAAGAGGCATTGCCATCATTCCAAAGTCCAACACTGTCCCA AGATTGTTGGAAAACAAGGACGTCAACAGCTTCGACTTGGACGAACAAGATTTCG CTGACATTGCCAAGTTGGACATCAACTTGAGATTCAACGACCCATGGGACTGGGA CAAGATTCCTATCTTCGTCTAA US 10,941,424 B2 143 144 - continued SEQUENCE LISTING SEQ ID NO : 83 Scheffersomyces stipitis D - xylose reductase xyli codon optimized NT sequence ATGCCATCTATCAAGTTAAATTCCGGTTACGACATGCCTGCTGTTGGTTTCGGTT GCTGGAAGGTTGATGTCGATACTTGTTCCGAGCAAATTTACCGTGCTATCAAGAC TGGTTACAGATTGTTCGATGGTGCTGAAGACTACGCCAACGAAAAGTTAGTCGGT GCTGGTGTTAAAAAGGCTATCGACGAAGGTATTGTTAAAAGAGAAGACTTGTTCT TGACTTCTAAGTTGTGGAACAACTACCACCATCCTGATAACGTCGAAAAAGCTTT GAACCGTACCTTGTCCGATTTGCAAGTCGATTACGTTGATTTGTTCTTGATTCAT TTCCCAGTTACCTTCAAGTTCGTTCCATTGGAAGAGAAGTATCCACCAGGTTTCT ACTGTGGTAAGGGTGATAACTTCGATTACGAAGATGTCCCAATCTTAGAAACCTG GAAGGCTTTAGAAAAGTTGGTTAAGGCTGGTAAGATCAGATCCATCGGTGTTTCT AACTTCCCAGGTGCCTTATTGTTAGACTTATTGAGAGGTGCTACCATTAAGCCTT CCGTTTTGCAAGTTGAACATCATCCTTACTTGCAACAACCAAGATTGATCGAATT CGCTCAATCTAGAGGTATCGCTGTTACTGCCTACTCTTCCTTCGGTCCACAATCT TTCGTTGAGTTGAACCAAGGTAGAGCTTTGAACACCTCTCCATTGTTCGAAAACG AAACTATTAAGGCCATTGCTGCTAAGCATGGTAAGTCTCCAGCCCAAGTTTTGTT GAGATGGTCTTCTCAAAGAGGTATCGCTATTATCCCAAAGTCTAATACTGTCCCA AGATTGTTGGAAAACAAGGACGTTAACTCCTTTGATTTGGATGAACAAGACTTTG CTGACATCGCTAAATTGGACATCAACTTGAGATTCAACGACCCATGGGACTGGGA CAAGATTCCAATTTTTGTTTAA SEQ ID NO : 84 Scheffersomyces stipitis D - xylose reductase xyli AA sequence MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQI YRAIKTGYRLFDGAEDYANEKLVG AGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQVDYVDLFLIH FPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKALEKLVKAGKIRSIGVS NFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLIEFAQSRGIAVTAYSSFGPQS FVELNQGRALNTSPLFENETIKAIAAKHGKSPAQVLLRWSSQRGIAIIPKSNTVP RLLENKDVNSFDLDEQDFADIAKLDINLRFNDPWDWDKIPIFV SEQ ID NO : 85 Saccharomyces cerevisiae aldose reductase GRE3 NT sequence ATGTCTTCACTGGTTACTCTTAATAACGGTCTGAAAATGCCCCTAGTCGGCTTAG GGTGCTGGAAAATTGACAAAAAAGTCTGTGCGAATCAAATTTATGAAGCTATCAA ATTAGGCTACCGTTTATTCGATGGTGCTTGCGACTACGGCAACGAAAAGGAAGTT GGTGAAGGTATCAGGAAAGCCATCTCCGAAGGTCTTGTTTCTAGAAAGGATATAT TTGTTGTTTCAAAGTTATGGAACAATTTTCACCATCCTGATCATGTAAAATTAGC TTTAAAGAAGACCTTAAGCGATATGGGACTTGATTATTTAGACCTGTATTATATT CACTTCCCAATCGCCTTCAAATATGTTCCATTTGAAGAGAAATACCCTCCAGGAT TCTATACGGGCGCAGATGACGAGAAGAAAGGTCACATCACCGAAGCACATGTACC AATCATAGATACGTACCGGGCTCTGGAAGAATGTGTTGATGAAGGCTTGATTAAG TCTATTGGTGTTTCCAACTTTCAGGGAAGCTTGATTCAAGATTTATTACGTGGTT GTAGAATCAAGCCCGTGGCTTTGCAAATTGAACACCATCCTTATTTGACTCAAGA ACACCTAGTTGAGTTTTGTAAATTACACGATATCCAAGTAGTTGCTTACTCCTCC TTCGGTCCTCAATCATTCATTGAGATGGACTTACAGTTGGCAAAAACCACGCCAA CTCTGTTCGAGAATGATGTAATCAAGAAGGTCTCACAAAACCATCCAGGCAGTAC CACTTCCCAAGTATTGCTTAGATGGGCAACTCAGAGAGGCATTGCCGTCATTCCA AAATCTTCCAAGAAGGAAAGGTTACTTGGCAACCTAGAAATCGAAAAAAAGTTCA CTTTAACGGAGCAAGAATTGAAGGATATTTCTGCACTAAATGCCAACAT CAGATT TAATGATCCATGGACCTGGTTGGATGGTAAATTCCCCACTTTTGCCTGA SEQ ID NO : 86 Saccharomyces cerevisiae aldose reductase GRE3 codon optimized NT sequence ATGTCTTCACTGGTTACTCTTAATAACGGTCTGAAAATGCCCCTAGTCGGCTTAG GGTGCTGGAAAATTGACAAAAAAGTCTGTGCGAATCAAATTTATGAAGCTATCAA ATTAGGCTACCGTTTATTCGATGGTGCTTGCGACTACGGCAACGAAAAGGAAGTT GGTGAAGGTATCAGGAAAGCCATCTCCGAAGGTCTTGTTTCTAGAAAGGATATAT TTGTTGTTTCAAAGTTATGGAACAATTTTCACCATCCTGATCATGTAAAATTAGC TTTAAAGAAGACCTTAAGCGATATGGGACTTGATTATTTAGACCTGTATTATATT CACTTCCCAATCGCCTTCAAATATGTTCCATTTGAAGAGAAATACCCTCCAGGAT TCTATACGGGCGCAGATGACGAGAAGAAAGGTCACATCACCGAAGCACATGTACC AATCATAGATACGTACCGGGCTCTGGAAGAATGTGTTGATGAAGGCTTGATTAAG TCTATTGGTGTTTCCAACTTTCAGGGAAGCTTGATTCAAGATTTATTACGTGGTT GTAGAATCAAGCCCGTGGCTTTGCAAATTGAACACCATCCTTATTTGACTCAAGA ACACCTAGTTGAGTTTTGTAAATTACACGATATCCAAGTAGTTGCTTACTCCTCC TTCGGTCCTCAATCATTCATTGAGATGGACTTACAGTTGGCAAAAACCACGCCAA CTCTGTTCGAGAATGATGTAATCAAGAAGGTCTCACAAAACCATCCAGGCAGTAC CACTTCCCAAGTATTGCTTAGATGGGCAACTCAGAGAGGCATTGCCGTCATTCCA AAATCTTCCAAGAAGGAAAGGTTACTTGGCAACCTAGAAATCGAAAAAAAGTTCA CTTTAACGGAGCAAGAATTGAAGGATATTTCTGCACTAAATGCCAACATCAGATT TAATGATCCATGGACCTGGTTGGATGGTAAATTCCCCACTTTTGCCTGA SEQ ID NO : 87 Saccharomyces cerevisiae aldose reductase GRE3 AA sequence MSSLVTLNNGLKMPLVGLGCWKIDKKVCANQIYEAIKLGYRLFDGACDYGNEKEV GEGIRKAISEGLVSRKDIFWSKLWNNFHHPDHVKLALKKTLSDMGLDYLDLYYI HFPIAFKYVPFEEKYPPGFYTGADDEKKGHITEAHVPIIDTYRALEECVDEGLIK US 10,941,424 B2 145 146 - continued SEQUENCE LISTING SIGVSNFQGSLIQDLLRGCRI KPVALQIEHHPYLTQEHLVEFCKLHDIQVVAYSS FGPQSFIEMDLQLAKTTPTLFENDVIKKVSQNHPGSTTSQVLLRWATQRGIAVIP KSSKKERLLGNLEIEKKFTLTEQELKDISALNANIRFNDPWTWLDGKFPTFA SEQ ID NO : 88 Scheffersomyces stipitis D - xylulose reductase xy12 NT sequence ATGACTGCTAACCCTTCCTTGGTGTTGAACAAGATCGACGACATTTCGTTCGAAA CTTACGATGCCCCAGAAATCTCTGAACCTACCGATGTCCTCGTCCAGGTCAAGAA AACCGGTATCTGTGGTTCCGACATCCACTTCTACGCCCATGGTAGAATCGGTAAC TTCGTTTTGACCAAGCCAATGGTCTTGGGTCACGAATCCGCCGGTACTGTTGTCC AGGTTGGTAAGGGTGTCACCTCTCTTAAGGTTGGTGACAACGTCGCTATCGAACC AGGTATTCCATCCAGATTCTCCGACGAATACAAGAGCGGTCACTACAACTTGTGT CCTCACATGGCCTTCGCCGCTACTCCTAACTCCAAGGAAGGCGAACCAAACCCAC CAGGTACCTTATGTAAGTACTTCAAGTCGCCAGAAGACTTCTTGGTCAAGTTGCC AGACCACGTCAGCTTGGAACTCGGTGCTCTTGTTGAGCCATTGTCTGTTGGTGTC CACGCCTCTAAGTTGGGTTCCGTTGCTTTCGGCGACTACGTTGCCGTCTTTGGTG CTGGTCCTGTTGGTCTTTTGGCTGCTGCTGTCGCCAAGACCTTCGGTGCTAAGGG TGTCATCGTCGTTGACATTTTCGACAACAAGTTGAAGATGGCCAAGGACATTGGT GCTGCTACTCACACCTTCAACTCCAAGACCGGTGGTTCTGAAGAATTGATCAAGG CTTTCGGTGGTAACGTGCCAAACGTCGTTTTGGAATGTACTGGTGCTGAACCTTG TATCAAGTTGGGTGTTGACGCCATTGCCCCAGGTGGTCGTTTCGTTCAAGT CGGT AACGCTGCTGGTCCAGTCAGCTTCCCAATCACCGTTTTCGCCATGAAGGAATTGA CTTTGTTCGGTTCTTTCAGATACGGATTCAACGACTACAAGACTGCTGTTGGAAT CTTTGACACTAACTACCAAAACGGTAGAGAAAATGCTCCAATTGACTTTGAACAA TTGATCACCCACAGATACAAGTTCAAGGACGCTATTGAAGCCTACGACTTGGTCA GAGCCGGTAAGGGTGCTGTCAAGTGTCTCATTGACGGCCCTGAGTAA SEQ ID NO : 89 Scheffersomyces stipitis D - xylulose reductase xy12 codon optimized NT sequence ATGACTGCTAACCCTTCCTTGGTGTTGAACAAGATCGACGACATTTCGTTCGAAA CTTACGATGCCCCAGAAATCTCTGAACCTACCGATGTCCTCGTCCAGGTCAAGAA AACCGGTATCTGTGGTTCCGACATCCACTTCTACGCCCATGGTAGAATCGGTAAC TTCGTTTTGACCAAGCCAATGGTCTTGGGTCACGAATCCGCCGGTACTGTTGTCC AGGTTGGTAAGGGTGTCACCTCTCTTAAGGTTGGTGACAACGTCGCTATCGAACC AGGTATTCCATCCAGATTCTCCGACGAATACAAGAGCGGTCACTACAACTTGTGT CCTCACATGGCCTTCGCCGCTACTCCTAACTCCAAGGAAGGCGAACCAAACCCAC CAGGTACCTTATGTAAGTACTTCAAGTCGCCAGAAGATTTCTTGGTCAAGTTGCC AGACCACGTCAGCTTGGAACTCGGTGCTCTTGTTGAGCCATTGTCTGTTGGTGTC CACGCCTCTAAGTTGGGTTCCGTTGCTTTCGGCGACTACGTTGCCGTCTTTGGAG CAGGTCCTGTTGGTCTTTTGGCTGCTGCTGTCGCCAAGACCTTCGGTGCTAAGGG TGTCATCGTCGTTGACATTTTCGACAACAAGTTGAAGATGGCCAAGGACATTGGA GCTGCTACTCACACCTTCAACTCCAAGACCGGTGGTTCTGAAGAATTGATCAAGG CTTTCGGTGGTAACGTGCCAAACGTCGTTTTGGAATGTACAGGTGCAGAACCTTG TATCAAGTTGGGTGTTGACGCCATTGCCCCAGGTGGTCGTTTCGTTCAAGTCGGT AACGCTGCTGGTCCAGTCAGCTTCCCAATCACCGTTTTCGCCATGAAGGAATTGA CTTTGTTCGGTTCTTTCAGATACGGATTCAACGACTACAAGACTGCTGTTGGAAT CTTTGACACTAACTACCAAAACGGTAGAGAAAATGCTCCAATTGACTTTGAACAA TTGATCACCCACAGATACAAGTTCAAGGACGCTATTGAAGCCTACGACTTGGTCA GAGCCGGTAAGGGTGCTGTCAAGTGTCTCATTGACGGCCCTGAGTAA SEQ ID NO : 90 Scheffersomyces stipitis D - xylulose reductase xy12 AA sequence MTANPSLVLNKIDDISFETYDAPEISEPTDVLVQVKKTGICGSDIHFYAHGRIGN FVLTKPMVLGHESAGTVQVGKGVTSLKVGDNVAIEPGIPSRFSDEYKSGHYNLC PHMAFAATPNSKEGEPNPPGTLCKYFKSPEDFLVKLPDHVSLELGALVEPLSVGV HASKLGSVAFGDYVAVFGAGPVGLLAAAVAKTFGAKGVIVVDIFDNKLKMAKDIG AATHTFNSKTGGSEELIKAFGGNVPNVVLECTGAEPCIKLGVDAIAPGGRFVQVG NAAGPVSFPITVFAMKELTLFGSFRYGFNDYKTAVGIFDTNYONGRENAPIDFEQ LITHRYKFKDAIEAYDLVRAGKGAVKCLIDGPE SEQ ID NO : 91 Trichoderma reesei Xylitol dehydrogenase xdh1 NT sequence ATGGCGACTCAAACGATCAACAAGGATGCGATCAGCAACCTCTCCTTCGTCCTCA ACAAGCCCGGCGACGTGACCTTTGAGGAGCGGCCGAAGCCGACCATCACGGACCC CAACGACGTCCTCGTCGCCGTCAACTACACGGGCATCTGCGGCTCCGACGTGCAC TACTGGGTGCACGGCGCCATCGGGCACTTCGTCGTCAAGGACCCGATGGTGCTGG GCCACGAGTCGGCCGGCACCGTCGTCGAGGTCGGCCCGGCCGTCAAGAGCCTCAA GCCCGGCGACCGCGTCGCCCTCGAGCCCGGCTACCCGTGCCGGCGGTGCTCCTTC TGCCGCGCCGGCAAATACAACCTGTGCCCGGACATGGTCTTCGCCGCCACGCCGC CGTACCACGGCACCCTGACGGGCCTGTGGGCGGCGCCCGCCGACTTCTGCTACAA GCTGCCGGACGGCGTGTCGCTGCAGGAGGGCGCGCTGATCGAGCCGCTGGCCGTG GCCGTCCACATTGTCAAGCAGGCCCGCGTCCAGCCGGGCCAGTCCGTCGTCGTCA TGGGCGCCGGCCCCGTCGGCCTGCTGTGCGCCGCCGTGGCCAAGGCGTACGGCGC CTCCACCATTGTCAGCGTCGACATCGTGCAGTCCAAGCTCGACTTTGCGCGCGGC TTCTGCTCGACGCACACGTACGTCTCGCAGCGCATCTCGGCTGAGGACAACGCAA AGGCCATCAAGGAGCTGGCGGGCCTGCCCGGCGGCGCCGACGTCGTGATTGACGC CAGCGGCGCGGAGCCGTCGATCCAGACGAGCATTCACGTCGTCCGCATGGGCGGC US 10,941,424 B2 147 148 - continued SEQUENCE LISTING ACGTACGTCCAGGGCGGCATGGGCAAGAGCGACATCACGTTCCCCATCATGGCCA TGTGCCTCAAGGAGGTGACGGTCCGGGGCTCGTTCCGCTACGGCGCCGGCGACTA CGAGCTGGCGGTCGAGCTGGTCCGGACGGGGCGGGTGGACGTCAAGAAGCTGATT ACGGGCACCGTCAGCTTCAAGCAGGCGGAGGAGGCGTTCCAAAAGGTCAAGTCTG GGGAGGCCATCAAGATTCTGATTGCCGGGCCCAACGAGAAGGTGTAA SEQ ID NO : 92 Trichoderma reesei Xylitol dehydrogenase xdh1 AA sequence MATOTINKDAISNLSFVLNKPGDVTFEERPKPTI TDPNDVLVAVNYTGICGSDVH YWVHGAIGHFVVKDPMVLGHESAGTWEVGPAVKSLKPGDRVALEPGYPCRRCSF CRAGKYNLCPDMVFAATPPYHGTLTGLWAAPADFCYKLPDGVSLQEGALIEPLAV AVHIVKQARVQPGQSVVVMGAGPVGLLCAAVAKAYGASTIVSVDIVQSKLDFARG FCSTHTYVSORISAEDNAKAI KELAGLPGGADVVIDASGAEPSIQTSIHVVRMGG TYVQGGMGKSDITFPIMAMCLKEVTVRGSFRYGAGDYELAVELVRTGRVDVKKLI TGTVSFKQAEEAFQKVKSGEAIKILIAGPNEKV SEQ ID NO : 93 Pyromyces sp . xylose isomerase xylA NT sequence ATGGCTAAGGAATATTTCCCACAAATTCAAAAGATTAAGTTCGAAGGTAAGGATT CTAAGAATCCATTAGCCTTCCACTACTACGATGCTGAAAAGGAAGTCATGGGTAA GAAAATGAAGGATTGGTTACGTTTCGCCATGGCCTGGTGGCACACTCTTTGCGCC GAAGGTGCTGACCAATTCGGTGGAGGTACAAAGTCTTTCCCATGGAACGAAGGTA CTGATGCTATTGAAATTGCCAAGCAAAAGGTTGATGCTGGTTTCGAAATCATGCA AAAGCTTGGTATTCCATACTACTGTTTCCACGATGTTGATCTTGTTTCCGAAGGT AACTCTATTGAAGAATACGAATCCAACCTTAAGGCTGTCGTTGCTTACCTCAAGG AAAAGCAAAAGGAAACCGGTATTAAGCTTCTCTGGAGTACTGCTAACGTCTTCGG TCACAAGCGTTACATGAACGGTGCCTCCACTAACCCAGACTTTGATGTTGTCGCC CGTGCTATTGTTCAAATTAAGAACGCCATAGACGCCGGTATTGAACTTGGTGCTG AAAACTACGTCTTCTGGGGTGGTCGTGAAGGTTACATGAGTCTCCTTAACACTGA CCAAAAGCGTGAAAAGGAACACATGGCCACTATGCTTACCATGGCTCGTGACTAC GCTCGTTCCAAGGGATTCAAGGGTACTTTCCTCATTGAACCAAAGCCAATGGAAC CAACCAAGCACCAATACGATGTTGACACTGAAACCGCTATTGGTTTCCTTAAGGC CCACAACTTAGACAAGGACTTCAAGGTCAACATTGAAGTTAACCACGCTACTCTT GCTGGTCACACTTTCGAACACGAACTTGCCTGTGCTGTTGATGCTGGTATGCTCG GTTCCATTGATGCTAACCGTGGTGACTACCAAAACGGTTGGGATACTGATCAATT CCCAATTGATCAATACGAACTCGTCCAAGCTTGGATGGAAATCATCCGTGGTGGT GGTTTCGTTACTGGTGGTACCAACTTCGATGCCAAGACTCGTCGTAACTCTACTG ACCTCGAAGACATCATCATTGCCCACGTTTCTGGTATGGATGCTATGGCTCGTGC TCTTGAAAACGCTGCCAAGCTCCTCCAAGAATCTCCATACACCAAGATGAAGAAG GAACGTTACGCTTCCTTCGACAGTGGTATTGGTAAGGACTTTGAAGATGGTAAGC TCACCCTCGAACAAGTTTACGAATACGGTAAGAAGAACGGTGAACCAAAGCAAAC TTCTGGTAAGCAAGAACTCTACGAAGCTATTGTTGCCATGTACCAATAA SEQ ID NO : 94 Pyromyces sp . xylose isomerase xylA codon optimized NT sequence ATGGCCAAGGAATACTTCCCACAAATCCAAAAGATTAAATTCGAAGGTAAAGATT CCAAGAACCCATTGGCTTTTCACTACTACGATGCTGAGAAGGAAGTTATGGGTAA GAAGATGAAGGATTGGTTGAGATTCGCTATGGCTTGGTGGCACACTTTGTGCGCT GAAGGTGCTGACCAATTCGGTGGTGGTACTAAGTCTTTCCCATGGAACGAAGGTA CTGATGCTATTGAAATCGCTAAGCAAAAAGTCGATGCTGGTTTTGAGATTATGCA AAAATTGGGTATCCCATACTACTGTTTCCACGACGTCGACTTGGTTTCTGAAGGT AATTCTATCGAAGAATACGAATCTAATTTGAAGGCTGTTGTCGCTTACTTAAAAG AAAAGCAAAAGGAGACTGGTATTAAGTTGTTGTGGTCCACCGCTAACGTCTTTGG TCATAAAAGATACATGAACGGTGCTTCCACCAACCCAGACTTCGATGTCGTCGCC AGAGCTATCGTTCAAATTAAAAACGCCATCGACGCTGGTATTGAATTGGGTGCTG AAAATTACGTCTTTTGGGGTGGTCGTGAAGGTTACATGTCTTTGTTGAACACTGA CCAAAAGAGAGAAAAAGAACACATGGCCACTATGTTGACCATGGCCAGAGATTAC GCCAGATCTAAGGGTTTCAAGGGTACCTTCTTAATTGAACCAAAACCTATGGAAC CAACTAAGCACCAATACGACGTTGACACTGAAACTGCTATCGGTTTTTTGAAGGC TCACAACTTGGATAAGGATTTTAAAGTCAACATTGAAGTTAACCATGCTACTTTG GCTGGTCACACTTTTGAACATGAATTGGCCTGTGCTGTTGATGCTGGTATGTTGG GTTCTATCGATGCTAATAGAGGTGACTATCAAAACGGTTGGGACACTGATCAATT CCCAATCGATCAATATGAATTAGTTCAAGCTTGGATGGAAATTATCAGAGGTGGT GGTTTCGTTACTGGTGGTACTAACTTCGATGCTAAGACCAGAAGAAACTCTACTG ATTTGGAAGATATTATCATTGCCCACGTTTCCGGTATGGATGCCATGGCCAGAGC TTTGGAAAACGCCGCCAAGTTATTGCAAGAGTCCCCATACACCAAGATGAAAAAG GAACGTTACGCTTCTTTCGACTCTGGTATCGGTAAAGACTTCGAAGATGGTAAGT TGACCTTGGAACAAGTTTACGAATACGGTAAGAAGAACGGTGAACCTAAACAAAC CTCTGGTAAACAAGAATTGTATGAAGCTATTGTTGCCATGTACCAATAA SEQ ID NO : 95 Pyromyces sp . xylose isomerase xyla AA sequence MAKEYFPQIQKIKFEGKDSKNPLAFHYYDAEKEVMGKKMKDWLRFAMAWWHTLCA EGADQFGGGTKSFPWNEGTDAIEIAKQKVDAGFEIMQKLGIPYYCFHDVDLVSEG NSIEEYESNLKAVVAYLKEKQKETGIKLLWSTANVFGHKRYMNGASTNPDFDVVA RAIVQIKNAIDAGIELGAENYVFWGGREGYMSLLNTDQKREKEHMATML TMARDY ARSKGFKGTFLIEPKPMEPTKHQYDVDTETAIGFLKAHNLDKDFKVNIEVNHATL AGHTFEHELACAVDAGMLGSIDANRGDYQNGWDTDQFPIDQYELVQAWMEIIRGG US 10,941,424 B2 149 150 - continued SEQUENCE LISTING GFVTGGTNFDAKTRRNSTDLEDIIIAHVSGMDAMARALENAAKLLQESPYTKMKK ERYAS FDSGIGKDFEDGKLTLEQVYEYGKKNGEPKQTSGKQELYEAIVAMYO SEQ ID NO : 96 Clostridium acetobutylicum butyrate - acetoacetate COA transferase , complex A ctfA NT sequence ATGAACTCTAAAATAATTAGATTTGAAAATTTAAGGTCATTCTTTAAAGATGGGA TGACAATTATGATTGGAGGTTTTTTAAACTGTGGCACTCCAACCAAATTAATTGA TTTTTTAGTTAATTTAAATATAAAGAATTTAACGATTATAAGTAATGATACATGT TATCCTAATACAGGTATTGGTAAGTTAATATCAAATAATCAAGTAAAAAAGCTTA TTGCTTCATATATAGGCAGCAACCCAGATACTGGCAAAAAACTTTTTAATAATGA ACTTGAAGTAGAGCTCTCTCCCCAAGGAACTCTAGTGGAAAGAATACGTGCAGGC GGATCTGGCTTAGGTGGTGTACTAACTAAAACAGGTTTAGGAACTTTGATTGAAA AAGGAAAGAAAAAAATATCTATAAATGGAACGGAATATTTGTTAGAGCTACCTCT TACAGCCGATGTAGCATTAATTAAAGGTAGTATTGTAGATGAGGCCGGAAACACC TTCTATAAAGGTACTACTAAAAACTTTAATCCCTATATGGCAATGGCAGCTAAAA CCGTAATAGTTGAAGCTGAAAATTTAGTTAGCTGTGAAAAACTAGAAAAGGAAAA AGCAATGACCCCCGGAGTTCTTATAAATTATATAGTAAAGGAGCCTGCATAA SEO ID NO : 97 Clostridium acetobutylicum butyrate - acetoacetate COA transferase , complex A ctfA AA sequence MNSKIIRFENLRSFFKDGMTIMIGGFLNCGTPTKLIDFLVNLNIKNLTIISNDTC YPNTGIGKLISNNQVKKLIASYIGSNPDTGKKLFNNELEVELSPQGTLVERIRAG GSGLGGVLTKTGLGTLIEKGKKKISINGTEYLLELPLTADVALI KGSIVDEAGNT FYKGTTKNFNPYMAMAAKTVIVEAENLVSCEKLEKEKAMTPGVLINYIVKEPA SEQ ID NO : 98 Clostridium acetobutylicum butyrate - acetoacetate COA transferase , subunit B ctfB NT sequence ATGATTAATGATAAAAACCTAGCGAAAGAAATAATAGCCAAAAGAGTTGCAAGAG AATTAAAAAATGGTCAACTTGTAAACTTAGGTGTAGGTCTTCCTACCATGGTTGC AGATTATATACCAAAAAATTTCAAAATTACTTTCCAATCAGAAAACGGAATAGTT GGAATGGGCGCTAGTCCTAAAATAAATGAGGCAGATAAAGATGTAGTAAATGCAG GAGGAGACTATACAACAGTACTTCCTGACGGCACATTTTTCGATAGCTCAGTTTC GTTTTCACTAATCCGTGGTGGTCACGTAGATGTTACTGTTTTAGGGGCTCTCCAG GTAGATGAAAAGGGTAATATAGCCAATTGGATTGTTCCTGGAAAAATGCTCTCTG GTATGGGTGGAGCTATGGATTTAGTAAATGGAGCTAAGAAAGTAATAATTGCAAT GAGACATACAAATAAAGGTCAACCTAAAATTTTAAAAAAATGTACACTTCCCCTC ACGGCAAAGTCTCAAGCAAATCTAATTGTAACAGAACTTGGAGTAATTGAGGTTA TTAATGATGGTTTACTTCTCACTGAAATTAATAAAAACACAACCATTGATGAAAT AAGGTCTTTAACTGCTGCAGATTTACTCATATCCAATGAACTTAGACCCATGGCT GTTTAG SEQ ID NO : 99 Clostridium acetobutylicum butyrate - acetoacetate COA transferase , subunit B ctfB AA sequence MINDKNLAKEIIAKRVARELKNGQLVNLGVGLPTMVADYIPKNFKITFQSENGIV GMGASPKINEADKDVVNAGGDYTTVLPDGTFFDSSVSFSLIRGGHVDVTVLGALQ VDEKGNIANWIVPGKMLSGMGGAMDLVNGAKKVIIAMRHTNKGQPKILKKCTLPL TAKSQANLIVTELGVIEVINDGLLLTEINKNTTIDEIRSLTAADLLISNELRPMA V SEO ID NO : 100 Escherichia coli ( strain K12 ) Acetyl - CoA : acetoacetate COA transferase subunit atoA NT sequence ATGGATGCGAAACAACGTATTGCGCGCCGTGTGGCGCAAGAGCTTCGTGATGGTG ACATCGTTAACTTAGGGATCGGTTTACCCACAATGGTCGCCAATTATTTACCGGA GGGTATTCATATCACTCTGCAATCGGAAAACGGCTTCCTCGGTTTAGGCCCGGTC ACGACAGCGCATCCAGATCTGGTGAACGCTGGCGGGCAACCGTGCGGTGTTTTAC CCGGTGCAGCCATGTTTGATAGCGCCATGTCATTTGCGCTAATCCGTGGCGGTCA TATTGATGCCTGCGTGCTCGGCGGTTTGCAAGTAGACGAAGAAGCAAACCTCGCG AACTGGGTAGTGCCTGGGAAAATGGTGCCCGGTATGGGTGGCGCGATGGATCTGG TGACCGGGTCGCGCAAAGTGATCATCGCCATGGAACATTGCGCCAAAGATGGTTC AGCAAAAATTTTGCGCCGCTGCACCATGCCACTCACTGCGCAACATGCGGTGCAT ATGCTGGTTACTGAACTGGCTGTCTTTCGTTTTATTGACGGCAAAATGTGGCTCA CCGAAATTGCCGACGGGTGTGATTTAGCCACCGTGCGTGCCAAAACAGAAGCTCG GTTTGAAGTCGCCGCCGATCTGAATACGCAACGGGGTGATTTATGA SEQ ID NO : 101 Escherichia coli ( strain K12 ) Acetyl - CoA : acetoacetate COA transferase subunit atoA AA sequence MDAKORIARRVAQELRDGDIVNLGIGLPTMVANYLPEGIHITLOSENGFLGLGPV TTAHPDLVNAGGQPCGVLPGAAMFDSAMSFALIRGGHIDACVLGGLQVDEEANLA NWVVPGKMVPGMGGAMDLVTGSRKVIIAMEHCAKDGSAKILRRCTMPLTAQHAVH MLVTELAVFRFIDGKMWLTEIADGCDLATVRAKTEARFEVAADLNTQRGDL SEQ ID NO : 102 Escherichia coli ( strain K12 ) Acetyl - CoA : acetoacetate COA transferase subunit atod NT uence ATGAAAACAAAATTGATGACATTACAAGACGCCACCGGCTTCTTTCGTGACGGCA TGACCATCATGGTGGGCGGATTTATGGGGATTGGCACTCCATCCCGCCTGGTTGA AGCATTACTGGAATCTGGTGTTCGCGACCTGACATTGATAGCCAATGATACCGCG TTTGTTGATACCGGCATCGGTCCGCTCATCGTCAATGGTCGAGTCCGCAAAGTGA US 10,941,424 B2 151 152 - continued SEQUENCE LISTING TTGCTTCACATATCGGCACCAACCCGGAAACAGGTCGGCGCATGATATCTGGTGA GATGGACGTCGTTCTGGTGCCGCAAGGTACGCTAATCGAGCAAATTCGCTGTGGT GGAGCTGGACTTGGTGGTTTTCTCACCCCAACGGGTGTCGGCACCGTCGTAGAGG AAGGCAAACAGACACTGACACTCGACGGTAAAACCTGGCTGCTCGAACGCCCACT GCGCGCCGACCTGGCGCTAATTCGCGCTCATCGTTGCGACACACTTGGCAACCTG ACCTATCAACTTAGCGCCCGCAACTTTAACCCCCTGATAGCCCTTGCGGCTGATA TCACGCTGGTAGAGCCAGATGAACTGGTCGAAACCGGCGAGCTGCAACCTGACCA TATTGTCACCCCTGGTGCCGTTATCGACCACATCATCGTTTCACAGGAGAGCAAA TAA SEQ ID NO : 103 Escherichia coli ( strain K12 ) Acetyl - COA : acetoacetate COA transferase subunit atod AA sequence MKTKLMTLQDATGFFRDGMTIMVGGFMGIGTPSRLVEALLESGVRDLTLIANDTA FVDTGIGPLIVNGRVRKVIASHIGTNPETGRRMISGEMDVVLVPOGTLIEQIRCG GAGLGGFLTPTGVGTVVEEGKQTLTLDGKTWLLERPLRADLALIRAHRCDTLGNL TYQLSARNFNPLIALAADITLVEPDELVETGELQPDHIVTPGAVIDHIIVSQESK SEQ ID NO : 104 Clostridium beijerinckii secondary alcohol dehydrogenase adh NT sequence ATGAAAGGTTTTGCAATGCTAGGTATTAATAAGTTAGGATGGATCGAAAAAGAAA GGCCAGTTGCGGGTTCATATGATGCTATTGTACGCCCATTAGCAGTATCTCCGTG TACATCAGATATACATACTGTTTTTGAGGGAGCTCTTGGAGATAGGAAGAATATG ATTTTAGGGCATGAAGCTGTAGGTGAAGTTGTTGAAGTAGGAAGTGAAGTGAAGG ATTTTAAACCTGGTGACAGAGTTATAGTTCCTTGTACAACTCCAGATTGGAGATC TTTGGAAGTTCAAGCTGGTTTTCAACAGCACTCAAACGGTATGCTCGCAGGATGG AAATTTTCAAATTTCAAGGATGGAGTTTTTGGTGAATATTTTCATGTAAATGATG CGGATATGAATCTTGCGATTCTACCTAAAGACATGCCATTAGAAAATGCTGTTAT GATAACAGATATGATGACTACTGGATTTCATGGAGCAGAACTTGCAGATATTCAA ATGGGTTCAAGTGTTGTGGTAATTGGCATTGGAGCTGTTGGCTTAATGGGAATAG CAGGTGCTAAATTACGTGGAGCAGGTAGAATAATTGGAGTGGGGAGCAGGCCGAT TTGTGTTGAGGCTGCAAAATTTTATGGAGCAACAGATATTCTAAATTATAAAAAT GGTCATATAGTTGATCAAGTTATGAAATTAACGAATGGAAAAGGCGTTGACCGCG TAATTATGGCAGGCGGTGGTTCTGAAACATTATCCCAAGCAGTATCTATGGTTAA ACCAGGAGGAATAATTTCTAATATAAATTATCATGGAAGTGGAGATGCTTTACTA ATACCACGTGTAGAATGGGGATGTGGAATGGCTCACAAGACTATAAAAGGAGGTC TTTGTCCTGGGGGACGTTTGAGAGCAGAAATGTTAAGAGATATGGTAGTATATAA TCGTGTTGATCTAAGTAAATTAGTTACACATGTATATCATGGATTTGATCACATA GAAGAAGCACTGTTATTAATGAAAGACAAGCCAAAAGACTTAATTAAAGCAGTAG TTATATTATAA SEO ID NO : 105 Clostridium beijerinckii secondary alcohol dehydrogenase adh codon optimized NT sequence ATGAAAGGGTTTGCCATGTTAGGTATCAATAAACTGGGCTGGATTGAAAAAGAGC GCCCGGTGGCGGGTTCATACGATGCAATTGTTCGTCCGCTGGCCGTCAGTCCGTG CACCAGCGACATCCATACAGTCTTTGAAGGTGCCCTGGGTGATCGGAAAAACATG ATTCTGGGCCATGAAGCCGTAGGCGAAGTAGTGGAAGTGGGCAGCGAGGTAAAGG ATTTCAAACCGGGTGATCGCGTAATTGTTCCTTGCACGACCCCAGATTGGCGCTC ACTGGAAGTTCAGGCTGGTTTTCAGCAGCATAGTAACGGTATGTTAGCAGGCTGG AAGTTTAGCAATTTTAAAGACGGGGTGTTCGGGGAGTATTTTCATGTCAACGATG CGGACATGAATCTGGCTATTTTACCTAAAGATATGCCGCTGGAGAACGCAGTGAT GATTACCGACATGATGACGACAGGCTTTCACGGTGCAGAACTGGCTGACATCCAA ATGGGCTCCAGTGTGGTGGTTATCGGTATTGGTGCGGTCGGGCTGATGGGTATCG CGGGCGCGAAATTACGGGGCGCTGGTCGCATCATCGGTGTCGGCAGCCGTCCAAT TTGCGTTGAAGCAGCTAAATTCTATGGTGCCACGGACATTCTGAACTATAAAAAT GGTCACATCGTCGATCAGGTGATGAAACTGACCAATGGCAAAGGTGTGGACCGCG TGATCATGGCGGGCGGCGGCTCAGAGACTTTATCTCAAGCGGTGTCTATGGTTAA ACCTGGGGGCATCATTTCTAATATTAACTATCATGGCTCCGGCGACGCATTACTG ATCCCGCGTGTTGAATGGGGCTGTGGGATGGCCCACAAAACCATTAAAGGGGGGT TATGTCCGGGTGGTCGCCTGCGTGCCGAAATGCTGCGTGACATGGTGGTTTACAA CCGTGTGGATCTGTCCAAACTGGTAACTCACGTATACCACGGTTTCGATCACATT GAAGAGGCGCTGCTGCTGATGAAGGATAAGCCAAAGGATCTGATTAAGGCGGTTG TTATCCTGTAA

SEQ ID NO : 106 Clostridium beijerinckii secondary alcohol dehydrogenase adh AA sequence MKGFAMLGINKLGWIEKERPVAGSYDAIVRPLAVSPCTSDIHTVFEGALGDRKNM ILGHEAVGEVVEVGSEVKDFKPGDRVIVPCTTPDWRSLEVQAGFQQHSNGMLAGW KFSNFKDGVFGEYFHVNDADMNLAILPKDMPLENAVMI TDMMTTGFHGAELADIQ MGSSVVVIGIGAVGLMGIAGAKLRGAGRIIGVGSRPICVEAAKFYGATDILNYKN GHIVDQVMKL TNGKGVDRVIMAGGGSETLSQAVSMVKPGGIISNINYHGSGDALL IPRVEWGCGMAHKTIKGGLCPGGRLRAEMLRDMVVYNRVDLSKLVTHVYHGFDHI EEALLLMKDKPKDLIKAVVIL SEO ID NO : 107 Clostridium carboxidivorans alcohol dehydrogenase adh NT sequence ATGAAGGTAACTAATGTTGAAGAACTGATGAAAAAAATGCAGGAAGTGCAAAATG CTCAAAAAAAATTTGGGAGTTTTACTCAGGAACAAGTAGATGAAATTTTCAGGCA US 10,941,424 B2 153 154 - continued SEQUENCE LISTING AGCAGCACTAGCAGCTAACAGTGCCAGAATAGATCTAGCTAAAATGGCAGTGGAA GAAACTAAAATGGGAATTGTAGAGGATAAGGTTATAAAAAATCATTTTGTTGCAG AATACATATATAATAAGTATAAAAATGAAAAAACTTGTGGGATTTTGGAAGAAGA TGAAGGCTTTGGAATGGTTAAAATTGCAGAACCTGTAGGTGTGATTGCAGCAGTA ATTCCAACAACAAATCCAACATCTACAGCAATATTTAAAGCATTATTAGCTTTGA AAACAAGAAATGGTATAATTTTTTCACCACATCCAAGAGCAAAAAAGTGTACTAT TGCAGCAGCTAAGTTAGTTCTTGATGCTGCAGTTAAAGCAGGTGCTCCTAAAGGA ATTATAGGTTGGATAGATGAACCTTCTATTGAACTTTCACAGATAGTAATGAAAG AAGCTGATATAATCCTTGCAACAGGTGGTCCAGGTATGGTTAAAGCAGCTTATTC TTCAGGTAAACCTGCTATAGGGGTTGGTCCTGGTAACACACCTGCTTTAATTGAT GAAAGTGCTGATATTAAAATGGCAGTAAATTCAATACTTCTTTCCAAAACTTTTG ATAATGGTATGATTTGTGCTTCAGAGCAGTCGGTAGTAGTTGTAGATTCAATATA TGAAGAAGTTAAGAAAGAATTTGCTCATAGAGGAGCTTATATTTTAAGTAAGGAT GAAACAACTAAAGTTGGAAAAATACTCTTAGTTAATGGTACATTAAATGCTGGTA TCGTTGGTCAGAGTGCTTATAAAATAGCAGAAATGGCAGGAGTTAAAGTTCCAGA AGATGCTAAAGTTCTTATAGGAGAAGTAAAATCAGTGGAGCATTCAGAAGAGCCA TTTTCACATGAAAAGTTATCTCCAGTTTTAGCTATGTATAGAGCTAAAAATTTTG ATGAAGCTCTTTTAAAAGCTGGAAGATTAGTTGAACTCGGTGGAATGGGTCATAC ATCTGTATTATATGTAAATGCAATAACTGAAAAAGTAAAAGTAGAAAAATTTAGA GAAACTATGAAGACTGGTAGAACATTAATAAATATGCCTTCAGCACAAGGTGCTA TAGGAGACATATATAACTTTAAACTAGCTCCTTCATTAACATTAGGTTGTGGTTC ATGGGGAGGAAACTCCGTATCAGAAAATGTTGGACCTAAACACTTATTAAATATA AAAAGTGTTGCTGAGAGGAGAGAAAATATGCTTTGGTTTAGAGTTCCTGAAAAGG TTTATTTTAAATATGGTAGTCTTGGAGTTGCATTAAAAGAATTAGATATTTTGGA TAAGAAAAAAGTATTTATAGTAACAGATAAAGTTCTTTATCAATTAGGTTATATA GATAGAGTTACAAAGATTCTTGAAGAATTGAAAATTTCATATAAAATATTTACAG ATGTAGAACCAGATCCAACCCTAGCTACAGCTAAAAAAGGTGCAGAAGAATTGTT ATCATTTAATCCAGATACTATTATAGCAGTTGGTGGTGGTTCAGCAATGGATGCT GCTAAGATTATGTGGGTAATGTATGAACATCCGGAAGTAAGATTTGAAGATTTAG CTATGAGATTTATGGATATAAGAAAGAGAGTATATACTTTTCCTAAGATGGGTGA AAAAGCAATGATGATTTCTGTTGCAACATCAGCAGGAACAGGATCAGAAGTAACA CCTTTTGCAGTAATTACTGATGAAAAAACAGGAGCTAAATATCCATTAGCTGATT ATGAATTAACTCCAAATATGGCTATAATTGATGCTGAACTTATGATGGGTATGCC AAAAGGATTAACAGCAGCTTCAGGAATAGATGCACTAACTCATGCAATAGAAGCT TATGTATCAATAATGGCTTCAGAATATACTAATGGATTAGCGTTAGAAGCAATAA GATTGATATTTAAGTATTTACCAATAGCTTACAGTGAAGGAACAACAAGTATAAA GGCAAGAGAAAAAATGGCGCATGCTTCAACAATAGCTGGTATGGCATTTGCTAAT GCATTTTTAGGAGTATGTCATTCAATGGCACATAAATTAGGATCAACTCATCACG TACCACATGGCATTGCCAATGCACTACTTATAAATGAAGTTATAAAATTTAATGC AGTAGAAAATCCAAGAAAACAAGCTGCATTTCCACAATATAAGTATCCAAATATA AAAAAGAGATATGCTAGAATAGCAGATTACCTTAACTTAGGTGGGTCAACAGACG ATGAAAAAGTACAATTATTAATAAATGCTATAGATGAATTAAAAGCTAAGATAAA TATTCCAGAAAGTATTAAAGAAGCAGGAGTAACAGAAGAAAAATTTTATGCTACT TTAGATAAAATGTCAGAATTAGCTTTTGATGATCAATGTACAGGTGCAAACCCTA GATATCCATTAATAAGTGAAATAAAACAAATGTATGTAAATGCATTTTAA SEQ ID NO : 108 Clostridium carboxidivorans alcohol dehydrogenase adh AA sequence MKVTNVEELMKKMQEVQNAQKKFGSFTQEQVDEIFRQAALAANSARIDLAKMAVE ETKMGIVEDKVIKNHFVAEYIYNKYKNEKTCGILEEDEGFGMVKIAEPVGVIAAV IPTTNPTSTAIFKALLALKTRNGIIFSPHPRAKKCTIAAAKLVLDAAVKAGAPKG IIGWIDEPSIELSQIVMKEADIILATGGPGMVKAAYSSGKPAIGVGPGNTPALID ESADIKMAVNSILLSKTFDNGMI CASEQSVVVVDSIYEEVKKEFAHRGAYILSKD ETTKVGKILLVNGTLNAGIVGQSAYKIAEMAGVKVPEDAKVLIGEVKSVEHSEEP FSHEKLSPVLAMYRAKNFDEALLKAGRLVELGGMGHTSVLYVNAITEKVKVEKFR ETMKTGRTLINMPSAQGAIGDIYNFKLAPSLTLGCGSWGGNSVSENVGPKHLLNI KSVAERRENMLWFRVPEKVYFKYGSLGVALKELDILDKKKVFIVTDKVLYQLGYI DRVTKILEELKISYKIFTDVEPDPTLATAKKGAEELLSFNPDTIIAVGGGSAMDA AKIMWVMYEHPEVRFEDLAMRFMDIRKRVYTFPKMGEKAMMISVATSAGTGSEVT PFAVITDEKTGAKYPLADYELTPNMAIIDAELMMGMPKGLTAASGIDALTHAIEA YVSIMASEYTNGLALEAIRLIFKYLPIAYSEGTTSIKAREKMAHASTIAGMAFAN AFLGVCHSMAHKLGSTHHVPHGIANALLINEVIKFNAVENPRKQAAFPQYKYPNI KKRYARIADYLNLGGSTDDEKVOLLINAIDELKAKINIPESIKEAGVTEEKFYAT LDKMSELAFDDQCTGANPRYPLISEIKOMYVNAF SEQ ID NO : 109 Escherichia coli soluble pyridine nucleotide transhydrogenase NT sequence ATGCCACATTCCTACGATTACGATGCCATAGTAATAGGTTCCGGCCCCGGCGGCGAAGGC GCTGCAATGGGCCTGGTTAAGCAAGGTGCGCGCGTCGCAGTTATCGAGCGTTATCAAAAT GTTGGCGGCGGTTGCACCCACTGGGGCACCATCCCGTCGAAAGCTCTCCGTCACGCCGTC AGCCGCATTATAGAATTCAATCAAAACCCACTTTACAGCGACCATTCCCGACTGCTCCGC TCTTCTTTTGCCGATATCCTTAACCATGCCGATAACGTGATTAATCAACAAACGCGCATG CGTCAGGGATTTTACGAACGTAATCACTGTGAAATATTGCAGGGAAACGCTCGCTTTGTT GACGAGCATACGTTGGCGCTGGATTGCCCGGACGGCAGCGTTGAAACACTAACCGCTGAA AAATTTGTTATTGCCTGCGGCTCTCGTCCATATCATCCAACAGATGTTGATTTCACCCAT CCACGCATTTACGACAGCGACTCAATTCTCAGCATGCACCACGAACCGCGCCATGTACTT ATCTATGGTGCTGGAGTGATCGGCTGTGAATATGCGTCGATCTTCCGCGGTATGGATGTA US 10,941,424 B2 155 156 - continued SEQUENCE LISTING AAAGTGGATCTGATCAACACCCGCGATCGCCTGCTGGCATTTCTCGATCAAGAGATGTCA GATTCTCTCTCCTATCACTTCTGGAACAGTGGCGTAGTGATTCGTCACAACGAAGAGTAC GAGAAGATCGAAGGCTGTGACGATGGTGTGATCATGCATCTGAAGTCGGGTAAAAAACTG AAAGCTGACTGCCTGCTCTATGCCAACGGTCGCACCGGTAATACCGATTCGCTGGCGTTA CAGAACATTGGGCTAGAAACTGACAGCCGCGGACAGCTGAAGGTCAACAGCATGTATCAG ACCGCACAGCCACACGTTTACGCGGTGGGCGACGTGATTGGTTATCCGAGCCTGGCGTCG GCGGCCTATGACCAGGGGCGCATTGCCGCGCAGGCGCTGGTAAAAGGCGAAGCCACCGCA CATCTGATTGAAGATATCCCTACCGGTATTTACACCATCCCGGAAATCAGCTCTGTGGGC AAAACCGAACAGCAGCTGACCGCAATGAAAGTGCCATATGAAGTGGGCCGCGCCCAGTTT AAACATCTGGCACGCGCACAAATCGTCGGCATGAACGTGGGCACGCTGAAAATTTTGTTC CATCGGGAAACAAAAGAGATTCTGGGTATTCACTGCTTTGGCGAGCGCGCTGCCGAAATT ATTCATATCGGTCAGGCGATTATGGAACAGAAAGGTGGCGGCAACACTATTGAGTACTTC GTCAACACCACCTTTAACTACCCGACGATGGCGGAAGCCTATCGGGTAGCTGCGTTAAAC GGTTTAAACCGCCTGTTTTAA SEQ ID NO : 110 Escherichia coli soluble pyridine nucleotide transhydrogenase AA sequence MPHSYDYDAIVIGSGPGGEGAAMGLVKQGARVAVIERYQNVGGGCTHWGTIPSKA LRHAVSRIIEFNQNPLYSDHSRLLRSSFADILNHADNVINQQTRMRQGFYERNHC EILOGNARFVDEHTLALDCPDGSVETLTAEKFVIACGSRPYHPTDVDFTHPRIYD SDSILSMHHEPRHVLIYGAGVIGCEYASIFRGMDVKVDLINTRDRLLAFLDQEMS DSLSYHFWNSGVVIRHNEEYEKIEGCDDGVIMHLKSGKKLKADCLLYANGRTGNT DSLALQNIGLETDSRGQLKVNSMYQTAQPHVYAVGDVIGYPSLASAAYDQGRIAA QALVKGEATAHLIEDIPTGIYTI PEISSVGKTEQOLTAMKVPYEVGRAQFKHLAR AQIVGMNVGTLKILFHRETKEILGIHCFGERAAEIIHIGQAIMEQKGGGNTIEYF VNTTFNYPTMAEAYRVAALNGLNRLF SEQ ID NO : 111 Forward primer to amplify fucA and fuco CCTTTAATAAGGAGATATACCATGGAACGAAATAAACTTGC SEQ ID NO : 112 Reverse primer to amplify fucA and fuco GGTTATTCCTCCTTATTTAGAGCTCTAAACGAATTCTTACCAGGCG GTATGGTAAA SEQ ID NO : 113 Forward primer to amplify fuck GAATTCGTTTAGAGCTCTAAATAAGGAGGAATAACCATGATGAAACAAGAAGTTA T

SEO ID NO : 114 Reverse primer to amplify fuck GAGCT CGGTACCCGGGGATCCAAAAAACCCCTCAAGACCC SEQ ID NO : 115 Forward primer to amplify thl CTGTTGTTATATTGTAATGATGTATGCAAGAGGGATAAA SEQ ID NO : 116 Reverse primer to amplify thl TATATCTCCTTCTTAAAGTTCATAAATCACCCCGTTGC SEQ ID NO : 117 Forward primer to amplify fuco ATGGCTAACAGAATGATTCTG SEQ ID NO : 118 Reverse primer to amplify fuco TTACCAGGCGGTATGGTAAAGCT SEQ ID NO : 119 Forward primer to amplify atoA / D CTGTTGTTATATTGTAATGATGTATGCAAGAGGGATAAA SEQ ID NO : 120 Reverse primer to amplify atoA / D TATATCTCCTTCTTAAAGTTCATAAATCACCCCGTTGC

The foregoing detailed description has been given for The disclosures, including the claims , figures and / or clearness of understanding only and no unnecessary limita- drawings, of each and every patent, patent application , and tions should be understood there from as modifications will 55 publication cited herein are hereby incorporated herein by be obvious to those skilled in the art. reference in their entireties. While the disclosure has been described in connection with specific embodiments thereof, it will be understood that NUMBERED EMBODIMENTS OF THE it is capable of further modifications and this application is DISCLOSURE intended to cover any variations, uses , or adaptations of the 60 disclosure following, in general, the principles of the dis- Particular subject matter contemplated by the present closure and including such departures from the present disclosure is set out in the below numbered embodiments . disclosure as come within known or customary practice 1. A recombinant microorganism capable of co -producing within the art to which the disclosure pertains and as may be 65 monoethylene glycol ( MEG ) and acetone from exog applied to the essential features hereinbefore set forth and as enous D - xylose , wherein the recombinant microorgan follows in the scope of the appended claims . ism expresses one or more of the following: US 10,941,424 B2 157 158 ( a ) at least one endogenous or exogenous nucleic acid ( a ) at least one endogenous or exogenous nucleic acid molecule encoding a D - tagatose 3 - epimerase that molecule encoding a D - xylulose 1 - kinase that cata catalyzes the conversion of D - xylulose to D - ribu lyzes the conversion of D - xylulose to D -xylulose - 1 lose ; phosphate ; ( b ) at least one endogenous or exogenous nucleic acid 5 ( b ) at least one endogenous or exogenous nucleic acid molecule encoding a D - ribulokinase that catalyzes molecule encoding a D -xylulose - 1 - phosphate aldo the conversion of D - ribulose from ( a ) to D - ribulose lase that catalyzes the conversion of D - xylulose - 1 1 - phosphate; phosphate from ( a ) to glycolaldehyde and dihy ( c ) at least one endogenous or exogenous nucleic acid droxyacetonephosphate ( DHAP ) ; molecule encoding a D - ribulose - 1 - phosphate aldo- 10 ( c ) at least one endogenous or exogenous nucleic acid lase that catalyzes the conversion of D -ribulose - 1 molecule encoding a glycolaldehyde reductase that phosphate from ( b ) to glycolaldehyde and dihy catalyzes the conversion of glycolaldehyde from ( b ) droxyacetonephosphate ( DHAP ) ; to MEG ; ( d ) at least one endogenous or exogenous nucleic acid ( d ) at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that 15 molecule encoding a thiolase that catalyzes the con catalyzes the conversion of glycolaldehyde from ( c ) version of acetyl - CoA to acetoacetyl - CoA ; to MEG ; ( e ) at least one endogenous or exogenous nucleic acid ( e ) at least one exogenous nucleic acid molecule encod molecule encoding an acetate : acetoacetyl - CoA ing a thiolase that catalyzes the conversion of acetyl- 20 transferase or hydrolase that catalyzes the conver COA to acetoacetyl - CoA ; sion of acetoacetyl - CoA from ( d ) to acetoacetate ; ( f) at least one endogenous or exogenous nucleic acid and / or molecule encoding an acetate :acetoacetyl - COA ( f) at least one endogenous or exogenous nucleic acid transferase or hydrolase that catalyzes the conver molecule encoding an acetoacetate decarboxylase sion of acetoacetyl - CoA from ( e ) to acetoacetate ; 25 that catalyzes the conversion of acetoacetate from ( e ) and / or to acetone; ( g ) at least one endogenous or exogenous nucleic acid wherein the produced intermediate DHAP is converted to molecule encoding an acetoacetate decarboxylase acetyl -CoA through the endogenous glycolysis path that catalyzes the conversion of acetoacetate from ( f) way in the microorganism , and wherein MEG and to acetone ; 30 acetone are co - produced . wherein the produced intermediate DHAP is converted 7. The recombinant microorganism of claim 6 , wherein to acetyl -CoA through the endogenous glycolysis the recombinant microorganism further comprises at pathway in the microorganism , and wherein MEG least one exogenous nucleic acid molecule encoding a and acetone are co - produced . secondary alcohol dehydrogenase that catalyzes the 2. The recombinant microorganism of claim 1 , wherein 35 conversion of acetone from ( f) to isopropanol. the recombinant microorganism further comprises at 8. The recombinant microorganism of claim 7 , wherein least one endogenous or exogenous nucleic acid mol- the recombinant microorganism further comprises at ecule encoding a secondary alcohol dehydrogenase that least one exogenous nucleic acid molecule encoding a catalyzes the conversion of acetone from ( g ) to isopro dehydratase that catalyzes the conversion of isopropa panol. 40 nol to propene . 3. The recombinant microorganism of claim 2 , wherein 9. The recombinant microorganism of any one of claims the recombinant microorganism further comprises at 6-8 , wherein the recombinant microorganism further least one endogenous or exogenous nucleic acid mol- comprises one or more modifications selected from the ecule encoding a dehydratase that catalyzes the con- group consisting of: version of isopropanol to propene . 45 ( a ) a deletion , insertion , or loss of function mutation in 4. The recombinant microorganism of any one of claims a gene encoding a D - xylulose - 5 - kinase that catalyzes 1-3 , wherein the recombinant microorganism further the conversion of D - xylulose to D -xylulose - 5 -phos comprises one or more modifications selected from the phate; group consisting of: ( b ) a deletion , insertion , or loss of function mutation in ( a ) a deletion , insertion , or loss of function mutation in 50 a gene encoding a glycolaldehyde dehydrogenase a gene encoding a D - xylulose - 5 - kinase that catalyzes that catalyzes the conversion of glycolaldehyde to the conversion of D - xylulose to D -xylulose - 5 -phos glycolic acid ; and phate ; ( c ) a deletion , insertion , or loss of function mutation in ( b ) a deletion , insertion , or loss of function mutation in a gene encoding a lactate dehydrogenase that cata a gene encoding a glycolaldehyde dehydrogenase 55 lyzes the conversion of pyruvate to lactate . that catalyzes the conversion of glycolaldehyde to 10. The recombinant microorganism of any one of claims glycolic acid ; and 6-9 , wherein an endogenous D -xylose isomerase cata ( c ) a deletion , insertion , or loss of function mutation in lyzes the conversion of D -xylose to D - xylulose . a gene encoding a lactate dehydrogenase that cata- 11. A recombinant microorganism capable of co -produc lyzes the conversion of pyruvate to lactate . 60 ing monoethylene glycol ( MEG ) and acetone from 5. The recombinant microorganism of any one of claims exogenous D - xylose and glucose , wherein the recom 1-4 , wherein an endogenous D - xylose isomerase cata- binant microorganism expresses one or more of the lyzes the conversion of D - xylose to D - xylulose . following: 6. A recombinant microorganism capable of co - producing ( a ) at least one exogenous nucleic acid molecule encod monoethylene glycol ( MEG ) and acetone from exog- 65 ing a xylose reductase or aldose reductase that cata enous D - xylose , wherein the recombinant microorgan lyzes the conversion of D - xylose to xylitol and at ism expresses one or more of the following: least one exogenous nucleic acid molecule encoding US 10,941,424 B2 159 160 a xylitol dehydrogenase that catalyzes the conver- exogenous D - xylose , wherein the recombinant micro sion of xylitol to D - xylulose ; organism expresses one or more of the following: ( b ) at least one exogenous nucleic acid molecule encod- ( a ) at least one endogenous or exogenous nucleic acid ing a D - xylose isomerase that catalyzes the conver molecule encoding a xylose dehydrogenase that sion of D -xylose to D - xylulose , and 5 catalyzes the conversion of D - xylose to D -xylono wherein the microorganism further expresses one or more lactone ; of the following: ( b ) at least one endogenous or exogenous nucleic acid ( c ) at least one endogenous or exogenous nucleic acid molecule encoding a xylonolactonase that catalyzes molecule encoding a D - tagatose 3 - epimerase that the conversion of D -xylonolactone from ( a ) to D -xy catalyzes the conversion of D - xylulose from ( a ) or 10 lonate ; ( b ) to D - ribulose ; ( c ) at least one endogenous or exogenous nucleic acid ( d ) at least one endogenous or exogenous nucleic acid molecule encoding a xylonate dehydratase that cata molecule encoding a D - ribulokinase that catalyzes lyzes the conversion of D -xylonate from ( b ) to the conversion of D - ribulose from ( c ) to D -ribulose 15 2 -keto - 3 - deoxy -xylonate ; 1 -phosphate ; ( d ) at least one endogenous or exogenous nucleic acid ( e ) at least one endogenous or exogenous nucleic acid molecule encoding a 2 -keto - 3 -deoxy - D -pentonate molecule encoding a D - ribulose - 1 -phosphate aldo aldolase that catalyzes the conversion of 2 -keto - 3 lase that catalyzes the conversion of D - ribulose - 1 deoxy - xylonate from ( c ) to glycolaldehyde and pyru phosphate from ( d ) to glycolaldehyde and dihy- 20 vate ; droxyacetonephosphate ( DHAP ) ; ( e ) at least one endogenous or exogenous nucleic acid ( f) at least one endogenous or exogenous nucleic acid molecule encoding a glycolaldehyde reductase that molecule encoding a glycolaldehyde reductase or catalyzes the conversion of glycolaldehyde from ( d ) methylglyoxal reductase that catalyzes the conver to MEG ; sion of glycolaldehyde from ( e ) to MEG ; 25 ( f) at least one exogenous nucleic acid molecule encod ( g ) at least one endogenous or exogenous nucleic acid ing a thiolase that catalyzes the conversion of acetyl molecule encoding a thiolase that catalyzes the con CoA to acetoacetyl -CoA ; version of acetyl - CoA to acetoacetyl - CoA ; ( g ) at least one endogenous or exogenous nucleic acid ( h ) at least one endogenous or exogenous nucleic acid molecule encoding an acetate : acetoacetyl - CoA molecule encoding an acetate : acetoacetyl - CoA 30 transferase or hydrolase that catalyzes the conver transferase or hydrolase that catalyzes the conver sion of acetoacetyl -CoA from ( f) to acetoacetate ; sion of acetoacetyl - CoA from ( g ) to acetoacetate ; and / or and / or ( h ) at least one exogenous nucleic acid molecule encod ( i ) at least one endogenous or exogenous nucleic acid ing an acetoacetate decarboxylase that catalyzes the molecule encoding an acetoacetate decarboxylase 35 conversion of acetoacetate from ( g ) to acetone ; that catalyzes the conversion of acetoacetate from ( h ) wherein the produced intermediate pyruvate is converted to acetone; to acetyl -CoA through the endogenous glycolysis path wherein the produced intermediate DHAP is converted to way in the microorganism , and wherein MEG and acetyl - CoA through the endogenous glycolysis path acetone are co -produced . way in the microorganism , and wherein MEG and 40 17. The recombinant microorganism of claim 16 , wherein acetone are co - produced . the recombinant microorganism further comprises at 12. The recombinant microorganism of claim 11 , wherein least one exogenous nucleic acid molecule encoding a the recombinant microorganism further comprises at secondary alcohol dehydrogenase that catalyzes the least one exogenous nucleic acid molecule encoding a conversion of acetone from ( h ) to isopropanol. secondary alcohol dehydrogenase that catalyzes the 45 18. The recombinant microorganism of claim 17 , wherein conversion of acetone from ( i ) to isopropanol. the recombinant microorganism further comprises at 13. The recombinant microorganism of claim 12 , wherein least one exogenous nucleic acid molecule encoding a the recombinant microorganism further comprises at dehydratase that catalyzes the conversion of isopropa least one exogenous nucleic acid molecule encoding a nol to propene. dehydratase that catalyzes the conversion of isopropa- 50 19. The recombinant microorganism of any one of claims nol to propene. 16-18 , wherein the recombinant microorganism further 14. The recombinant microorganism of any one of claims comprises one or more modifications selected from the 11-13 , wherein the recombinant microorganism further group consisting of: comprises one or more modifications selected from the ( a ) a deletion , insertion, or loss of function mutation in group consisting of: 55 a gene encoding a D - xylose isomerase that catalyzes ( a ) a deletion , insertion , or loss of function mutation in the conversion of D - xylose to D - xylulose ; a gene encoding a D - xylulose - 5 - kinase that catalyzes ( b ) a deletion, insertion , or loss of function mutation in the conversion of D - xylulose to D -xylulose -5 -phos a gene encoding a glycolaldehyde dehydrogenase phate ; and that catalyzes the conversion of glycolaldehyde to ( b ) a deletion , insertion , or loss of function mutation in 60 glycolic acid ; and a gene encoding an alkaline phosphatase that cata ( c ) a deletion , insertion , or loss of function mutation in lyzes the conversion of D - xylulose - 5 - phosphate to a gene encoding a lactate dehydrogenase that cata D - xylulose . lyzes the conversion of pyruvate to lactate . 15. The recombinant microorganism of any one of claims 20. A recombinant microorganism capable of co - produc 11-14 , wherein the microorganism is a fungus. 65 ing monoethylene glycol ( MEG ) and acetone from 16. A recombinant microorganism capable of co - produc exogenous D - xylose , wherein the recombinant micro ing monoethylene glycol ( MEG ) and acetone from organism expresses one or more of the following: US 10,941,424 B2 161 162 ( a ) at least one endogenous or exogenous nucleic acid 27. The recombinant microorganism of claim 1 or claim molecule encoding a xylose dehydrogenase that 11 , wherein the D - ribulokinase is encoded by one or catalyzes the conversion of D - xylose to D - xylonate ; more nucleic acid molecules obtained from E. coli . ( b ) at least one endogenous or exogenous nucleic acid 28. The recombinant microorganism of claim 27 , wherein molecule encoding a xylonate dehydratase that cata 5 the one or more nucleic acid molecules is fucK . lyzes the conversion of D - xylonate from ( a ) to 29. The recombinant microorganism of claim 1 or claim 2 -keto - 3 - deoxy -xylonate ; 11 , wherein the D - ribulose - 1 - phosphate aldolase is ( c ) at least one endogenous or exogenous nucleic acid encoded by one or more nucleic acid molecules molecule encoding a 2 -keto - 3 -deoxy - D -pentonate obtained from E. coli . 10 30. The recombinant microorganism of claim 29 , wherein aldolase that catalyzes the conversion of 2 -keto - 3 the one or more nucleic acid molecules is fucA . deoxy -xylonate from ( b ) to glycolaldehyde and 31. The recombinant microorganism of any one of claim pyruvate ; 1 , 6 , 11 , 16 or 20 , wherein the glycolaldehyde reductase ( d ) at least one exogenous nucleic acid molecule encod is encoded by one or more nucleic acid molecules ing a glycolaldehyde reductase that catalyzes the 15 obtained from a microorganism selected from E. coli or conversion of glycolaldehyde from ( c ) to MEG ; S. cerevisiae . ( e ) at least one exogenous nucleic acid molecule encod 32. The recombinant microorganism of claim 31 , wherein ing a thiolase that catalyzes the conversion of acetyl the one or more nucleic acid molecules is selected from COA to acetoacetyl- CoA ; fuco , yqhD , dkgA ( yqhE ), dkgB ( yafB ), yeaE , yghz , ( f) at least one endogenous or exogenous nucleic acid 20 gldA and /or GRE2 . molecule encoding an acetate :acetoacetyl - CoA 33. The recombinant microorganism of any one of claim transferase or hydrolase that catalyzes the conver- 1 , 6 , 11 , 16 or 20 , wherein the thiolase is encoded by sion of acetoacetyl -CoA from ( e ) to acetoacetate ; one or more nucleic acid molecules obtained from a and / or microorganism selected from Clostridium sp . , Bacillus ( g ) at least one exogenous nucleic acid molecule encod- 25 sp . , E. coli and Marinobacter sp . ing an acetoacetate decarboxylase that catalyzes the 34. The recombinant microorganism of claim 33 , wherein conversion of acetoacetate from ( f) to acetone ; the microorganism is selected from Clostridium aceto wherein the produced intermediate pyruvate is converted butylicum , Clostridium thermosaccharolyticum , Bacil to acetyl- CoA through the endogenous glycolysis path lus cereus , E. coli and Marinobacter hydrocarbono way in the microorganism , and wherein MEG and 30 clasticus . acetone are co -produced . 35. The recombinant microorganism of claim 33 , wherein 21. The recombinant microorganism of claim 20 , wherein the one or more nucleic acid molecules is th1A and / or the recombinant microorganism further comprises at atoB . least one exogenous nucleic acid molecule encoding a 36. The recombinant microorganism of any one of claim secondary alcohol dehydrogenase that catalyzes the 35 1 , 6 , 11 , 16 or 20 , wherein the acetate :acetoacetyl - CoA conversion of acetone from ( g ) to isopropanol. transferase or hydrolase is encoded by one or more 22. The recombinant microorganism of claim 21 , wherein nucleic acid molecules obtained from a microorganism the recombinant microorganism further comprises at selected from Clostridium sp . or E. coli . least one exogenous nucleic acid molecule encoding a 37. The recombinant microorganism of claim 36 , wherein dehydratase that catalyzes the conversion of isopropa- 40 the microorganism is Clostridium acetobutylicum . nol to propene . 38. The recombinant microorganism of claim 36 , wherein 23. The recombinant microorganism of any one of claims the one or more nucleic acid molecules encoding the 20-22 , wherein the recombinant microorganism further acetate :acetoacetyl - CoA transferase is atoA and / or comprises one or more modifications selected from the atoD . group consisting of: 45 39. The recombinant microorganism of claim 36 , wherein ( a ) a deletion , insertion , or loss of function mutation in the one or more nucleic acid molecules encoding the a gene encoding a D - xylose isomerase that catalyzes acetate :acetoacetyl - CoA hydrolase is ctfA and /or ctfB . the conversion of D - xylose to D - xylulose ; 40. The recombinant microorganism of any one of claim ( b ) a deletion , insertion, or loss of function mutation in 1 , 6 , 11 , 16 or 20 , wherein the acetoacetate decarboxy a gene encoding a glycolaldehyde dehydrogenase 50 lase is encoded by one or more nucleic acid molecules that catalyzes the conversion of glycolaldehyde to obtained from a microorganism selected from glycolic acid ; and Clostridium sp . , Bacillus sp . , Chromobacterium sp . and ( c ) a deletion , insertion , or loss of function mutation in Pseudomonas sp . a gene encoding a lactate dehydrogenase that cata- 41. The recombinant microorganism of claim 40 , wherein lyzes the conversion of pyruvate to lactate . 55 the microorganism is selected from Clostridium aceto 24. The recombinant microorganism of claim 1 or claim butylicum , Clostridium beijerinckii , Clostridium cel 11 , wherein the D - tagatose 3 - epimerase is encoded by lulolyticum , Bacillus polymyxa , Chromobacterium vio one or more nucleic acid molecules obtained from a laceum and Pseudomonas putida. microorganism selected from Pseudomonas sp . , 42. The recombinant microorganism of claim 40 , wherein Mesorhizobium sp . or Rhodobacter sp . 60 the one or more nucleic acid molecules encoding the 25. The recombinant microorganism of claim 24 , wherein acetoacetate decarboxylase is adc . the microorganism is selected from Pseudomonas 43. The recombinant microorganism of any one of claim cichorii, Pseudomonas sp . ST- 24 , Mesorhizobium loti 2 , 7 , 12 , 17 or 21 , wherein the secondary alcohol or Rhodobacter sphaeroides. dehydrogenase is encoded by one or more nucleic acid 26. The recombinant microorganism of claim 24 , wherein 65 molecules obtained from a microorganism selected the one or more nucleic acid molecules is dte and / or C1 from Burkholderia sp , Alcaligenes sp . , Clostridium sp . , KKR1 . Thermoanaerobacter sp . , Phytomonas sp . , Rhodococ US 10,941,424 B2 163 164 cus sp . , Methanobacterium sp . , Methanogenium sp . , one or more nucleic acid molecules obtained from a Entamoeba sp . , Trichomonas sp . , and Tritrichomonas microorganism selected from Caulobacter sp . , Halo sp . arcula sp . , Haloferax sp . , Halorubrum sp . and Tricho 44. The recombinant microorganism of claim 43 , wherein derma sp . the microorganism is selected from Burkholderia sp . 5 59. The recombinant microorganism of claim 58 , wherein AIU 652 , Alcaligenes eutrophus, Clostridium ragsda the microorganism is selected from Caulobacter cres centus, Haloarcula marismortui, Haloferax volcanii , lei, Clostridium beijerinckii, Thermoanaerobacter Halorubrum lacusprodundi and Trichoderma reesei . brockii, Thermoanaerobacter ethanolicus ( Clostridium 60. The recombinant microorganism of claim 58 , wherein thermohydrosulfuricum ), Rhodococcus ruber, Metha the one or more nucleic acid molecules encoding the nobacterium palustre , methanogenic archaea Methano- 10 xylose dehydrogenase is selected from xylB , xdh or genium liminatans, parasitic protist Entamoeba his xydl . tolytica , parasitic protozoan Tritrichomonas foetus and 61. The recombinant microorganism of claim 16 , wherein human parasite Trichomonas vaginalis. the xylonolactonase is encoded by one or more nucleic 45. The recombinant microorganism of claim 43 , wherein acid molecules obtained from a microorganism the one or more nucleic acid molecules encoding the 15 selected from Caulobacter sp . and Haloferax sp . secondary alcohol dehydrogenase is adhB or EhAdh1 . 62. The recombinant microorganism of claim 61 , wherein 46. The recombinant microorganism of claim 6 , wherein the microorganism is selected from Caulobacter cres the D - xylulose 1 - kinase is encoded by one or more centus, Haloferax volcanii and Haloferax gibbonsii . nucleic acid molecules obtained from Homo sapiens. 63. The recombinant microorganism of claim 61 , wherein 47. The recombinant microorganism of claim 46 , wherein 20 the one or more nucleic acid molecules encoding the the one or more nucleic acid molecules encoding the xylonolactonase is xylC . D - xylulose 1 - kinase is ketohexokinase C ( khk - C ). 64. The recombinant microorganism of claim 16 or claim 48. The recombinant microorganism of claim 6 , wherein 20 , wherein the xylonate dehydratase is encoded by one the D - xylulose - 1 - phosphate aldolase is encoded by one or more nucleic acid molecules obtained from a micro or more nucleic acid molecules obtained from Homo 25 organism selected from Caulobacter sp . , Haloferax sp . , sapiens. Sulfolobus sp . and E. coli . 49. The recombinant microorganism of claim 48 , wherein 65. The recombinant microorganism of claim 64 , wherein the one or more nucleic acid molecules encoding the the microorganism is selected from Caulobacter cres D -xylulose - 1 - phosphate aldolase is aldolase B ( AL centus, Haloferax volcanii, E. coli and Sulfolobus sol DOB ) . 30 fataricus. 50. The recombinant microorganism of claim 11 , wherein 66. The recombinant microorganism of claim 64 , wherein the xylose reductase or aldose reductase is encoded by the one or more nucleic acid molecules encoding the one or more nucleic acid molecules obtained from a xylonate dehydratase is selected from xylD , yjhG , yagF microorganism selected from Hypocrea sp . , Saccharo and xad . myces sp . , Pachysolen sp . , Pichia sp . , Candida sp . , 35 67. The recombinant microorganism of claim 16 or claim Aspergillus sp ., Neurospora sp . , and Cryptococcus sp . is20 , encodedwherein bythe one2 - keto or -more 3 - deoxy nucleic - D -pentonate acid molecules aldolase 51. The recombinant microorganism of claim 50 , wherein obtained from a microorganism selected from the microorganism is selected from Hypocrea jecorina , Pseudomonas sp . and E. coli . S. cerevisiae , Pachysolen tannophilus, Pichia stipitis, 68. The recombinant microorganism of claim 67 , wherein Pichia quercuum , Candida shehatae, Candida tenuis, 40 the microorganism is E. coli . Candida tropicalis, Aspergillus niger, Neurospora 69. The recombinant microorganism of claim 67 , wherein crassa and Cryptococcus lactativorus . the one or more nucleic acid molecules encoding the 52. The recombinant microorganism of claim 50 , wherein 2 -keto - 3 -deoxy - D -pentonate aldolase is selected from yjhH and yagE . the one or more nucleic acid molecules encoding the 70. The recombinant microorganism of any one of claim xylose reductase or aldose reductase is xyll or GRE3 . 45 1 , 6 , 11 , 16 or 20 , wherein MEG is produced through 53. The recombinant microorganism of claim 11 , wherein the conversion of glycolaldehyde in a C - 2 branch the xylitol dehydrogenase is encoded by one or more pathway and acetone is produced through the conver nucleic acid molecules obtained from a microorganism sion of DHAP or pyruvate in a C - 3 branch pathway. selected from Pichia sp . , Saccharomyces sp . , Glucono 71. The recombinant microorganism of any one of claim bacter sp . , Galactocandida sp . , Neurospora sp . , and 50 2 , 7 , 12 , 17 or 21 , wherein MEG is produced through Serratia sp . the conversion of glycolaldehyde in a C - 2 branch 54. The recombinant microorganism of claim 53 , wherein pathway and IPA is produced through the conversion of the microorganism is selected from Pichia stipitis, S. DHAP or pyruvate in a C - 3 branch pathway. cerevisiae, Gluconobacter oxydans, Galactocandida 72. The recombinant microorganism of any one of claim mastotermitis, Neurospora crassa and Serratia marc- 55 3 , 8 , 13, 18 or 22 , wherein MEG is produced through escens . the conversion of glycolaldehyde in a C - 2 branch 55. The recombinant microorganism of claim 53 , wherein pathway and propene is produced through the conver the one or more nucleic acid molecules encoding the sion of DHAP or pyruvate in a C - 3 branch pathway. xylitol dehydrogenase is xyl2 or xdh1. 73. The recombinant microorganism of any one of claims 56. The recombinant microorganism of claim 11 , wherein 60 70-72 , wherein at least a portion of the excess NADH the xylose isomerase is encoded by one or more nucleic produced in the C - 3 branch is used as a source of acid molecules obtained from E. coli . reducing equivalents in the C - 2 branch . 57. The recombinant microorganism of claim 56 , wherein 74. The recombinant microorganism of any one of claims the one or more nucleic acid molecules encoding the 70-72 , wherein at least a portion of the excess NADH xylose isomerase is xylA . 65 produced in the C - 3 branch is used to produce ATP . 58. The recombinant microorganism of claim 16 or claim 75. The recombinant microorganism of any one of claim 20 , wherein the xylose dehydrogenase is encoded by 1 , 6 , 11 , 16 or 20 , wherein the co - produced MEG and