I 1111111111111111 1111111111 11111 1111111111 11111 1111111111111111 IIII IIII IIII US010011854B2 c12) United States Patent (IO) Patent No.: US 10,011,854 B2 San et al. (45) Date of Patent: Jul. 3, 2018 (54) FATTY ACID PRODUCTIVITY WO W02012052468 4/2012 WO WO 2012-087963 * 6/2012 (71) Applicant: WILLIAM MARSH RICE WO WO 2012-109221 * 8/2012 WO W02013059218 4/2013 UNIVERSITY, Houston, TX (US) WO W02013096665 6/2013 (72) Inventors: Ka-Yiu San, Houston, TX (US); Wei OTHER PUBLICATIONS Li, Houston, TX (US) Whisstock et al. Quaterly Reviews of Biophysics, 2003, "Prediction (73) Assignee: William Marsh Rice University, of protein function from protein sequence and structure", 36(3): Houston, TX (US) 307-340.* Witkowski et al. Conversion of a beta-ketoacyl synthase to a ( *) Notice: Subject to any disclaimer, the term ofthis malonyl decarboxylase by replacement of the active-site cysteine patent is extended or adjusted under 35 with glutamine, Biochemistry. Sep. 7, 1999;38(36)11643-50.* U.S.C. 154(b) by O days. Kisselev L., Polypeptide release factors in prokaryotes and eukaryotes: same function, different structure. Structure, 2002, vol. 10: 8-9.* (21) Appl. No.: 15/095,158 Gurvitz Aner, The essential mycobacterial genes, fabG 1 and fabG4, encode 3-oxoacyl-thioester reductases that are functional in yeast (22) Filed: Apr. 11, 2016 mitochondrial fatty acid synthase type 2, Mo! Genet Genomics (2009), 282: 407-416.* (65) Prior Publication Data Bergler H, et a., Protein EnvM is the NADH-dependent enoyl-ACP reductase (Fahl) of Escherichia coli, J Biol Chem. 269(8):5493-6 US 2016/0215309 Al Jul. 28, 2016 (1994). Bokinsky G. et al., Synthesis of three advanced biofuels from ionic Related U.S. Application Data liquid-pretreated switchgrass using engineered Escherichia coli, PNAS, 108(50): 19949-19954 (2011). (63) Continuation-in-part of application No. Caughey I, Kekwick RG, The characteristics of some components PCT/US2014/059319, filed on Oct. 6, 2014. of the fatty acid synthetase system in the plastids fom the mesocarp ofavocado (Persea americana) fruit, Eur J Biochem. 123(3):553-61 (60) Provisional application No. 61/889,166, filed on Oct. (1982). 10, 2013. Dutta D, et al., Crystal structure of FabG4 from Mycobacterium tuberculosis reveals the importance of C-terminal residues in (51) Int. Cl. ketoreductase activity, J Struct Biol. 174(1):147-55 (2011). C12N 1120 (2006.01) Fuhrer T, Sauer U. Different biochemical mechanisms ensure net­ C12P 7164 (2006.01) work-wide balancing of reducing equivalents in microbial metabo­ lism, J Bacteriol. 191(7):2112-21 (2009). C12P 7162 (2006.01) Jing et al., Phylogenetic and experimental characterization of an C12N 9/00 (2006.01) acyl-ACP thioesterase family reveals significant diversity in enzy­ C12N 9102 (2006.01) matic specificity and activity, BMC Biochemistry 2011, 12:44. C07H 21104 (2006.01) Martinez I, et al., Replacing Escherichia coli NAO-dependent C12N 9/04 (2006.01) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facili­ C12N 9/16 (2006.01) tates NADPH dependent pathways, Metab Eng. 10(6):352-9 (2008). (52) U.S. Cl. Murarka A., et al., Fermentative Utilization of Glycerol by CPC C12P 716409 (2013.01); C12N 9/0006 Escherichia coli and Its Implications for the Production of Fuels and (2013.01); C12N 9/16 (2013.01); C12Y Chemicals, Appl Environ Microbiol. Feb. 2008; 74(4): 1124-1135. 1011011 (2013.01); C12Y 101/0133 (2015.07); Radakovits, R., Eduafo, P.M. and Posewitz, M.C. Genetic engineer­ ing of fatty acid chain length in Phaeodactylum tricornutum. Metab. C12Y 301/02014 (2013.01); Cl2Y 101/01 Engin., 13, 89-95 (2011). (2013.01) (Continued) ( 58) Field of Classification Search None Primary Examiner - Iqbal H Chowdhury See application file for complete search history. (74) Attorney, Agent, or Firm - Boulware & Valoir (56) References Cited U.S. PATENT DOCUMENTS (57) ABSTRACT The present disclosure relates to an engineered microbe Al* 2013/0040340 2/2013 Dauner Cl2P 7/6436 capable of improved productivity of fatty acid or fatty acid 435/42 2014/0004580 Al* 1/2014 Roberts . Cl2N 9/0008 derivative. An NAD+-dependent 3-oxoacyl-ACP reductase 435/134 or NAD+-dependent 3-oxoacyl-CoA reductase replaces or 2014/0093921 Al 4/2014 San et al. supplements the native NADP+-dependent 3-oxoacyl-ACP 2014/0193867 Al 7/2014 San et al. 2014/0212935 Al 7/2014 San et al. reductase so as to utilize the higher availability of NAD+ rather than NADP+ in the cell. Higher production, yield and FOREIGN PATENT DOCUMENTS titer of fatty acids are therefore obtained. Such microbes can be combined with other mutations to further improve yield WO W02010142522 12/2010 WO W02011064183 6/2011 of fatty acids or fatty acid derivatives. WO WO 2011-116279 * 9/2011 WO W02011116279 9/2011 7 Claims, 17 Drawing Sheets US 10,011,854 B2 Page 2 (56) References Cited OTHER PUBLICATIONS Ruffing, A.M. & Jones, H.D.T. Physiological effects of free fatty acid production in genetically engineered Synechococcus elongatus PCC 7942. Biotechnology and Bioengineering, 2012; 109 (9): 2190. Sanchez AM, et al., Effect of overexpression of a soluble pyridine nucleotide transhydrogenase (UdhA) on the production of poly(3- hydroxybutyrate) in Escherichia coli, Biotechnol Prog. 22(2):420-5 (2006). Wang Y, at al, Improvement of NADPH bioavailability in Escherichia coli by replacing NAD+-dependent glyceraldehyde-3- phosphate dehydrogenase GapA with NADP +-dependent GapB from Bacillus subtilis and addition ofNAD kinase, J Ind Microbiol Biotechnol. 2013. PMID: 24048943. Zhang X, Li M, Agrawal A, San KY, Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases, Metab Eng. 13(6):713-22 (2011). * cited by examiner U.S. Patent Jul. 3, 2018 Sheet 1 of 17 US 10,011,854 B2 FIGURE IA Glucose 1 PEP 1 Pyruvate ---.. Acetate ! _/ H•, HCO:;, ATP ADP, p+ Acety! CoA--------__:::,,._,-,::;c/ '--- / ___ .. Malony!-CoA Ace Acy!-CoA ! p ACP-SH "}- - bD r AC -SH Fatty --------. Fab ,..a '---.. Co-A-SH ..,, OAA ~ acids Acy!-ACP ./ --......, thioesterase Ma!onyl-ACP Ma!ate ! Citrate NAD(P) Acyl-ACP ~ FumarateI \ [:] !socitrate NAD 'P)Hr-noyl-ACP l reductase CO + ACP beta-ketoacy!-AC 2 \ J synthase 1/U Succinate o.-ketoglutara!e Enoy!-ACP 3-Ketoacyl-ACP "'Succiny!-CoA / droxyacyl-ACP 7( NADPH and/or H20 dralase 3-oxoacyl-ACP NADH reductase -- 3 D Hyd y! ACP NADP+ and/or - - roxyac - NAD• Fatty acid elongation cycle U.S. Patent Jul. 3, 2018 Sheet 2 of 17 US 10,011,854 B2 FIGURE 1B 0 0 II 11 -o-C-CH2-C-S-CoA 0 II R-C-S-ACP FabD l 0 0 U II FabB -o-C-CH2-C-S-ACP H++ NADH FabF 0 FabH II CHrC-S-CoA 0 0 0 II II II R-CH=CH-C-S-ACP R-C-CH2-C-S-ACP 0 0 11 II FabA CHs-C-CHrC-S-ACP FabZ OH 0 I II R-CH-CHrC-S-ACP NADP+ U.S. Patent Jul. 3, 2018 Sheet 3 of 17 US 10,011,854 B2 FGUREIC Descriptio Step Enzyme Reaction n Activates acetyl CoA Acetyl ~) 0 for reaction (a) CoA:ACP with transacylase G.}·i;'JL.•. ::-1-0:iA (:cH,/L.. ,S-ACP malonyl­ ACP Activates malonyl Malonyl CoA for (b) CoA:ACP reaction transacylase with acetyl­ ACP Reacts priming 3-ketoacyl­ ,i acetyl-ACP ...... _......... : (c) ACP .. with chain­ synthetase {C} extending malonyl­ ACP. NADPH+H;J) Reduces the carbon 3-ketoacyl­ \ ,-,,="' 3 ketone (d) ACP . \-~..,:::'.":.:: ........ toa reductase hydroxyl group 3- H i'' Hydroxyacyl Removes (e) ACP Cl·(;'°L::,1)CS~ACP water dehydrase H NADPH + H © H 0 Enoyl-ACP (f} reductase 01{1.:y)t.. S·"ACP H U.S. Patent Jul. 3, 2018 Sheet 4 of 17 US 10,011,854 B2 trr;. prom11t~r Ras RBS FlGURE2 Abbreviations: RBS, ribosomal binding site; rcTE, acyl-ACP thioesterase from R. communis; MtGl, a NAD"'-dependent 3-oxoacyl-ACP reductase from lvf. tuberculosis; lacl, regulator gene of trc promoter system; AmpR, ampicillin resistant gene; T 1 tem1inator and T2 tenninator, transcriptional terminator of rrnB; pBR322 origin, origin of replication. U.S. Patent Jul. 3, 2018 Sheet 5 of 17 US 10,011,854 B2 /tt:. µr~mt~ttir RSS pXZ18G2 RES Amp,R 12 terminatar FIGURE 3A Abbreviations: RBS, ribosomal binding site; rcTE, acyl-ACP thioesterase from R. communis; MtG2, a NAD+-dependent 3-oxoacyl-ACP reductase from M. tuberculosis with an omission of the first 16 amino acids; lacl, regulator gene of trc promoter system; AmpR, ampicillin resistant gene; Tl terminator and T2 tem1inator, transcriptional terminator of rrnB; pBR322 origin, origin of replication. U.S. Patent Jul. 3, 2018 Sheet 6 of 17 US 10,011,854 B2 trc promoter l / RBS / /" } / t ···-··········"""-.. ,._ 'l '· t'fr' ~tl>a l ( :s. 5 3.3) pXZ18DG2 kt==- MttabG2 Amp~:~~ "~' t2 terminatnr_ .~~:com {3c17:5) _:j'J .··.·_ir:~:,.,.,.,.... .····:·:·:·:»?''*'''':~;Z@fW" . T1 te:rminator- · / . .,\. / \ Hin d1H {4-5 64}° mtfab-G2 FIGURE 3B U.S. Patent Jul. 3, 2018 Sheet 7 of 17 US 10,011,854 B2 FIGURE 4 SEQ ID NO: 1 (Ricinus communis palrnitoyl-acyl carrier protein thioestcrasc (rcTE) from XM - 0025l5518fXP- 002515564 MVATAAAATSSFFPVPSQSADANFDKAPASLGGIKLKSTSCSRG LQVKANAQAPPKINGSSVGFTTSVETVKNDGDMPLPPPPRTFINQLPDWSMLLAAITT IFLAAEKQWMMLDWKPRRPDMLIDPFGIGRIVQDGLIFRQNFSIRSYEIGADRTASIE TLMNHLQETALNHVKTAGLLGDGFGSTPEMSKRNLIWVVTRMQVLVDRYPTWGDVVQV DTWVSKSGKNGMRRDWCVRDSRTGETLTRASSVWVMMNKLTRRLSKIPEEVRGEIEPY FLNSDPIVDEDSRKLPKLDDSNADYVRKGLTPRWSDLDINQHVNNVKYIGWILESAPL PILESHELSAITLEYRRECGRDSVLQSLTAVSGNGIGNLGNAGDIECQHLLRLEDGAE IVRGRTEWRPKYSSNFGIMGQIPVESA FIGURE 5 SEQ ID NO: 2 3-oxoacyl-ACP rcductasc FabG [EC:1.1.1.100] from Mycobactcrium tuberculosis MAPKRSSDLFSQVVNSGPGSFLARQLGVPQPETLRRYRAGEPPLTGSLLIGGAGRVVEPL RAALEKDYDLVGNNLGGRWADSFGGLVFDATGITEPAGLKGLHEFFTPVLRNLGRCGRVV VVGGTPEAAASTNERIAQRALEGFTRSLGKELRRGATTALVYLSPDAKPAATGLESTMRF