USOO8883464B2

(12) United States Patent (10) Patent No.: US 8,883,464 B2 Lynch et al. (45) Date of Patent: Nov. 11, 2014

(54) METHODS FOR PRODUCING (56) References Cited 3-HYDROXYPROPIONCACID AND OTHER U.S. PATENT DOCUMENTS PRODUCTS M 2.408,889 A 10, 1946 Short (75) Inventors: Michael D. Lynch, Boulder, CO (US); 2.464,768 A 3, 1949 Redmon et al. Ryan T. Gill, Denver, CO (US); Tanya 2.469,701 A 5/1949 Redmon E. W. Lipscomb, Boulder, CO (US) 2,798,0533,904,685. A 9/19757, 1957 ShahidiBrown et et al. al. (73) Assignees: OPX Biotechnologies, Inc., Boulder, 4,029,5773,915,921 A 10/19756, 1977 Schlatzer,E. Jr. s al. CO (US); The Regents of the 4,268,641 A 5/1981 Koenig et al. University of Colorado, a Body 1945. A 13. Mister et al. Corporate, Denver, CO (US) 4,666,983.I - J. A 5/1987 Tsubakimoto et al. 4,685,915 A 8, 1987 Hasse et al. (*) Notice: Subject to any disclaimer, the term of this 4,708,997 A 1 1/1987 Stanley, Jr. et al. patent is extended or adjusted under 35 3. A s 3. Its et al. U.S.C. 154(b) by 0 days. 4,952.505. A 8/1990 Chomelir et al. 4,985,518 A 1/1991 Alexander et al. (21) Appl. No.: 13/498,468 5,009,653 A 4/1991 Osborn, III 5,093,472 A 3, 1992 Bresciani (22) PCT Filed: Sep. 27, 2010 5,135,677 A 8/1992 Yamaguchi et al. 5,145,906 A 9, 1992 Chambers et al. (Under 37 CFR 1.47) 5, 180,798. A 1/1993 Nakamura et al. 5,252.474. A 10, 1993 Gewain et al. (86). PCT No.: PCT/US2O1 O/OSO436 5.331,059 A 7/1994 Engelhardt et al. 5,342,899 A 8, 1994 Graham et al. S371 (c)(1), 5,350,799 A 9/1994 Woodrum et al. (2), (4) Date: Feb. 12, 2013 5.426,199 A 6/1995 Lundquist 5,470,928 A 11/1995 Harwood et al. (87) PCT Pub. No.: WO2011/038364 5,510,307. A 4, 1996 Narayanan et al. 5,510,526 A 4, 1996 Baniel et al. PCT Pub. Date: Mar. 31, 2011 5,558,656 A 9/1996 Bergman 5,723,639 A 3, 1998 Datta et al. (65) Prior Publication Data (Continued) US 2013 FOOT1893 A1 Mar. 21, 2013 FOREIGN PATENT DOCUMENTS

EP 1124789 B1 9, 2004 Related U.S. Application Data EP 1036190 B1 5, 2005 (60) Provisional application No. 61/246,141, filed on Sep. (Continued) 27, 2009, provisional application No. 61/298,844, OTHER PUBLICATIONS filed on Jan. 27, 2010, provisional application No. -- 61/321,480, filed on Apr. 6, 2010. Chica et al. Curr Opin Biotechnol. Aug. 2005:16(4):378-84.* Sen et al. Appl Biochem Biotechnol. Dec. 2007: 143(3):212-23.* (51) Int. Cl. Asano, et al. A new enzymatic method of acrylamide production. CI2P 7/40 (2006.01) Agricultural and Biological Chemistry. 1982; 46(5): 1183-1190. Brown, et al. Synthesis of labeled acrylamide and CI2N 9/00 (2006.01) N-methylolacrylamide (NMA): 15N-acrylamide, 13C-NMA, 15N CI2N L/20 (2006.01) NMA, and 13C, 15N-NMA. Journal of labelled compounds & CI2M I/00 (2006.01) radiopharmaceuticals. 2005; 48(14): 1031-1039. CI2N IS/00 (2006.01) Kurcok, et al. Reactions off-lactones with potassium alkoxides and C7H 2L/04 (2006.01) their complexes with 18-crown-6 in aprotic solvents. Journal of CI2N 9/02 (2006.01) Organic Chemistry. 1993; 58(16):4219-4220. A6IL I5/24 (2006.01) Langlois, et al. A new preparation of trifluoromethanesulfinate salts. CI2P 7/42 (2006.01) Journal of Fluorine Chemistry. 2007: 128(7):851-856. CI2P 7/52 (2006.01) (Continued) CI2N 15/63 (2006.01) (52) U.S. Cl. Primary Examiner — Christian Fronda CPC, C12N 15/63 (2013.01); C12P 7/40 (2013.01); (74) Attorney, Agent, or Firm — Wilson Sonsini Goodrich & CI2N 9/0008 (2013.01); A61L 15/24 Rosati (2013.01): CI2P(2013.01); 742 (2013,0): CI2P 752 CI2M (2013.01) 4712 (57) ABSTRACT USPC ..... 435/136: 435/183:435/252.3 435/294.1 This invention relates to metabolically engineered microor s s 435 ?320.1 5361232 ganism strains, such as bacterial strains, in which there is an increased utilization of malonyl-CoA for production of a (58) Field of Classification Search chemical product, which includes 3-hydroxypropionic acid. None See application file for complete search history. 31 Claims, 65 Drawing Sheets US 8,883,464 B2 Page 2

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Purification and characterization of 2-oxoglutarate NCBI Reference Sequence: ZP 05125944.1 (Sep. 15, 2008). decarboxylase of Leuconostoc oenos. Journal of General Microbiol Nexant, Inc. Chemsystems Perp Program, Acrylic Acid, 08/09-3, Jul. ogy 136 (1990), 1497-1499. 2010. Kern, et al. Engineering primary metabolic pathways of industrial Office action dated Apr. 29, 2011 for U.S. Appl. No. 12/328,588. micro-organisms. J Biotechnol. Mar. 30, 2007: 129(1):6-29. Epub Office action dated Jul. 4, 2011 for EP Applilcation No. 08727619.2. Dec. 2, 2006. Office action dated Sep. 17, 2010 for U.S. Appl. No. 12/328,588. Kim et al. Extractive Recovery of Products from Fermentation Ohmiya, et al. Structure of cellulases and their applications. Broths. Biotechnol. Bioprocess Eng., 1999; 4:1-11. Biotechnol Genet Eng Rev. 1997:14:365-414. Kim, et al. The Rutpathway for pyrimidine degradation: novel chem Ohnishi, et al. A novel methodology employing Corynebacterium istry and toxicity problems. 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Average vs. 3HP Concentration Day 1: E. Coli BW25113, Aerobic

Figure 5A

Average AOD vs. 3HP Concentration Day 1: E. Coli BW25113, Aerobic

Figure 15B U.S. Patent Nov. 11, 2014 Sheet 49 of 65 US 8,883,464 B2

Average Na vs. 3HP Concentration Day 1: E. Coli BW25113, Aerobic

Figure 15C

Average vs. 3HP Concentration Day 1: E. Coli BW25113, Anaerobic

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Average AOD vs. 3HP Concentration Day 1: E. Coli BW25113, Anaerobic

3HP) (g/L)

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Average N. vs. 3HP Concentration Day 1: E. Coli BW25113, Anaerobic

Figure 15F U.S. Patent Nov. 11, 2014 Sheet 51 of 65 US 8,883,464 B2

Average vs. 3HP Concentration Day 1: B. Subtilis, Aerobic

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Average AOD vs. 3HP Concentration Day 1: B. Subtilis, Aerobic

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Average vs. 3HP Concentration Day 1: S. Cervisiae S288C, Aerobic

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Average AOD vs. 3HP Concentration Day 1: S. Cervisiae S288C, Anaerobic

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phosphoenolpyruvate oxaloacetate oad-2 NAD-- ACP actate CO NADH Ap malonate semialdehyde pyruvate poxB a UQ COA acetate UCH2 pfie CO formate acetyl-coA

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U.S. Patent Nov. 11, 2014 Sheet 65 of 65 US 8,883,464 B2

FIG. 25A DRY FIBRGUS PBYMER AND FBER MARIAS FBRS MAERIA NAIA

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POLYETY ENE BOTTOM SEE US 8,883,464 B2 1. 2 METHODS FOR PRODUCING net conversion in a fermentative microorganism cell to 3-HYDROXYPROPIONCACID AND OTHER desired target chemical products employing commercially PRODUCTS viable fermentation methods. More particularly, among prob lems remaining to be solved are how to improve specific CROSS-REFERENCE TO RELATED productivity and Volumetric productivity, such as to economi APPLICATIONS cally important levels, in modified microorganisms that are adapted to produce a chemical product having malonyl-CoA This is a national phase entry under 35 U.S.C. 371 of PCT as a Substrate in the microbial production pathway of that Application No. PCT/US2010/050436, filed on Sep. 27, chemical product, such as 3-hydroxypropionic acid (3-HP). 2010, which claims priority from U.S. Provisional Applica 10 tion 61/246,141, filed Sep. 27, 2009, U.S. Provisional Appli SUMMARY OF THE INVENTION cation 61/298.844, filed Jan. 27, 2010, and U.S. Provisional Application 61/321,480, filed Apr. 6, 2010. The entire con According to one embodiment, the invention is directed to tents of each application are hereby incorporated by reference a method for producing an acrylic acid-based consumer prod in their entirety. 15 uct, said method comprising i) combining a carbon source and a microorganism cell culture to produce 3-hydroxypro FIELD OF THE INVENTION pionic acid, whereina) said cell culture comprises an inhibi tor offatty acid synthase or said microorganism is genetically This invention relates to metabolically engineered micro modified for reduced enzymatic activity in the organism’s organisms, such as bacterial strains, in which there is an fatty acid synthase pathway; or b) wherein said microorgan increased utilization of malonyl-CoA for production of a ism is genetically modified for increased enzymatic activity chemical product, which may include the chemical 3-hydrox in the organism's malonyl-CoA reductase (mcr) pathway by ypropionic acid (3-HP) and products made from 3-HP. The introduction of a heterologous nucleic acid sequence coding metabolically engineered microorganisms may be adapted to for a polypeptide having mono-functional malonyl-CoA exhibit increased tolerance to 3-HP. Also, genetic modifica 25 reductase activity; or c) said 3-hydroxypropionic acid is pro tions may be made to provide one or more 3-HP biosynthesis duced at a specific productivity of greater than 0.05 grams per pathways such as in microorganisms comprising one or more gram of microorganism cell on a dry weight basis per hour or genetic modifications of a complex identified as the 3-HP at a volumetric productivity of greater than 0.50 grams per toleragenic complex. liter per hour; ii) converting the 3-hydroxypropionic acid to 30 acrylic acid; and iii) processing the acrylic acid into a con SEQUENCE LISTING Sumer product. In various aspects, the carbon Source has a ratio of carbon-14 to carbon-12 of about 1.0x10' or greater. The instant application contains a Sequence Listing which The carbon Source according to the invention may be pre has been submitted in ASCII format via EFS-Web and is dominantly glucose, Sucrose, fructose, dextrose, lactose, or a hereby incorporated by reference in its entirety. Said ASCII 35 combination thereof. Alternatively, the carbon Source is glyc copy, created on May 2, 2013, is named 34246-744-831 erol. SL.txt and is 2,177 Kilobytes in size. In certain embodiments, the cell culture comprises an inhibitor of fatty acid synthase or said microorganism is BACKGROUND OF THE INVENTION genetically modified for reduced enzymatic activity in the 40 organism's fatty acid synthase pathway. For example, the With increasing acceptance that petroleum hydrocarbon inhibitor of a fatty acid synthase may be selected from the Supplies are decreasing and their costs are ultimately increas group consisting of thiolactomycin, triclosan, cerulenin, ing, interest has increased for developing and improving thienodiazaborine, isoniazid, and analogs thereof. industrial microbial systems for production of chemicals and The microorganism of the invention may be genetically fuels. Such industrial microbial systems could completely or 45 modified for increased enzymatic activity in the organism’s partially replace the use of petroleum hydrocarbons for pro malonyl-CoA reductase (mcr) pathway by introduction of a duction of certain chemicals. heterologous nucleic acid sequence coding for a polypeptide Numerous chemicals are produced through such means, having mono-functional malonyl-CoA reductase activity. In ranging from antibiotic and anti-malarial pharmaceutical various embodiments, the mono-functional malonyl-CoA products to fine chemicals to fuels such as ethanol. Commer 50 reductase is NADPH-independent. cial objectives for microbial fermentation include the In various embodiments, the 3-hydroxypropionic acid is increase of titer, production rate, and yield of a target chemi produced according to the invention at a specific productivity cal product. When the overall specific productivity in a fer of greater than 0.05 grams per gram of microorganism cell on mentation event is elevated, this may positively affect yield in a dry weight basis per hour or at a volumetric productivity of addition to production rate and other economic factors, such 55 greater than 0.05 grams per liter per hour. as capital costs. Included within the invention are embodiments where the One candidate chemical for such production is 3-hydrox cell culture comprises a genetically modified microorganism. ypropionic acid (“3-HP, CAS No. 503-66-2), which may be The genetically modified microorganism can be modified for converted to a number of basic building blocks for polymers a trait selected from reduced enzymatic activity in the organ used in a wide range of industrial and consumer products. 60 ism's fatty acid synthase pathway, increased enzymatic activ Unfortunately, previous efforts to microbially synthesize ity in the organism’s malonyl-CoA reductase pathway, 3-HP to achieve commercially viable titers have revealed that increased tolerance to 3-hydroxypropionic acid, increased the microbes being used were inhibited by concentrations of enzymatic activity in the organism's NADPH-dependent 3-HP far below a determined commercially viable titer. transhydrogenase pathway, increased intracellular bicarbon In spite of strong interest to improve microbial fermenta 65 ate levels, increased enzymatic activity in the organism’s tion economics by improving yield and/or productivity for acetyl-CoA carboxylase pathway, and combinations thereof. certain chemical products, there remains a need to increase For example, the genetically modified microorganism can be US 8,883,464 B2 3 4 modified for reduced enzymatic activity in the organism’s ganism cell on a dry weight basis per hour or at a Volumetric fatty acid synthase pathway. Alternatively, the reduced enzy productivity of greater than 0.50 grams per liter per hour. matic activity is a reduction in enzymatic activity in an The method of the invention may include production of a enzyme selected from the group consisting of beta-ketoacyl consumer product, Such as diapers, carpet, paint, adhesives, ACP reductase, 3-hydroxyacyl-CoA dehydratase, enoyl and acrylic glass. The invention includes biologically-pro ACP reductase, and thioesterase. In various aspects, the duced 3-hydroxypropionic acid, where the 3-hydroxypropi reduced enzymatic activity in the organism's fatty acid syn onic acid is produced according to the method of the inven thase pathway occurs via introduction of a heterologous tion. Such 3-hydroxypropionic acid may be essentially free of nucleic acid sequence coding for an inducible promoter oper chemical catalyst, including a molybdenum and/or vanadium ably linked to a sequence coding fora enzyme in the fatty acid 10 based catalyst. The 3-hydroxypropionic acid is produced synthase pathway or homolog thereof, or a heterologous according to the method of the invention may have a ratio of nucleic acid sequence coding for an enzyme in the fatty acid carbon-14 to carbon-12 of about 1.0x10' or greater. In synthase pathway or homolog thereof with reduced activity. various aspects, the 3-hydroxypropionic acid contains less In various aspects, the enzyme in the fatty acid synthase than about 10% carbon derived from petroleum. In addition, pathway or homolog thereof is a polypeptide with tempera 15 3-hydroxypropionic acid according to the invention may con ture-sensitive beta-ketoacyl-ACP or temperature-sensitive tain a residual amount of organic material related to its enoyl-ACP reductase activity. Variously, the genetically method of production. In various embodiments, the 3-hy modified microorganism is modified for increased enzymatic droxypropionic acid contains a residual amount of organic activity in the organism's malonyl-CoA reductase pathway. material in an amount between 1 and 1,000 parts per million In certain embodiments, the increase in enymatic activity of the 3-hydroxypropionic acid. in the malonyl-CoA reductase (mcr) pathway occurs by intro Acrylic acid and a polymer produced from acrylic acid, duction of a heterologous nucleic acid sequence coding for a where such are produced according to the method of the polypeptide having bi-functional malonyl-CoA reductase invention, are also included within the invention. Products, enzymatic activity or mono-functional malonyl-CoA reduc including commercial and consumer products, obtained from tase activity. The heterologous nucleic acid sequence may be 25 the polymers are also encompassed. For example, diapers, selected from a sequence having at least 70% homology with carpet, paint, adhesives, and acrylic glass are encompassed. a sequence selected from SEQID NO. 780-789. In addition, the invention encompasses a system for bio In various embodiments, the genetically modified micro production of acrylic acid according to claim 40, said system organism is modified for increased tolerance to 3-hydrox comprising: a tank for Saccharification of biomass; a line for ypropionic acid. The increase in tolerance to 3-hydroxypro 30 passing the product of saccharification to a fermentation tank pionic acid may occur in one or more components of the 3-HP optionally via a pre-fermentation tank; a fermentation tank toleragenic complex (3HPTGC) complex, or wherein said suitable for microorganism cell culture; a line for discharging increase in tolerance to 3-hydroxypropionic acid results from contents from the fermentation tank to an extraction and/or providing at least one genetic modification of each of Group separation vessel; an extraction and/or separation vessel Suit A and Group B of the 3HPTGC. The one or more components 35 able for removal of 3-hydroxypropionic acid from cell culture may be selected from CynS, CynT. AroG, Spe), SpeE, SpeF, waste; a line for transferring 3-hydroxypropionic acid to a ThrA, Asd, CysM, IroK, Ilv A, and homologs thereof. In dehydration vessel; and a dehydration vessel suitable for con various embodiments, the modification is a disruption of one version of 3-hydroxypropionic acid to acrylic acid. In various or more 3HPTGC repressor genes. The repressor genes may embodiments, the system further comprises one or more pre be selected from tyrR, trpR, metJ, purR, lysR, nrdR, and 40 fermentation tanks, distillation columns, centrifuge vessels, homologs thereof. back extraction columns, mixing vessels, or combinations Increased enzymatic activity in the organism's NADPH thereof. In various embodiments, the system has a minimum dependent transhydrogenase pathway may occur by introduc production capacity of at least 1 ton acrylic acid per year. tion of a heterologous nucleic acid sequence coding for a Within the scope of the invention are genetically modified polypeptide having at least 70% homology with a sequence 45 microorganism, wherein the microorganism is capable of selected from SEQ ID NO. 776 or 778. In various embodi producing 3-hydroxypropionate at a specific rate selected ments, the increased intracellular bicarbonate levels occurs from the rates of greater than 0.05 g/g)CW-hr, 0.08 by introduction of a heterologous nucleic acid sequence cod g/gDCW-hr, greater than 0.1 g/g|DCW-hr, greater than 0.13 ing for a polypeptide having cyanase and/or carbonic anhy g/gDCW-hr, greater than 0.15g/gDCW-hr, greater than 0.175 drase activity. Heterologous nucleic acid sequence may be 50 g/gDCW-hr, greater than 0.2 g/g|DCW-hr, greater than 0.25 selected from a sequence having at least 70% homology with g/gDCW-hr, greater than 0.3 g/g|DCW-hr, greater than 0.35 a sequence selected from SEQID NO. 337. g/gDCW-hr, greater than 0.4 g/g|DCW-hr, greater than 0.45 In various embodiments, an increased enzymatic activity g/gDCW-hr, or greater than 0.5 g/g)CW-hr. in the organism’s acetyl-CoA carboxylase pathway occurs by The genetically modified microorganism may comprise introduction of a heterologous nucleic acid sequence coding 55 genetic modifications to increase malonyl-coA reductase for a polypeptide having at least 70% homology with a activity and acetyl-coA carboxylase activity, and genetic sequence selected from SEQID NO. 768-775. modifications to reduce enoyl-ACP reductase activity, lactate The genetically modified bacteria may be further modified dehydrogenase activity and acetate kinase activity. Variously, to decrease activity of lactate dehydrogenase, phosphate the microorganism comprises genetic modifications to acetyltransferase, pyruvate oxidase, or pyruvate-formate 60 increase malonyl-coA reductase activity and acetyl-coA car lyase, and combinations thereof. boxylase activity, and genetic modifications to reduce enoyl The method according to the invention may further com ACP reductase activity, lactate dehydrogenase activity and prise separating and/or purifying 3-hydroxypropionic acid acetylphosphate transferase activity. In addition, the micro from said cell culture by extraction of 3-hydroxypropionic organism may comprise genetic modifications to increase acid from said culture in the presence of a tertiary amine. 65 malonyl-coA reductase activity and acetyl-coA carboxylase Variously, 3-hydroxypropionic acid is produced at a specific activity, and genetic modifications to reduce enoyl-ACP productivity of greater than 0.05 grams per gram of microor reductase activity, lactate dehydrogenase activity, acetate US 8,883,464 B2 5 6 kinase activity and acetylphosphate transferase activity. In activity, increases 2-dehydro-3-deoxyphosphoheptonate various aspects, the microorganism comprises genetic modi aldolase activity, and/or increases D-3-phosphoglycerate fications to increase malonyl-coA reductase activity and dehydrogenase activity. acetyl-coA carboxylase activity, and genetic modifications to In various embodiments, the invention includes a culture reduce enoyl-ACP reductase activity, lactate dehydrogenase system comprising a carbon Source in an aqueous medium activity and pyruvate formate lyase activity. In various and a genetically modified microorganism according to any embodiments, the microorganism comprises genetic modifi one of claims 48-92, wherein said genetically modified cations to increase malonyl-coA reductase activity and organism is present in an amount selected from greater than acetyl-coA carboxylase activity, and genetic modifications to 0.05 g|DCW/L, 0.1 g|DCW/L, greater than 1 g|DCW/L, greater reduce enoyl-ACP reductase activity, lactate dehydrogenase 10 than 5 g|DCW/L, greater than 10 g|DCW/L, greater than 15 activity and pyruvate oxidase activity. Also included are gDCW/L or greater than 20 g|DCW/L, such as when the microorganisms comprising genetic modifications to Volume of the aqueous medium is selected from greater than increase malonyl-coA reductase activity and acetyl-coA car 5 mL, greater than 100 mL, greater than 0.5 L, greater than 1 boxylase activity, and genetic modifications to reduce enoyl L. greater than 2 L, greater than 10 L, greater than 250 L. ACP reductase activity, lactate dehydrogenase activity and 15 greater than 1000 L, greater than 10,000 L, greater than methylglyoxal synthase activity. In addition, microorganisms 50,000 L, greater than 100,000 L or greater than 200,000 L, according to the invention may comprise genetic modifica and Such as when the Volume of the aqueous medium is tions to increase malonyl-coA reductase activity and acetyl greater than 250 L and contained within a steel vessel. coA carboxylase activity, and genetic modifications to Variously, the carbon Source for Such culture systems is increase B-ketoacyl-ACP synthase activity, and decrease lac selected from dextrose, Sucrose, a pentose, a polyol, a hexose, tate dehydrogenase activity and methylglyoxal synthase both a hexose and a pentose, and combinations thereof, the activity, and/or the microorganism may comprise genetic pH of the aqueous medium is less than 7.5, the culture system modifications to increase malonyl-coA reductase activity and is aerated, such as at an oxygen transfer rate selected from i) acetyl-coA carboxylase activity, and genetic modifications to greater than 5 mmole/L-hr of oxygen and less than 200 reduce enoyl-ACP reductase activity, guanosine 3'-diphos 25 mmole/L-hr oxygen; ii) greater than 5 mmole/L-hr of oxygen phate 5'-triphosphate synthase activity, and guanosine and less than 100 mmole/L-hr oxygen; iii) greater than 5 3'-diphosphate 5'-diphosphate synthase activity. Also, in mmole/L-hr of oxygen and less than 80 mmole/L-hr oxygen; Some microorganisms enoyl-CoA reductase, is reduced and iv) greater than 5 mmole/L-hr of oxygen and less than 50 instead of or in addition to doing such for enoyl-ACP reduc mmole/L-hr oxygen. tase activity. 30 In various embodiments, the invention is an aqueous broth In various embodiments, a further genetic modification has obtained from a culture system according to any one of claims been made that increases NADH/NADPH transhydrogenase 93-99, wherein said aqueous broth comprises i) a concentra activity. For example, the transhydrogenase activity may be tion of 3-hydroxypropionate selected from greater than 5g/L, soluble, may be membrane bound, may have a further genetic greater than 10 g/L, greater than 15 g/L, greater than 20 g/L. modification that has been made that increases cyanase activ 35 greater than 25 g/L, greater than 30 g/L, greater than 35 g/L. ity, may include a further genetic modification that increases greater than 40 g/L, greater than 50 g/L, greater than 60 g/L. carbonic anhydrase activity, and/or may include a further greater than 70 g/L, greater than 80 g/L, greater than 90 g/L. genetic modification that increases pyruvate dehydrogenase or greater than 100 g/L 3-hydroxypropionate; and ii) a con activity. centration of 1,3-propanediol selected from less than 30 g/L.; In various embodiments, a further genetic modification has 40 less than 20 g/L, less than 10 g/L, less than 5g/L, less than 1 been made that decreases guanosine 3'-diphosphate 5'-triph g/L, or less than 0.5 g/L. In some aspects, the aqueous broth osphate synthase activity, and guanosine 3'-diphosphate comprises an amount of biomass selected from less than 20 5'-diphosphate synthase activity. Also included is when a gDCW/L biomass, less than 15 g|DCW/L biomass, less than genetic modification has been made that increases the 10 g|DCW/L biomass, less than 5 gDCW/L biomass or less NADH/NAD+ ratio in an aerated environment. Further, a 45 than 1 g)CW/L biomass. Alternatively, the aqueous broth genetic modification may be made that decreases B-ketoacyl according to the invention is such that the 3-HP/succinate ACP synthase activity, decreases 3-hydroxypropionate ratio (g3-HP/g Succinate) is greater than 3, greater than 10 reductase activity, decreases NAD+ dependant 3-hydrox greater than 30, greater than 60, greater than 100, greater than ypropionate dehydrogenase activity, decreases NAD+ depen 150 or greater than 200. In various aspects, the 3-HP/fumarate dant 3-hydroxypropionate dehydrogenase activity, increases 50 ratio (g3-HP/g fumarate) is greater than 3, greater than 10 tolerance to 3-hydroxypropionic acid, increases activity of greater than 30, greater than 60, greater than 100, greater than any enzyme in the 3-HPtoleragenic complex, increases pyru 150 or greater than 200, or the 3-HP/glycerol ratio (g3-HP/g vate dehydrogenase activity, increases cyanase activity, glycerol) is greater than 3, greater than 10, greater than 30, increases carbonic anhydrase activity, increases aspartate greater than 60, greater than 100, greater than 150 or greater kinase activity, increases threonine dehydratase activity, 55 than 200, or the 3-HP/acetate ratio (g3-HP/g acetate) is increases 2-dehydro-3-deoxyphosphoheptonate aldolase greater than 1.5, greater than 3, greater than 10, greater than activity, increases cysteine synthase activity, increases 30, greater than 60, greater than 100, greater than 150 or ribose-phosphate diphosphokinase activity, increases ribo greater than 200, or the 3-HP/alanine ratio (g3-HP/galanine) nucleoside-diphosphate reductase activity, increases L-cys is greater than 3, greater than 10, greater than 30, greater than teine desulfhydrase activity, increases lysine decarboxylase 60 60, greater than 100, greater than 150 or greater than 200, or activity, increases homocysteine transmethylase activity, the 3-HP/beta-alanine ratio (g3-HP/g beta-alanine) is greater increases dihydrofolate reductase activity, increases than 1.5, greater than 3, greater than 10, greater than 30, N-acetylglutamylphosphate reductase activity, increases greater than 60, greater than 100, greater than 150 or greater acetylglutamate kinase activity, increases argininosuccinate than 200, or the 3-HP/glutamate ratio (g3-HP/gglutamate) is lyase activity, increases acetylornithine deacetylase activity, 65 greater than 3, greater than 10, greater than 30, greater than increases chorismate mutase activity, increases prephenate 60, greater than 100, greater than 150 or greater than 200, or dehydratase activity, increases prephenate dehydrogenase the 3-HP/glutamine ratio (g3-HP/gglutamine) is greater than US 8,883,464 B2 7 8 3, greater than 10, greater than 30, greater than 60, greater FIG.9D, sheets 1-7, provides a multi-sheet depiction of the than 100, greater than 150 or greater than 200, or the 3-HP/ 3HPTGC for Cupriavidus necator (previously, Ralstonia 3-hydroxypropionaldehyderatio (g3-HP/g3-hydroxypropio eutropha). Sheet 1 provides a general Schematic depiction of aldehyde) is greater than 1.5, greater than 3, greater than 10, the arrangement of the remaining sheets. greater than 30, greater than 60, greater than 100, greater than 5 FIG. 10 provides a representation of the glycine cleavage 150 or greater than 200, or the 3-HP/1,3-propanediol ratio pathway. (g3-HP/g 1,3-propanediol) is greater than 1.5, greater than 3, FIG. 11 provides, from a prior art reference, a summary of greater than 10, greater than 30, greater than 60, greater than a known 3-HP production pathway from glucose to pyruvate 100, greater than 150 or greater than 200, and/or the 3-HP/ to acetyl-CoA to malonyl-CoA to 3-HP. lactate ratio (g3-HP/g lactate) is greater than 3, greater than 10 FIG. 12 provides, from a prior art reference, a summary of 10, greater than 30, greater than 60, greater than 100, greater a known 3-HP production pathway from glucose to phospho than 150 or greater than 200. enolpyruvate (PEP) to oxaloacetate (directly or via pyruvate) to aspartate to B-alanine to malonate semialdehyde to 3-HP. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 13 provides, from a prior art reference, a summary of 15 known 3-HP production pathways. The novel features of the invention are set forth with par FIGS. 14A and B provide a schematic diagram of natural ticularity in the claims. A better understanding of the features mixed fermentation pathways in E. coli. and advantages of the present invention will be obtained by FIG. 15A-O provides graphic data of control microorgan reference to the following detailed description that sets forth isms responses to 3-HP, and FIG. 15P provides a comparison illustrative embodiments, in which the principles of the with one genetic modification of the 3HPTG.C. invention are utilized, and the accompanying drawings of FIG. 16 depicts a known chemical reaction catalyzed by which: alpha-ketoglutarate encoded by the kgd gene from M. tuber FIG. 1 depicts metabolic pathways of a microorganism culosis. related to aspects of the present invention, more particularly 25 FIG. 17depicts a new enzymatic function, the decarboxy related to 3-HP production, with gene names of E. coli shown lation of Oxaloacetate to malonate semialdehyde that is to be at certain enzymatic steps, the latter for example and not achieved by modification of the kgd gene. meant to be limiting. FIG. 18 shows a proposed selection approach for kgd FIG. 2A depicts metabolic pathways of a microorganism mutantS. related to aspects of the present invention, with gene names of 30 FIG. 19 shows a screening protocol related to the proposed E. coli shown at certain enzymatic steps, the latter for selection approach depicted in FIG. 18. example and not meant to be limiting. FIG. 20 provides a comparison regarding the IroK peptide FIG. 2B provides a more detailed depiction of representa Sequence. tive enzymatic conversions and exemplary E. coli genes of the FIG. 21 provides a calibration curve for 3-HP conducted fatty acid synthetase system that was more generally depicted 35 with HPLC. in FIG. 2A. FIG. 22 provides a calibration curve for 3-HP conducted FIG. 3 provides an exemplary multiple sequence align for GC/MS. ment, comparing carbonic anhydrase polypeptides FIG. 23 provides a representative standard curve for the (CLUSTAL 2.0.12 multiple sequence alignment of Carbonic enaymatic assay for 3-HP. Anhydrase Polypeptides). 40 FIGS. 24 A, B, and C and FIGS. 25 A and B show a FIG. 4A provides an exemplary sequence alignment: Com schematic of the entire process of converting biomass to a parison of DNA sequences of fablf (JP1111 (SEQ ID No.: finished product such as a diaper. 769)) and wildtype (BW25113 (SEQ ID No.:827)) E. coli Tables also are provided herein and are part of the specifi fabl genes DNA mutation: C722T. cation. FIG. 4B provides an exemplary sequence alignment: Com 45 parison of protein sequences of fabl (JP1111 (SEQID No.: DETAILED DESCRIPTION OF THE INVENTION 770) and wildtype (BW25113 (SEQID No.:828)) E. coli fabl genes Amino Acid-S241F. The present invention is related to various production FIGS.5, 6 and 7 provide data and results from Example 11. methods and/or genetically modified microorganisms that FIG. 8 depicts metabolic pathways of a microorganism 50 have utility for fermentative production of various chemical with multiple genetic modifications related to aspects of the products, to methods of making Such chemical products that present invention, more particularly related to 3-HP produc utilize populations of these microorganisms in vessels, and to tion, with gene names of E. coli shown at certain enzymatic systems for chemical production that employ these microor steps, the latter for example and not meant to be limiting. ganisms and methods. Among the benefits of the present FIG.9A, sheets 1-7 is a multi-sheet depiction of portions of 55 invention is increased specific productivity when Such micro metabolic pathways, showing pathway products and organisms produce a chemical product during a fermentation enzymes, that together comprise the 3-HP toleragenic com event or cycle. The present invention provides production plex (3HPTGC) in E. coli. Sheet 1 provides a general sche techniques and/or genetically modified microorganisms to matic depiction of the arrangement of the remaining sheets. produce a chemical product of interest, such as 3-hydrox FIG.9B, sheets 1-7, provides a multi-sheet depiction of the 60 ypropionic acid (3-HP) with one or more means for modulat 3HPTGC for Bacillus subtilis. Sheet 1 provides a general ing conversion of malonyl-CoA to fatty acyl molecules schematic depiction of the arrangement of the remaining (which thereafter may be converted to fatty acids, for example sheets. fatty acyl-ACP molecules), wherein the production pathway FIG.9C, sheets 1-7, provides a multi-sheet depiction of the comprises an enzymatic conversion step that uses malonyl 3HPTGC for Saccharomyces cerevisiae. Sheet 1 provides a 65 CoA as a substrate. The means for modulating conversion of general Schematic depiction of the arrangement of the malonyl-CoA to fatty acyl molecules, such as fatty acyl-ACP remaining sheets. molecules, is effective to balance carbon flow to microbial US 8,883,464 B2 9 10 biomass with carbon flow to chemical product, and Surpris art. For example, enzyme activity assays can be used to iden ingly affords achievement of elevated specific productivity tify cells having reduced enzyme activity. See, for example, rates. Enzyme Nomenclature, Academic Press, Inc., New York As noted herein, various aspects of the present invention 2007. are directed to a microorganism cell comprises a metabolic The term "heterologous DNA."heterologous nucleic acid pathway from malonyl-CoA to 3-HP, and means for modu sequence.” and the like as used herein refers to a nucleic acid lating conversion of malonyl-CoA to fatty acyl molecules sequence wherein at least one of the following is true: (a) the (which thereafter may be converted to fatty acids) also are sequence of nucleic acids is foreign to (i.e., not naturally provided. Then, when the means for modulating modulate to found in) a given host microorganism; (b) the sequence may decrease Such conversion, a proportionally greater number of 10 be naturally found in a given host microorganism, but in an malonyl-CoA molecules are 1) produced and/or 2) converted unnatural (e.g., greater than expected) amount; or (c) the via the metabolic pathway from malonyl-CoA to 3-HP. In sequence of nucleic acids comprises two or more Subse various embodiments, additional genetic modifications may quences that are not found in the same relationship to each be made. Such as to 1) increase intracellular bicarbonate other in nature. For example, regarding instance (c), a heter levels, such as by increasing carbonic anhydrase, 2) increase 15 ologous nucleic acid sequence that is recombinantly pro enzymatic activity of acetyl-CoA carboxylase, and NADPH duced will have two or more sequences from unrelated genes dependent transhydrogenase. arranged to make a new functional nucleic acid. Unexpected increases in specific productivity by a popu The term "heterologous is intended to include the term lation of a genetically modified microorganism may be “exogenous” as the latter term is generally used in the art. achieved in methods and systems in which that microorgan With reference to the host microorganism's genome prior to ism has a microbial production pathway from malonyl-CoA the introduction of a heterologous nucleic acid sequence, the to a selected chemical product as well as a reduction in the nucleic acid sequence that codes for the enzyme is heterolo enzymatic activity of a selected enzyme of the microorgan gous (whether or not the heterologous nucleic acid sequence ism's fatty acid synthase system (more particularly, its fatty is introduced into that genome). acid elongation enzymes). In various embodiments, specific 25 As used herein, the term "gene disruption, or grammatical Supplements to a bioreactor vessel comprising Such microor equivalents thereof (and including “to disrupt enzymatic ganism population may also be provided to further improve function.” “disruption of enzymatic function.” and the like), is the methods and systems. intended to mean a genetic modification to a microorganism Additionally, for one chemical product, 3-hydroxypropi that renders the encoded gene product as having a reduced onic acid (3-HP), genetic modifications for production path 30 polypeptide activity compared with polypeptide activity in or ways are provided, and a toleragenic complex is described for from a microorganism cell not so modified. The genetic modi which genetic modifications, and/or culture system modifi fication can be, for example, deletion of the entire gene, cations, may be made to increase microorganism tolerance to deletion or other modification of a regulatory sequence 3-HP. Moreover, genetic modifications to increase expression required for transcription or translation, deletion of a portion and/or enzymatic activity of carbonic anhydrase and/or cya 35 of the gene which results in a truncated gene product (e.g., nase may provide dual-functions to advantageously improve enzyme) or by any of various mutation strategies that reduces both 3-HP production and 3-HP tolerance. activity (including to no detectable activity level) the encoded Other additional genetic modifications are disclosed herein gene product. A disruption may broadly include a deletion of for various embodiments. all or part of the nucleic acid sequence encoding the enzyme, 40 and also includes, but is not limited to other types of genetic DEFINITIONS modifications, e.g., introduction of stop codons, frame shift mutations, introduction or removal of portions of the gene, As used in the specification and the claims, the singular and introduction of a degradation signal, those genetic modi forms “a,” “an and “the include plural referents unless the fications affecting mRNA transcription levels and/or stability, context clearly dictates otherwise. Thus, for example, refer 45 and altering the promoter or repressor upstream of the gene ence to an "expression vector” includes a single expression encoding the enzyme. vector as well as a plurality of expression vectors, either the In various contexts, a gene disruption is taken to mean any same (e.g., the same operon) or different; reference to “micro genetic modification to the DNA, mRNA encoded from the organism' includes a single microorganism as well as a plu DNA, and the corresponding amino acid sequence that results rality of microorganisms; and the like. 50 in reduced polypeptide activity. Many different methods can As used herein, dry cell weight (DCW) for E. coli strains is be used to make a cell having reduced polypeptide activity. calculated as 0.33 times the measured ODoo value, based on For example, a cell can be engineered to have a disrupted baseline DCW to ODoo determinations. regulatory sequence or polypeptide-encoding sequence using As used herein, “reduced enzymatic activity.” “reducing common mutagenesis or knock-out technology. See, e.g., enzymatic activity, and the like is meant to indicate that a 55 Methods in Yeast Genetics (1997 edition), Adams et al., Cold microorganism cells, oran isolated enzyme, exhibits a lower Spring Harbor Press (1998). One particularly useful method level of activity than that measured in a comparable cell of the of gene disruption is complete gene deletion because it same species or its native enzyme. That is, enzymatic conver reduces or eliminates the occurrence of genetic reversions in sion of the indicated substrate(s) to indicated product(s) the genetically modified microorganisms of the invention. under known standard conditions for that enzyme is at least 60 Accordingly, a disruption of a gene whose product is an 10, at least 20, at least 30, at least 40, at least 50, at least 60, enzyme thereby disrupts enzymatic function. Alternatively, at least 70, at least 80, or at least 90 percent less than the antisense technology can be used to reduce the activity of a enzymatic activity for the same biochemical conversion by a particular polypeptide. For example, a cell can be engineered native (non-modified) enzyme under a standard specified to contain a cDNA that encodes an antisense molecule that condition. This term also can include elimination of that 65 prevents a polypeptide from being translated. Further, gene enzymatic activity. A cell having reduced enzymatic activity silencing can be used to reduce the activity of a particular of an enzyme can be identified using any method known in the polypeptide. US 8,883,464 B2 11 12 The term “antisense molecule' as used herein encom protocols. “Hybridizing specifically to” or “specifically passes any nucleic acid molecule or nucleic acid analog (e.g., hybridizing to’ or like expressions refer to the binding, peptide nucleic acids) that contains a sequence that corre duplexing, or hybridizing of a molecule Substantially to or sponds to the coding strandofan endogenous polypeptide. An only to a particular nucleotide sequence or sequences under antisense molecule also can have flanking sequences (e.g., stringent conditions when that sequence is present in a com regulatory sequences). Thus, antisense molecules can be plex mixture (e.g., total cellular) DNA or RNA. ribozymes or antisense oligonucleotides. The term “identified enzymatic functional variant’ means As used herein, a ribozyme can have any general structure a polypeptide that is determined to possess an enzymatic including, without limitation, hairpin, hammerhead, or activity and specificity of an enzyme of interest but which has axhead structures, provided the molecule cleaves RNA. 10 an amino acid sequence different from Such enzyme of inter The term “reduction' or “to reduce' when used in such est. A corresponding “variant nucleic acid sequence' may be phrase and its grammatical equivalents are intended to constructed that is determined to encode such an identified encompass a complete elimination of such conversion(s). enzymatic functional variant. For a particular purpose. Such Bio-production, as used herein, may be aerobic, as increased tolerance to 3-HP via genetic modification to microaerobic, or anaerobic. 15 increase enzymatic conversion at one or more of the enzy As used herein, the language “sufficiently homologous' matic conversion steps of the 3HPTGC in a microorganism, refers to proteins or portions thereof that have amino acid one or more genetic modifications may be made to provide sequences that include a minimum number of identical or one or more heterologous nucleic acid sequence(s) that equivalent amino acid residues when compared to an amino encode one or more identified 3HPTGC enzymatic functional acid sequence of the amino acid sequences provided in this variant(s). That is, each Such nucleic acid sequence encodes a application (including the SEQ ID Nos./sequence listings) polypeptide that is not exactly the known polypeptide of an such that the protein or portion thereof is able to achieve the enzyme of the 3HPTGC, but which nonetheless is shown to respective enzymatic reaction and/or other function. To deter exhibit enzymatic activity of such enzyme. Such nucleic acid mine whether a particular protein or portion thereof is suffi sequence, and the polypeptide it encodes, may not fall within ciently homologous may be determined by an assay of enzy 25 a specified limit of homology or identity yet by its provision matic activity, such as those commonly known in the art. in a cell nonetheless provide for a desired enzymatic activity Descriptions and methods for sequence identity and and specificity. The ability to obtain such variant nucleic acid homology are intended to be exemplary and it is recognized sequences and identified enzymatic functional variants is that these concepts are well-understood in the art. Further, it Supported by recent advances in the states of the art in bioin is appreciated that nucleic acid sequences may be varied and 30 formatics and protein engineering and design, including still encode an enzyme or other polypeptide exhibiting a advances in computational, predictive and high-throughput desired functionality, and such variations are within the scope methodologies. Functional variants more generally include of the present invention. enzymatic functional variants, and the nucleic acids Further to nucleic acid sequences, “hybridization” refers to sequences that encode them, as well as variants of non-enzy the process in which two single-stranded polynucleotides 35 matic polypeptides, wherein the variant exhibits the function bind non-covalently to form a stable double-stranded poly of the original (target) sequence. nucleotide. The term “hybridization” may also refer to triple The use of the phrase “segment of interest' is meant to stranded hybridization. The resulting (usually) double include both a gene and any other nucleic acid sequence stranded polynucleotide is a “hybrid” or “duplex.” segment of interest. One example of a method used to obtain “Hybridization conditions' will typically include salt con 40 a segment of interest is to acquire a culture of a microorgan centrations of less than about 1M, more usually less than ism, where that microorganism's genome includes the gene about 500 mM and less than about 200 mM. Hybridization or nucleic acid sequence segment of interest. temperatures can be as low as 5°C., but are typically greater When the genetic modification of a gene product, i.e., an than 22°C., more typically greater than about 30° C., and enzyme, is referred to herein, including the claims, it is under often are in excess of about 37° C. Hybridizations are usually 45 stood that the genetic modification is of a nucleic acid performed under Stringent conditions, i.e. conditions under sequence. Such as or including the gene, that normally which a probe will hybridize to its target subsequence. Strin encodes the stated gene product, i.e., the enzyme. gent conditions are sequence-dependent and are different in In some embodiments a truncated respective polypeptide different circumstances. Longer fragments may require has at least about 90% of the full length of a polypeptide higher hybridization temperatures for specific hybridization. 50 encoded by a nucleic acid sequence encoding the respective As other factors may affect the stringency of hybridization, native enzyme, and more particularly at least 95% of the full including base composition and length of the complementary length of a polypeptide encoded by a nucleic acid sequence Strands, presence of organic solvents and extent of base mis encoding the respective native enzyme. By a polypeptide matching, the combination of parameters is more important having an amino acid sequence at least, for example, 95% than the absolute measure of any one alone. Generally, strin 55 “identical to a reference amino acid sequence of a polypep gent conditions are selected to be about 5° C. lower than the tide is intended that the amino acid sequence of the claimed T, for the specific sequence at a defined ionic strength and polypeptide is identical to the reference sequence except that pH. Exemplary stringent conditions include salt concentra the claimed polypeptide sequence can include up to five tion of at least 0.01 M to no more than 1 MNaion concen amino acid alterations per each 100 amino acids of the refer tration (or other salts) at a pH 7.0 to 8.3 and a temperature of 60 ence amino acid of the polypeptide. In other words, to obtain at least 25° C. For example, conditions of 5xSSPE (750 mM a polypeptide having an amino acid sequence at least 95% NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a identical to a reference amino acid sequence, up to 5% of the temperature of 25-30°C. are suitable for allele-specific probe amino acid residues in the reference sequence can be deleted hybridizations. For stringent conditions, see for example, or Substituted with another amino acid, or a number of amino Sambrook and Russell and Anderson “Nucleic Acid Hybrid 65 acids up to 5% of the total amino acid residues in the reference ization 1 Ed., BIOS Scientific Publishers Limited (1999), sequence can be inserted into the reference sequence. These which are hereby incorporated by reference for hybridization alterations of the reference sequence can occurat the amino or US 8,883,464 B2 13 14 carboxy terminal positions of the reference amino acid Although it is contemplated that all of the above mentioned sequence or anywhere between those terminal positions, carbon substrates and mixtures thereof are suitable in the interspersed either individually among residues in the refer present invention as a carbon Source, common carbon Sub ence sequence or in one or more contiguous groups within the strates used as carbon Sources are glucose, fructose, and reference sequence. In other embodiments truncation may be Sucrose, as well as mixtures of any of these Sugars. Other more substantial, as described elsewhere herein. suitable substrates include xylose, arabinose, other cellulose Species and other phylogenic identifications are according based C-5 Sugars, high-fructose corn syrup, and various other to the classification known to a person skilled in the art of Sugars and Sugar mixtures as are available commercially. microbiology. Sucrose may be obtained from feedstocks such as Sugarcane, Where methods and steps described herein indicate certain 10 Sugar beets, cassava, bananas or other fruit, and Sweet Sor events occurring in certain order, those of ordinary skill in the ghum. Glucose and dextrose may be obtained through sac art will recognize that the ordering of certain steps may be charification of starch based feedstocks including grains such modified and that Such modifications are in accordance with as corn, wheat, rye, barley, and oats. Also, in some embodi the variations of the invention. Additionally, certain steps may ments all or a portion of the carbon source may be glycerol. be performed concurrently in a parallel process when pos 15 Alternatively, glycerol may be excluded as an added carbon sible, as well as performed sequentially. SOUC. Prophetic examples provided herein are meant to be In one embodiment, the carbon source is selected from broadly exemplary and not limiting in any way. This applies glucose, fructose. Sucrose, dextrose, lactose, glycerol, and to the examples regarding separation and purification of mixtures thereof. Variously, the amount of these components 3-HP, and conversions of 3-HP to downstream compounds, in the carbon source may be greater than about 50%, greater since there are numerous possible approaches to Such steps than about 60%, greater than about 70%, greater than about and conversions, including those disclosed in references 80%, greater than about 90%, or more, up to 100% or essen recited and incorporated herein. tially 100% of the carbon source. The meaning of abbreviations is as follows: “C” means In addition, methylotrophic organisms are known to utilize Celsius or degrees Celsius, as is clear from its usage, DCW 25 a number of other carbon containing compounds such as means dry cell weight, 's' means second(s), 'min' means methylamine, glucosamine and a variety of amino acids for minute(s), “h.” “hr,” or “hrs’ means hour(s), “psi' means metabolic activity. For example, methylotrophic yeast are pounds per square inch, “nm” means nanometers, 'd' means known to utilize the carbon from methylamine to form treha day(s), “Lu” or “ull' or “ul' means microliter(s), “mL' lose or glycerol (Bellion et al., Microb. Growth C1 Compd. means milliliter(s), “L” means liter(s), “mm” means millime 30 (Int. Symp.), 7th (1993), 415-32. Editor(s): Murrell, J. Collin: ter(s), “nm’ means nanometers, “mM” means millimolar, Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, “uM” or “uM” means micromolar, “M” means molar, various species of Candida will metabolizealanine or oleic “mmol” means millimole(s), “umol” or “uMol' means acid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). micromole(s)', 'g' means gram(s), 'ug' or “ug' means Hence it is contemplated that the source of carbon utilized in microgram(s) and “ng” means nanogram(s), "PCR means 35 embodiments of the present invention may encompass a wide polymerase chain reaction, “OD' means optical density, variety of carbon-containing Substrates. “ODoo” means the optical density measured at a photon In addition, fermentable Sugars may be obtained from cel wavelength of 600 nm, “kDa' means kilodaltons, “g means lulosic and lignocellulosic biomass through processes of pre the gravitation constant, “bp” means base pair(s), “kbp' treatment and saccharification, as described, for example, in means kilobase pair(s), “96 w/v’ means weight/volume per 40 U.S. Patent Publication No. 2007/0031918A1, which is cent, “% V/v’ means volume/volume percent, “IPTG' means herein incorporated by reference. Biomass refers to any cel isopropyl-L-D-thiogalactopyranoiside, “RBS’ means ribo lulosic or lignocellulosic material and includes materials Some binding site, “rpm’ means revolutions per minute, comprising cellulose, and optionally further comprising “HPLC' means high performance liquid chromatography, hemicellulose, lignin, starch, oligosaccharides and/or and "GC’ means gas chromatography. As disclosed herein, 45 monosaccharides. Biomass may also comprise additional “3-HP' means 3-hydroxypropionic acid and “3HPTGC components, such as protein and/or lipid. Biomass may be means the 3-HPtoleragenic complex. Also, 105 and the like derived from a single source, or biomass can comprise a are taken to mean 10 and the like. mixture derived from more than one source; for example, I. Carbon Sources biomass could comprise a mixture of corn cobs and corn Bio-production media, which is used in the present inven 50 stover, or a mixture of grass and leaves. Biomass includes, but tion with recombinant microorganisms having a biosynthetic is not limited to, bioenergy crops, agricultural residues, pathway for 3-HP, must contain suitable carbon sources or municipal Solid waste, industrial Solid waste, sludge from substrates for the intended metabolic pathways. Suitable sub paper manufacture, yard waste, wood and forestry waste. strates may include, but are not limited to, monosaccharides Examples of biomass include, but are not limited to, corn Such as glucose and fructose, oligosaccharides Such as lactose 55 grain, corn cobs, crop residues such as corn husks, corn or Sucrose, polysaccharides Such as starch or cellulose or stover, grasses, wheat, wheat Straw, barley, barley Straw, hay, mixtures thereof and unpurified mixtures from renewable rice straw, Switchgrass, waste paper, Sugarcane bagasse, Sor feedstocks such as cheese whey permeate, cornsteep liquor, ghum, Soy, components obtained from milling of grains, Sugar beet molasses, and barley malt. Additionally the carbon trees, branches, roots, leaves, wood chips, sawdust, shrubs Substrate may also be one-carbon Substrates Such as carbon 60 and bushes, vegetables, fruits, flowers and animal manure. dioxide, carbon monoxide, or methanol for which metabolic Any such biomass may be used in a bio-production method or conversion into key biochemical intermediates has been dem system to provide a carbon Source. Various approaches to onstrated. In addition to one and two carbon Substrates breaking down cellulosic biomass to mixtures of more avail methylotrophic organisms are also known to utilize a number able and utilizable carbon molecules, including Sugars, of other carbon containing compounds such as methylamine, 65 include: heating in the presence of concentrated or dilute acid glucosamine and a variety of amino acids for metabolic activ (e.g., <1% sulfuric acid); treating with ammonia; treatment ity. with ionic salts; enzymatic degradation; and combinations of US 8,883,464 B2 15 16 these. These methods normally follow mechanical separation one or more of the present inventor(s) and/or subject to and milling, and are followed by appropriate separation pro assignment to the owner of the present patent application. CCSSCS. The examples describe specific modifications and evalua In various embodiments, any of a wide range of Sugars, tions to certain bacterial and yeast microorganisms. The including, but not limited to Sucrose, glucose, Xylose, cellu scope of the invention is not meant to be limited to such lose or hemicellulose, are provided to a microorganism, Such species, but to be generally applicable to a wide range of as in an industrial system comprising a reactor vessel in which Suitable microorganisms. Generally, a microorganism used a defined media (Such as a minimal salts media including but for the present invention may be selected from bacteria, not limited to M9 minimal media, potassium sulfate minimal cyanobacteria, filamentous fungi and yeasts. media, yeast synthetic minimal media and many others or 10 For some embodiments, microbial hosts initially selected variations of these), an inoculum of a microorganism provid for 3-HP toleragenic bio-production should also utilize sug ing one or more of the 3-HP biosynthetic pathway alterna ars including glucose at a high rate. Most microbes are tives, and the a carbon Source may be combined. The carbon capable of utilizing carbohydrates. However, certain environ source enters the cell and is catabolized by well-known and mental microbes cannot utilize carbohydrates to high effi common metabolic pathways to yield common metabolic 15 ciency, and therefore would not be suitable hosts for such intermediates, including phosphoenolpyruvate (PEP). (See embodiments that are intended for glucose or other carbohy Molecular Biology of the Cell, 3rd Ed., B. Alberts et al. drates as the principal added carbon source. Garland Publishing, New York, 1994, pp. 42-45, 66-74, incor As the genomes of various species become known, the porated by reference for the teachings of basic metabolic present invention easily may be applied to an ever-increasing catabolic pathways for Sugars; Principles of Biochemistry, range of Suitable microorganisms. Further, given the rela 3rd Ed., D. L. Nelson & M. M. Cox, Worth Publishers, New tively low cost of genetic sequencing, the genetic sequence of York, 2000, pp 527-658, incorporated by reference for the a species of interest may readily be determined to make teachings of major metabolic pathways; and Biochemistry, application of aspects of the present invention more readily 4th Ed., L. Stryer, W. H. Freeman and Co., New York, 1995, obtainable (based on the ease of application of genetic modi pp. 463-650, also incorporated by reference for the teachings 25 fications to an organism having a known genomic sequence). of major metabolic pathways.) More particularly, based on the various criteria described Bio-based carbon can be distinguished from petroleum herein, suitable microbial hosts for the bio-production of based carbon according to a variety of methods, including 3-HP that comprise tolerance aspects provided herein gener without limitation ASTM D6866, or various other tech ally may include, but are not limited to, any gram negative niques. For example, carbon-14 and carbon-12 ratios differin 30 organisms, more particularly a member of the family Entero bio-based carbon Sources versus petroleum-based sources, bacteriaceae, Such as E. coli, or Oligotropha carboxi where higher carbon-14 ratios are found in bio-based carbon dovorans, or Pseudomononas sp.: any gram positive micro Sources. In various embodiments, the carbon Source is not organism, for example Bacillus subtilis, Lactobaccillus sp. or petroleum-based, or is not predominantly petroleum based. In Lactococcus sp.: a yeast, for example Saccharomyces cerevi various embodiments, the carbon Source is greater than about 35 siae, Pichia pastoris or Pichia stipitis; and other groups or 50% non-petroleum based, greater than about 60% non-pe microbial species. More particularly, suitable microbial hosts troleum based, greater than about 70% non-petroleum based, for the bio-production of 3-HP generally include, but are not greater than about 80% non-petroleum based, greater than limited to, members of the genera Clostridium, Zymomonas, about 90% non-petroleum based, or more. In various embodi Escherichia, Salmonella, Rhodococcus, Pseudomonas, ments, the carbon source has a carbon-14 to carbon-12 ratio 40 Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Kleb of about 1.0x10" or greater. siella, Paenibacillus, Arthrobacter, Corynebacterium, Brevi Various components may be excluded from the carbon bacterium, Pichia, Candida, Hansenula and Saccharomyces. Source. For example, in some embodiments, acrylic acid, Hosts that may be particularly of interest include: Oligotro 1,4-butanediol, and/or glycerol are excluded or essentially pha carboxidovorans (such as strain OM5), Escherichia coli, excluded from the carbon Source. As such, the carbon Source 45 Alcaligenes eutrophus (Cupriavidus necator), Bacillus according to some embodiments of the invention may be less licheniformis, Paenibacillus macerans, Rhodococcus eryth than about 50% glycerol, less than about 40% glycerol, less ropolis, Pseudomonas putida, Lactobacillus plantarum, than about 30% glycerol, less than about 20% glycerol, less Enterococcus faecium, Enterococcus gallinarium, Entero than about 10% glycerol, less than about 5% glycerol, less coccus faecalis, Bacillus subtilis and Saccharomyces cerevi than about 1% glycerol, or less. For example, the carbon 50 Siae. Source may be essentially glycerol-free. By essentially glyc More particularly, suitable microbial hosts for the bio erol-free is meant that any glycerol that may be present in a production of 3-HP generally include, but are not limited to, residual amount does not contribute substantially to the pro members of the genera Clostridium, Zymomonas, Escheri duction of the target chemical compound. chia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, II. Microorganisms 55 Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paeni Features as described and claimed herein may be provided bacillus, Arthrobacter, Corynebacterium, Brevibacterium, in a microorganism selected from the listing herein, or Pichia, Candida, Hansenula and Saccharomyces. another Suitable microorganism, that also comprises one or Hosts that may be particularly of interest include: Oligotro more natural, introduced, or enhanced 3-HP bio-production pha carboxidovorans (such as strain OM57), Escherichia pathways. Thus, in Some embodiments the microorganism 60 coli, Alcaligenes eutrophus (Cupriavidus necator), Bacillus comprises an endogenous 3-HP production pathway (which licheniformis, Paenibacillus macerans, Rhodococcus eryth may, in Some Such embodiments, be enhanced), whereas in ropolis, Pseudomonas putida, Lactobacillus plantarum, other embodiments the microorganism does not comprise an Enterococcus faecium, Enterococcus gallinarium, Entero endogenous 3-HP production pathway. coccus faecalis, Bacillus subtilis and Saccharomyces cerevi Varieties of these genetically modified microorganisms 65 siae. Also, any of the known strains of these species may be may comprise genetic modifications and/or other system utilized as a starting microorganism, as may any of the fol alterations as may be described in other patent applications of lowing species including respective strains thereof Cupria US 8,883,464 B2 17 18 vidus basilensis, Cupriavidus campinensis, Cupriavidus components, for example less than 10, 5, 2 or 1 g/L of a gilardi, Cupriavidus laharsis, Cupriavidus metallidurans, complex nitrogen Source including but not limited to yeast Cupriavidus Oxalaticus, Cupriavidus pauculus, Cupriavidus extract, peptone, tryptone, soy flour, corn steep liquor, or pinatubonensis, Cupriavidus respiraculi, and Cupriavidus casein. These minimal medias may also have limited Supple taiwanensis. mentation of vitamin mixtures including biotin, vitamin B12 In some embodiments, the recombinant microorganism is a and derivatives of vitamin B12, thiamin, pantothenate and gram-negative bacterium. In some embodiments, the recom other vitamins. Minimal medias may also have limited simple binant microorganism is selected from the genera Zymomo inorganic nutrient sources containing less than 28, 17, or 2.5 nas, Escherichia, Pseudomonas, Alcaligenes, and Klebsiella. mM phosphate, less than 25 or 4 mM sulfate, and less than In some embodiments, the recombinant microorganism is 10 130 or 50 mM total nitrogen. selected from the species Escherichia coli, Cupriavidus neca Bio-production media, which is used in embodiments of tor, Oligotropha carboxidovorans, and Pseudomonas putida. the present invention with genetically modified microorgan In some embodiments, the recombinant microorganism is an isms, must contain Suitable carbon Substrates for the intended E. coli strain. metabolic pathways. As described hereinbefore, suitable car In some embodiments, the recombinant microorganism is a 15 bon Substrates include carbon monoxide, carbon dioxide, and gram-positive bacterium. In some embodiments, the recom various monomeric and oligomeric Sugars. binant microorganism is selected from the genera Suitable pH ranges for the bio-production are between pH Clostridium, Salmonella, Rhodococcus, Bacillus, Lactoba 3.0 to pH 10.0, where pH 6.0 to pH 8.0 is a typical pH range cillus, Enterococcus, Paenibacillus, Arthrobacter, Coryne for the initial condition. However, the actual culture condi bacterium, and Brevibacterium. In some embodiments, the tions for a particular embodiment are not meant to be limited recombinant microorganism is selected from the species by these pH ranges. Bacillus licheniformis, Paenibacillus macerans, Rhodococ Bio-productions may be performed under aerobic, cus erythropolis, Lactobacillus plantarum, Enterococcus microaerobic, or anaerobic conditions, with or without agita faecium, Enterococcus gallinarium, Enterococcus faecalis, tion. and Bacillus subtilis. In particular embodiments, the recom 25 The amount of 3-HP or other product(s) produced in a binant microorganism is a B. subtilis Strain. bio-production media generally can be determined using a In some embodiments, the recombinant microorganism is a number of methods known in the art, for example, high per yeast. In some embodiments, the recombinant microorgan formance liquid chromatography (HPLC), gas chromatogra ism is selected from the genera Pichia, Candida, Hansenula phy (GC), or GC/Mass Spectroscopy (MS). Specific HPLC and Saccharomyces. In particular embodiments, the recom 30 methods for the specific examples are provided herein. binant microorganism is Saccharomyces cerevisiae. IV. Bio-Production Reactors and Systems It is further appreciated, in view of the disclosure, that any Fermentation systems utilizing methods and/or composi of the above microorganisms may be used for production of tions according to the invention are also within the scope of chemical products other than 3-HP. the invention. The ability to genetically modify the host is essential for 35 Any of the recombinant microorganisms as described and/ the production of any recombinant microorganism. The mode or referred to herein may be introduced into an industrial of gene transfer technology may be by electroporation, con bio-production system where the microorganisms convert a jugation, transduction or natural transformation. A broad carbon source into 3-HP in a commercially viable operation. range of host conjugative plasmids and drug resistance mark The bio-production system includes the introduction of such ers are available. The cloning vectors are tailored to the host 40 a recombinant microorganism into a bioreactor vessel, with a organisms based on the nature of antibiotic resistance mark carbon Source Substrate and bio-production media Suitable ers that can function in that host. for growing the recombinant microorganism, and maintain III. Media and Culture Conditions ing the bio-production system within a suitable temperature In addition to an appropriate carbon Source, such as range (and dissolved oxygen concentration range if the reac selected from one of the herein-disclosed types, bio-produc 45 tion is aerobic or microaerobic) for a suitable time to obtain a tion media must contain Suitable minerals, salts, cofactors, desired conversion of a portion of the substrate molecules to buffers and other components, known to those skilled in the 3-HP. Industrial bio-production systems and their operation art, suitable for the growth of the cultures and promotion of are well-known to those skilled in the arts of chemical engi the enzymatic pathway necessary for 3-HP production, or neering and bioprocess engineering. other products made under the present invention. 50 Bio-productions may be performed under aerobic, Another aspect of the invention regards media and culture microaerobic, or anaerobic conditions, with or without agita conditions that comprise genetically modified microorgan tion. The operation of cultures and populations of microor isms of the invention and optionally Supplements. ganisms to achieve aerobic, microaerobic and anaerobic con Typically cells are grown at a temperature in the range of ditions are known in the art, and dissolved oxygen levels of a about 25°C. to about 40°C. in an appropriate medium, as 55 liquid culture comprising a nutrient media and Such microor well as up to 70° C. for thermophilic microorganisms. Suit ganism populations may be monitored to maintain or confirm able growth media in the present invention are common com a desired aerobic, microaerobic oranaerobic condition. When mercially prepared media such as Luria Bertani (LB) broth, syngas is used as a feedstock, aerobic, microaerobic, or M9 minimal media, Sabouraud Dextrose (SD) broth, Yeast anaerobic conditions may be utilized. When Sugars are used, medium (YM) broth, (Ymin) yeast synthetic minimal media, 60 anaerobic, aerobic or microaerobic conditions can be imple and minimal media as described herein, such as M9 minimal mented in various embodiments. media. Other defined or synthetic growth media may also be Any of the recombinant microorganisms as described and/ used, and the appropriate medium for growth of the particular or referred to herein may be introduced into an industrial microorganism will be known by one skilled in the art of bio-production system where the microorganisms convert a microbiology or bio-production science. In various embodi 65 carbon source into 3-HP, and optionally in various embodi ments a minimal media may be developed and used that does ments also to one or more downstream compounds of 3-HP in not comprise, or that has a low level of addition of various a commercially viable operation. The bio-production system US 8,883,464 B2 19 20 includes the introduction of such a recombinant microorgan nology: A Textbook of Industrial Microbiology, Second Edi ism into a bioreactor vessel, with a carbon Source Substrate tion (1989) Sinauer Associates, Inc., Sunderland, Mass. and bio-production media Suitable for growing the recombi Deshpande, MukundV., Appl. Biochem. Biotechnol. 36:227, nant microorganism, and maintaining the bio-production sys (1992), and Biochemical Engineering Fundamentals, 2" Ed. tem within a Suitable temperature range (and dissolved oxy J. E. Bailey and D. F. Ollis, McGraw Hill, New York, 1986, gen concentration range if the reaction is aerobic or herein incorporated by reference for general instruction on microaerobic) for a suitable time to obtain a desired conver bio-production. sion of a portion of the substrate molecules to 3-HP. Although embodiments of the present invention may be In various embodiments, syngas components or Sugars are performed in batch mode, or in fed-batch mode, it is contem provided to a microorganism, Such as in an industrial system 10 plated that the invention would be adaptable to continuous comprising a reactor vessel in which a defined media (Such as bio-production methods. Continuous bio-production is con a minimal salts media including but not limited to M9 mini sidered an “open' system where a defined bio-production mal media, potassium sulfate minimal media, yeast synthetic medium is added continuously to a bioreactor and an equal minimal media and many others or variations of these), an amount of conditioned media is removed simultaneously for inoculum of a microorganism providing an embodiment of 15 processing. Continuous bio-production generally maintains the biosynthetic pathway(s) taught herein, and the carbon the cultures within a controlled density range where cells are source may be combined. The carbon source enters the cell primarily in log phase growth. Two types of continuous biore and is catabolized by well-known and common metabolic actor operation include a chemostat, wherein fresh media is pathways to yield common metabolic intermediates, includ fed to the vessel while simultaneously removing an equal rate ing phosphoenolpyruvate (PEP). (See Molecular Biology of of the vessel contents. The limitation of this approach is that the Cell, 3' Ed., B. Alberts et al. Garland Publishing, New cells are lost and high cell density generally is not achievable. York, 1994, pp. 42-45, 66-74, incorporated by reference for In fact, typically one can obtain much higher cell density with the teachings of basic metabolic catabolic pathways for Sug a fed-batch process. Another continuous bioreactor utilizes ars: Principles of Biochemistry, 3" Ed., D. L. Nelson & M. M. perfusion culture, which is similar to the chemostat approach Cox, Worth Publishers, New York, 2000, pp. 527-658, incor 25 except that the stream that is removed from the vessel is porated by reference for the teachings of major metabolic Subjected to a separation technique which recycles viable pathways; and Biochemistry, 4" Ed., L. Stryer, W. H. Free cells back to the vessel. This type of continuous bioreactor man and Co., New York, 1995, pp. 463-650, also incorporated operation has been shown to yield significantly higher cell by reference for the teachings of major metabolic pathways.). densities than fed-batch and can be operated continuously. Further to types of industrial bio-production, various 30 Continuous bio-production is particularly advantageous for embodiments of the present invention may employ a batch industrial operations because it has less downtime associated type of industrial bioreactor. A classical batch bioreactor sys with draining, cleaning and preparing the equipment for the tem is considered “closed’ meaning that the composition of next bio-production event. Furthermore, it is typically more the medium is established at the beginning of a respective economical to continuously operate downstream unit opera bio-production event and not subject to artificial alterations 35 tions, such as distillation, than to run them in batch mode. and additions during the time period ending Substantially Continuous bio-production allows for the modulation of with the end of the bio-production event. Thus, at the begin one factor or any number of factors that affect cell growth or ning of the bio-production event the medium is inoculated end product concentration. For example, one method will with the desired organism or organisms, and bio-production maintain a limiting nutrient such as the carbon Source or is permitted to occur without adding anything to the system. 40 nitrogen level at a fixed rate and allow all other parameters to Typically, however, a “batch' type of bio-production event is moderate. In other systems a number of factors affecting batch with respect to the addition of carbon source and growth can be altered continuously while the cell concentra attempts are often made at controlling factors such as pH and tion, measured by media turbidity, is kept constant. Methods oxygen concentration. In batch systems the metabolite and of modulating nutrients and growth factors for continuous biomass compositions of the system change constantly up to 45 bio-production processes as well as techniques for maximiz the time the bio-production event is stopped. Within batch ing the rate of product formation are well known in the art of cultures cells moderate through a static lag phase to a high industrial microbiology and a variety of methods are detailed growth log phase and finally to a stationary phase where by Brock, Supra. growth rate is diminished or halted. Ifuntreated, cells in the It is contemplated that embodiments of the present inven stationary phase will eventually die. Cells in log phase gen 50 tion may be practiced using either batch, fed-batch or con erally are responsible for the bulk of production of a desired tinuous processes and that any known mode of bio-produc end product or intermediate. tion would be suitable. It is contemplated that cells may be A variation on the standard batch system is the fed-batch immobilized on an inert scaffold as whole cell catalysts and system. Fed-batch bio-production processes are also Suitable subjected to suitable bio-production conditions for 3-HP pro in the present invention and comprise a typical batch system 55 duction, or be cultured in liquid media in a vessel. Such as a with the exception that the nutrients, including the Substrate, culture vessel. Thus, embodiments used in Such processes, are added in increments as the bio-production progresses. and in bio-production systems using these processes, include Fed-Batch systems are useful when catabolite repression is a population of genetically modified microorganisms of the apt to inhibit the metabolism of the cells and where it is present invention, a culture system comprising Such popula desirable to have limited amounts of substrate in the media. 60 tion in a media comprising nutrients for the population, and Measurement of the actual nutrient concentration in Fed methods of making 3-HP and thereafter, a downstream prod Batch systems may be measured directly, Such as by sample uct of 3-HP analysis at different times, or estimated on the basis of the Embodiments of the invention include methods of making changes of measurable factors such as pH, dissolved oxygen 3-HP in a bio-production system, some of which methods and the partial pressure of waste gases Such as CO. Batch and 65 may include obtaining 3-HP after such bio-production event. fed-batch approaches are common and well known in the art For example, a method of making 3-HP may comprise: pro and examples may be found in Thomas D. Brock in Biotech viding to a culture vessel a media comprising Suitable nutri US 8,883,464 B2 21 22 ents; providing to the culture vessel an inoculum of a geneti nant) microorganism may comprise modifications other than cally modified microorganism comprising genetic via plasmid introduction, including modifications to its modifications described herein such that the microorganism genomic DNA. produces 3-HP from Syngas and/or a Sugar molecule; and It has long been recognized in the art that some amino acids maintaining the culture vessel under Suitable conditions for 5 in amino acid sequences can be varied without significant the genetically modified microorganism to produce 3-HP. effect on the structure or function of proteins. Variants It is within the scope of the present invention to produce, included can constitute deletions, insertions, inversions, and to utilize in bio-production methods and systems, includ repeats, and type substitutions so long as the indicated ing industrial bio-production systems for production of 3-HP. enzyme activity is not significantly adversely affected. Guid a recombinant microorganism genetically engineered to 10 ance concerning which amino acid changes are likely to be modify one or more aspects effective to increase tolerance to phenotypically silent can be found, interalia, in Bowie, J.U., 3-HP (and, in some embodiments, also 3-HP bio-production) et al., “Deciphering the Message in Protein Sequences: Tol by at least 20 percent over control microorganism lacking the erance to Amino Acid Substitutions. Science 247: 1306-1310 one or more modifications. (1990). This reference is incorporated by reference for such In various embodiments, the invention is directed to a 15 teachings, which are, however, also generally known to those system for bioproduction of acrylic acid as described herein, skilled in the art. said system comprising: a tank for saccharification of biom In various embodiments polypeptides obtained by the ass; a line for passing the product of saccharification to a expression of the polynucleotide molecules of the present fermentation tank optionally via a pre-fermentation tank; a invention may have at least approximately 50%, 60%, 70%, fermentation tank Suitable for microorganism cell culture; a 80%, 90%. 95%, 96%, 97%, 98%, 99% or 100% identity to line for discharging contents from the fermentation tank to an one or more amino acid sequences encoded by the genes extraction and/or separation vessel; an extraction and/or sepa and/or nucleic acid sequences described herein for the 3-HP ration vessel suitable for removal of 3-hydroxypropionic acid tolerance-related and biosynthesis pathways. from cell culture waste; a line for transferring 3-hydroxypro As a practical matter, whether any particular polypeptide is pionic acid to a dehydration vessel; and a dehydration vessel 25 at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, suitable for conversion of 3-hydroxypropionic acid to acrylic 97%, 98% or 99% identical to any reference amino acid acid. In various embodiments, the system includes one or sequence of any polypeptide described herein (which may more pre-fermentation tanks, distillation columns, centrifuge correspond with a particular nucleic acid sequence described vessels, back extraction columns, mixing vessels, or combi herein). Such particular polypeptide sequence can be deter nations thereof. 30 mined conventionally using known computer programs such The following published resources are incorporated by the Bestfit program (Wisconsin Sequence Analysis Package, reference herein for their respective teachings to indicate the Version 8 for Unix, Genetics Computer Group, University level of skill in these relevant arts, and as needed to support a Research Park, 575 Science Drive, Madison, Wis. 53711). disclosure that teaches how to make and use methods of When using Bestfit or any other sequence alignment program industrial bio-production of 3-HP, or other product(s) pro 35 to determine whether a particular sequence is, for instance, duced under the invention, from Sugar sources, and also 95% identical to a reference sequence according to the industrial systems that may be used to achieve Such conver present invention, the parameters are set such that the per sion with any of the recombinant microorganisms of the centage of identity is calculated over the full length of the present invention (Biochemical Engineering Fundamentals, reference amino acid sequence and that gaps in homology of 2' Ed. J. E. Bailey and D. F. Ollis, McGraw Hill, New York, 40 up to 5% of the total number of amino acid residues in the 1986, entire book for purposes indicated and Chapter 9, pages reference sequence are allowed. 533-657 in particular for biological reactor design; Unit For example, in a specific embodiment the identity Operations of Chemical Engineering, 5' Ed., W. L. McCabe between a reference sequence (query sequence, i.e., a et al., McGraw Hill, New York 1993, entire book for purposes sequence of the present invention) and a subject sequence, indicated, and particularly for process and separation tech 45 also referred to as a global sequence alignment, may be deter nologies analyses; Equilibrium Staged Separations, P. C. mined using the FASTDB computer program based on the Wankat, Prentice Hall, Englewood Cliffs, N.J. USA, 1988, algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 entire book for separation technologies teachings). Generally, (1990)). Preferred parameters for a particular embodiment in it is further appreciated, in view of the disclosure, that any of which identity is narrowly construed, used in a FASTDB the above methods and systems may be used for production of 50 amino acid alignment, are: Scoring Scheme=PAM (Percent chemical products other than 3-HP. Accepted Mutations) 0, k-tuple=2, Mismatch Penalty=1, V. Genetic Modifications, Nucleotide Sequences, and Joining Penalty=20, Randomization Group Length=0, Cutoff Amino Acid Sequences Score=1, Window Size=sequence length, Gap Penalty=5, Embodiments of the present invention may result from Gap Size Penalty=0.05, Window Size=500 or the length of introduction of an expression vector into a host microorgan 55 the Subject amino acid sequence, whichever is shorter. ism, wherein the expression vector contains a nucleic acid According to this embodiment, if the Subject sequence is sequence coding for an enzyme that is, or is not, normally shorter than the query sequence due to N- or C-terminal found in a host microorganism. deletions, not because of internal deletions, a manual correc The ability to genetically modify a host cell is essential for tion is made to the results to take into consideration the fact the production of any genetically modified (recombinant) 60 that the FASTDB program does not account for N- and C-ter microorganism. The mode of gene transfer technology may minal truncations of the Subject sequence when calculating be by electroporation, conjugation, transduction, or natural global percent identity. For subject sequences truncated at the transformation. A broad range of host conjugative plasmids N- and C-termini, relative to the query sequence, the percent and drug resistance markers are available. The cloning vec identity is corrected by calculating the number of residues of tors are tailored to the host organisms based on the nature of 65 the query sequence that are lateral to the N- and C-terminal of antibiotic resistance markers that can function in that host. the Subject sequence, which are not matched/aligned with a Also, as disclosed herein, a genetically modified (recombi corresponding Subject residue, as a percent of the total bases US 8,883,464 B2 23 24 of the query sequence. A determination of whether a residue promoter (Haldimann et al., 1998, J. Bacteriol. 180: 1277 is matched/aligned is determined by results of the FASTDB 1286). Other promoters are described in “Useful proteins sequence alignment. This percentage is then Subtracted from from recombinant bacteria' in Scientific American, 1980, the percent identity, calculated by the FASTDB program 242: 74-94; and in Sambrook and Russell, “Molecular Clon using the specified parameters, to arrive at a final percent 5 ing: A Laboratory Manual. Third Edition 2001 (volumes identity score. This final percent identity score is what is used 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Har for the purposes of this embodiment. Only residues to the N bor, N.Y. and C-termini of the Subject sequence, which are not The control sequence may also be a suitable transcription matched/aligned with the query sequence, are considered for terminator sequence, a sequence recognized by a host cell to the purposes of manually adjusting the percent identity score. 10 terminate transcription. The terminator sequence is operably That is, only query residue positions outside the farthest N linked to the 3' terminus of the nucleotide sequence encoding and C-terminal residues of the Subject sequence are consid the polypeptide. Any terminator that is functional in an E. coli ered for this manual correction. For example, a 90 amino acid cell may be used in the present invention. It may also be residue subject sequence is aligned with a 100 residue query desirable to add regulatory sequences that allow regulation of sequence to determine percent identity. The deletion occurs at 15 the expression of the polypeptide relative to the growth of the the N-terminus of the subject sequence and therefore, the host cell. Examples of regulatory systems are those that cause FASTDB alignment does not show a matching/alignment of the expression of the gene to be turned on or offin response to the first 10 residues at the N-terminus. The 10 unpaired resi a chemical or physical stimulus, including the presence of a dues represent 10% of the sequence (number of residues at the regulatory compound. Regulatory systems in prokaryotic N- and C-termini not matched/total number of residues in the systems include the lac, tac, and trp operator systems. query sequence) so 10% is Subtracted from the percent iden For various embodiments of the invention the genetic tity score calculated by the FASTDB program. If the remain manipulations may be described to include various genetic ing 90 residues were perfectly matched the final percent iden manipulations, including those directed to change regulation tity would be 90%. In another example, a 90 residue subject of and therefore ultimate activity of an enzyme or enzymatic sequence is compared with a 100 residue query sequence. 25 activity of an enzyme identified in any of the respective path This time the deletions are internal deletions so there are no ways. Such genetic modifications may be directed to tran residues at the N- or C-termini of the subject sequence which Scriptional, translational, and post-translational modifica are not matched/aligned with the query. In this case the per tions that result in a change of enzyme activity and/or cent identity calculated by FASTDB is not manually cor selectivity under selected and/or identified culture conditions rected. Once again, only residue positions outside the N- and 30 and/or to provision of additional nucleic acid sequences Such C-terminal ends of the Subject sequence, as displayed in the as to increase copy number and/or mutants of an enzyme FASTDB alignment, which are not matched/aligned with the related to 3-HP production. Specific methodologies and query sequence are manually corrected for. approaches to achieve Such genetic modification are well More generally, nucleic acid constructs can be prepared knownto one skilled in the art, and include, but are not limited comprising an isolated polynucleotide encoding a polypep 35 to: increasing expression of an endogenous genetic element; tide having enzyme activity operably linked to one or more decreasing functionality of a repressor gene; introducing a (several) control sequences that direct the expression of the heterologous genetic element; increasing copy number of a coding sequence in a microorganism, such as E. coli, under nucleic acid sequence encoding a polypeptide catalyzing an conditions compatible with the control sequences. The iso enzymatic conversion step to produce 3-HP; mutating a lated polynucleotide may be manipulated to provide for 40 genetic element to provide a mutated protein to increase expression of the polypeptide. Manipulation of the poly specific enzymatic activity; over-expressing; under-express nucleotide's sequence prior to its insertion into a vector may ing; over-expressing a chaperone; knocking out a protease; be desirable or necessary depending on the expression vector. altering or modifying feedback inhibition; providing an The techniques for modifying polynucleotide sequences uti enzyme variant comprising one or more of an impaired bind lizing recombinant DNA methods are well established in the 45 ing site for a repressor and/or competitive inhibitor, knocking art. out a repressor gene; evolution, selection and/or other The control sequence may be an appropriate promoter approaches to improve mRNA stability as well as use of sequence, a nucleotide sequence that is recognized by a host plasmids having an effective copy number and promoters to cell for expression of a polynucleotide encoding a polypep achieve an effective level of improvement. Random mutagen tide of the present invention. The promoter sequence contains 50 esis may be practiced to provide genetic modifications that transcriptional control sequences that mediate the expression may fall into any of these or other stated approaches. The of the polypeptide. The promoter may be any nucleotide genetic modifications further broadly fall into additions (in sequence that shows transcriptional activity in the host cell of cluding insertions), deletions (such as by a mutation) and choice including mutant, truncated, and hybrid promoters, Substitutions of one or more nucleic acids in a nucleic acid of and may be obtained from genes encoding extracellular or 55 interest. In various embodiments a genetic modification intracellular polypeptides either homologous or heterologous results in improved enzymatic specific activity and/or turn to the host cell. Examples of suitable promoters for directing over number of an enzyme. Without being limited, changes transcription of the nucleic acid constructs, especially in an E. may be measured by one or more of the following: K. K. coli host cell, are the lac promoter (Gronenborn, 1976, MoI. and Kavidia. Gen. Genet. 148: 243-250), tac promoter (DeBoer et al., 60 In various embodiments, to function more efficiently, a 1983, Proceedings of the National Academy of Sciences USA microorganism may comprise one or more gene deletions. 80: 21-25), trc promoter (Brosius et al., 1985, J. Biol. Chem. For example, in E. coli, the genes encoding the lactate dehy 260:3539-3541), T7 RNA polymerase promoter (Studier and drogenase (ldh A), phosphate acetyltransferase (pta), pyru Moffatt, 1986, J. MoI. Biol. 189: 113-130), phage promoter vate oxidase (poXB), and pyruvate-formate lyase (pflB) may p. (Elvin et al., 1990, Gene 87: 123-126), tetA primoter 65 be disrupted, including deleted. Such gene disruptions, (Skerra, 1994, Gene 151: 131-135), araBAD promoter (Guz including deletions, are not meant to be limiting, and may be man et al., 1995, J. Bacteriol. 177: 4121-4130), and rhaP. implemented in various combinations in various embodi US 8,883,464 B2 25 26 ments. Gene deletions may be accomplished by mutational well within the skill of the person of ordinary skill in the art, gene deletion approaches, and/or starting with a mutant strain and microorganisms comprising these, also are within the having reduced or no expression of one or more of these Scope of various embodiments of the invention, as are meth enzymes, and/or other methods known to those skilled in the ods and systems comprising Such sequences and/or microor art. Gene deletions may be effectuated by any of a number of 5 ganisms. In various embodiments, nucleic acid sequences known specific methodologies, including but not limited to encoding Sufficiently homologous proteins or portions the RED/ET methods using kits and other reagents sold by thereofare within the scope of the invention. More generally, Gene Bridges (Gene Bridges GmbH, Dresden, Germany, nucleic acids sequences that encode a particular amino acid ). sequence employed in the invention may vary due to the More particularly as to the latter method, use of Red/ET 10 degeneracy of the genetic code, and nonetheless fall within recombination, is known to those of ordinary skill in the art the scope of the invention. The following table provides a and described in U.S. Pat. Nos. 6,355,412 and 6,509,156, Summary of similarities among amino acids, upon which issued to Stewart et al. and incorporated by reference herein conservative and less conservative Substitutions may be for its teachings of this method. Material and kits for such based, and also various codon redundancies that reflect this method are available from Gene Bridges (Gene Bridges 15 degeneracy. GmbH, Dresden, Germany, ). and the method may proceed by following the manufacturers TABLE 1. instructions. The method involves replacement of the target gene by a selectable marker via homologous recombination Relation performed by the recombinase from w-phage. The host organ Amino Acid ships DNA codons ism expressing w-red recombinase is transformed with a lin Alanine N, Ali GCT, GCC, GCA, GCG ear DNA product coding for a selectable marker flanked by the terminal regions (generally ~50 bp, and alternatively up to Proline N CCT, CCC, CCA, CCG about -300 bp) homologous with the target gene. The marker Waline N, Ali GTT, GTC GTA, GTG could then be removed by another recombination step per 25 formed by a plasmid vector carrying the FLP-recombinase, or Leucine N, Ali CTT, CTC, CTA, CTG, TTA, TTG another recombinase, such as Cre. Targeted deletion of parts of microbial chromosomal DNA Isoleucine N, Ali ATT, ATC, ATA or the addition of foreign genetic material to microbial chro Methionine N ATG momes may be practiced to alter a host cell's metabolism so 30 as to reduce or eliminate production of undesired metabolic Phenyl- N. Aro TTT, TTC products. This may be used in combination with other genetic alanine modifications such as described herein in this general Tryptophan N TGG example. In this detailed description, reference has been Glycine PU GGT, GGC, GGA, GGG made to multiple embodiments and to the accompanying 35 drawings in which is shown by way of illustration specific Serine PU TCT, TCC, TCA, TCG, AGT, AGC exemplary embodiments in which the invention may be prac ticed. These embodiments are described in sufficient detail to Threonine PU ACT, ACC, ACA, ACG enable those skilled in the art to practice the invention, and it Asparagine PU Ami AAT. AAC is to be understood that modifications to the various disclosed 40 embodiments may be made by a skilled artisan. Glutamine PU Ami CAA, CAG Further, for 3-HP production, such genetic modifications may be chosen and/or selected for to achieve a higher flux rate Cysteine PU TGT, TGC through certain enzymatic conversion steps within the respec Aspartic NEG, A GAT, GAC tive 3-HP production pathway and so may affect general 45 acid cellular metabolism in fundamental and/or major ways. Glutamic NEG, A GAA, GAG It will be appreciated that amino acid “homology” includes acid conservative Substitutions, i.e. those that Substitute a given amino acid in a polypeptide by another amino acid of similar Arginine POS, B CGT, CGC, CGA, CGG, AGA, AGG characteristics. Typically seen as conservative Substitutions 50 are the following replacements: replacements of an aliphatic Lysine POS, B AAA AAG amino acid Such as Ala, Val, Leu and Ile with anotheraliphatic Histidine POS CAT CAC amino acid; replacement of a Ser with a Thr or vice versa; replacement of an acidic residue Such as Asp or Glu with Tyrosine Aro TAT, TAC another acidic residue; replacement of a residue bearing an 55 amide group, such as ASnor Gln, with another residuebearing Stop Codons TAA, TAG, TGA an amide group; exchange of a basic residue Such as Lys or Legend: Side groups and other related properties: A = acidic; B = basic; Arg with another basic residue; and replacement of an aro Ali = aliphatic; Ami = amine; Arc = aromatic; N = nonpolar; PU = polar uncharged; NEG = negatively charged; POS = positively matic residue such as Phe or Tyr with another aromatic resi charged. due. 60 For all nucleic acid and amino acid sequences provided Also, variants and portions of particular nucleic acid herein, it is appreciated that conservatively modified variants sequences, and respective encoded amino acid sequences of these sequences are included, and are within the scope of recited herein may be exhibit a desired functionality, e.g., the invention in its various embodiments. Functionally enzymatic activity at a selected level, when such nucleic acid equivalent nucleic acid and amino acid sequences (functional 65 sequence variant and/or portion contains a 15 nucleotide variants), which may include conservatively modified Vari sequence identical to any 15 nucleotide sequence set forth in ants as well as more extensively varied sequences, which are the nucleic acid sequences recited herein including, without US 8,883,464 B2 27 28 limitation, the sequence starting at nucleotide number 1 and level, which may require multiple substitutions). More sub ending at nucleotide number 15, the sequence starting at stitutions may be made in regions recognized or believed to nucleotide number 2 and ending at nucleotide number 16, the not be involved with an active site or other binding or struc sequence starting at nucleotide number 3 and ending at nucle tural motif. In accordance with Table 1, for example, substi otide number 17, and so forth. It will be appreciated that the tutions may be made of one polar uncharged (PU)amino acid invention also provides isolated nucleic acid that contains a for a polar uncharged amino acid of a listed sequence, option nucleotide sequence that is greater than 15 nucleotides (e.g., ally considering size/molecular weight (i.e., Substituting a 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more serine for a threonine). Guidance concerning which amino nucleotides) in length and identical to any portion of the acid changes are likely to be phenotypically silent can be sequence set forth in nucleic acid sequences recited herein. 10 found, inter alia, in Bowie, J. U., et Al., “Deciphering the For example, the invention provides isolated nucleic acid that Message in Protein Sequences: Tolerance to Amino Acid contains a 25 nucleotide sequence identical to any 25 nucle Substitutions.” Science 247: 1306-1310 (1990). This refer otide sequence set forth in any one or more (including any ence is incorporated by reference for Such teachings, which grouping of) nucleic acid sequences recited herein including, are, however, also generally known to those skilled in the art. without limitation, the sequence starting at nucleotide num 15 Recognized conservative amino acid Substitutions comprise ber 1 and ending at nucleotide number 25, the sequence (Substitutable amino acids following each colon of a set): starting at nucleotide number 2 and ending at nucleotide alaser, arg:lys; asn:gln or his; asp:glu; cys:ser, gln.asn., glu: number 26, the sequence starting at nucleotide number 3 and asp; gly: pro; his:asin or gln, ile:leu or val; leucile or val; lys: ending at nucleotide number 27, and so forth. Additional arg or gln or glu, met:leu or ile; phe:met or leu or tyr; ser:thr; examples include, without limitation, isolated nucleic acids thr:ser; trp:tyr; tyr: trp orphe; val:ile or leu. that contain a nucleotide sequence that is 50 or more nucle It is noted that codon preferences and codon usage tables otides (e.g., 100, 150, 200, 250, 300, or more nucleotides) in for a particular species can be used to engineer isolated length and identical to any portion of any of the sequences nucleic acid molecules that take advantage of the codon usage disclosed herein. Such isolated nucleic acids can include, preferences of that particular species. For example, the iso without limitation, those isolated nucleic acids containing a 25 lated nucleic acid provided herein can be designed to have nucleic acid sequence represented in any one section of dis codons that are preferentially used by a particular organism of cussion and/or examples, such as regarding 3-HP production interest. Numerous Software and sequencing services are pathways, nucleic acid sequences encoding enzymes of the available for Such codon-optimizing of sequences. fatty acid synthase system, or 3-HP tolerance. For example, The invention provides polypeptides that contain the entire the invention provides an isolated nucleic acid containing a 30 amino acid sequence of an amino acid sequence listed or nucleic acid sequence listed herein that contains a single otherwise disclosed herein. In addition, the invention pro insertion, a single deletion, a single substitution, multiple vides polypeptides that contain a portion of an amino acid insertions, multiple deletions, multiple Substitutions, or any sequence listed or otherwise disclosed herein. For example, combination thereof (e.g., single deletion together with mul the invention provides polypeptides that contain a 15 amino tiple insertions). Such isolated nucleic acid molecules can 35 acid sequence identical to any 15 amino acid sequence of an share at least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99 amino acid sequence listed or otherwise disclosed herein percent sequence identity with a nucleic acid sequence listed including, without limitation, the sequence starting at amino herein (i.e., in the sequence listing). acid residue number 1 and ending at amino acid residue Additional examples include, without limitation, isolated number 15, the sequence starting at amino acid residue num nucleic acids that contain a nucleic acid sequence that 40 ber 2 and ending at amino acid residue number 16, the encodes an amino acid sequence that is 50 or more amino acid sequence starting at amino acid residue number 3 and ending residues (e.g., 100, 150, 200, 250, 300, or more amino acid at amino acid residue number 17, and so forth. It will be residues) in length and identical to any portion of an amino appreciated that the invention also provides polypeptides that acid sequence listed or otherwise disclosed herein. contain an amino acid sequence that is greater than 15 amino In addition, the invention provides isolated nucleic acid 45 acid residues (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, that contains a nucleic acid sequence that encodes an amino 27, 28, 29, 30 or more amino acid residues) in length and acid sequence having a variation of an amino acid sequence identical to any portion of an amino acid sequence listed or listed or otherwise disclosed herein. For example, the inven otherwise disclosed herein For example, the invention pro tion provides isolated nucleic acid containing a nucleic acid vides polypeptides that contain a 25 amino acid sequence sequence encoding an amino acid sequence listed or other 50 identical to any 25 amino acid sequence of an amino acid wise disclosed herein that contains a single insertion, a single sequence listed or otherwise disclosed herein including, with deletion, a single Substitution, multiple insertions, multiple out limitation, the sequence starting at amino acid residue deletions, multiple Substitutions, or any combination thereof number 1 and ending at amino acid residue number 25, the (e.g., single deletion together with multiple insertions). Such sequence starting at amino acid residue number 2 and ending isolated nucleic acid molecules can contain a nucleic acid 55 at amino acid residue number 26, the sequence starting at sequence encoding an amino acid sequence that shares at amino acid residue number 3 and ending at amino acid resi least 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, or 99 percent due number 27, and so forth. Additional examples include, sequence identity with an amino acid sequence listed or oth without limitation, polypeptides that contain an amino acid erwise disclosed herein. sequence that is 50 or more amino acid residues (e.g., 100, Examples of properties that provide the bases for conser 60 150, 200,250,300 or more amino acid residues) in length and Vative and other amino acid Substitutions are exemplified in identical to any portion of an amino acid sequence listed or Table 1. Accordingly, one skilled in the art may make numer otherwise disclosed herein. Further, it is appreciated that, per ous Substitutions to obtain an amino acid sequence variant above, a 15 nucleotide sequence will provide a 5 amino acid that exhibits a desired functionality. BLASTP, CLUSTALP. sequence, so that the latter, and higher-length amino acid and other alignment and comparison tools may be used to 65 sequences, may be defined by the above-described nucleotide assess highly conserved regions, to which fewer Substitutions sequence lengths having identity with a sequence provided may be made (unless directed to alter activity to a selected herein. US 8,883,464 B2 29 30 In addition, the invention provides polypeptides that an tuted for (or by) a hydrophobic residue, e.g., leucine, isoleu amino acid sequence having a variation of the amino acid cine, phenylalanine, Valine or alanine; (b) a cysteine or pro sequence set forth in an amino acid sequence listed or other line is substituted for (or by) any other residue; (c) a residue wise disclosed herein. For example, the invention provides having an electropositive side chain, e.g., lysine, arginine, or polypeptides containing an amino acid sequence listed or 5 histidine, is substituted for (or by) an electronegative residue, otherwise disclosed herein that contains a single insertion, a e.g., glutamic acid or aspartic acid; or (d) a residue having a single deletion, a single Substitution, multiple insertions, bulky side chain, e.g., phenylalanine, is Substituted for (orby) multiple deletions, multiple Substitutions, or any combination one not having a side chain, e.g., glycine. The effects of these thereof (e.g., single deletion together with multiple inser amino acid substitutions (or other deletions or additions) can tions). Such polypeptides can containanamino acid sequence 10 be assessed for polypeptides having enzymatic activity by that shares at least 60, 65, 70, 75, 80, 85,90, 95, 97,98 or 99 analyzing the ability of the polypeptide to catalyze the con percent sequence identity with an amino acid sequence listed version of the same substrate as the related native polypeptide or otherwise disclosed herein. A particular variantamino acid to the same product as the related native polypeptide. Accord sequence may comprise any number of variations as well as ingly, polypeptides having 5, 10, 20, 30, 40, 50 or less con any combination of types of variations. 15 servative substitutions are provided by the invention. The invention includes, in various embodiments, an amino Polypeptides and nucleic acids encoding polypeptides can acid sequence having a variation of any of the polynucleotide be produced by standard DNA mutagenesis techniques, for and polypeptide sequences disclosed herein. As one example, example, M13 primer mutagenesis. Details of these tech variations are exemplified for the carbonic anhydrase (E. coli niques are provided in Sambrook and Russell, 2001. Nucleic cynT) amino acid sequence set forth in SEQID NO:544. FIG. acid molecules can contain changes of a coding region to fit 3 provides a CLUSTAL multiple sequence alignment of the the codon usage bias of the particular organism into which the E. coli carbonic anhydrase aligned with carbonic anhydrases molecule is to be introduced. of eleven other species that had relatively high homology, Alternatively, the coding region can be altered by taking based on low E values, in a BLASTP comparison. SEQ ID advantage of the degeneracy of the genetic code to alter the NO:544 is the fifth sequence shown. Multiple conservative 25 coding sequence in Such a way that, while the nucleic acid and less conservative substitutions are shown (i.e., by the “: sequence is substantially altered, it nevertheless encodes a and “” designations, respectively), which can lead to addi polypeptide having an amino acid sequence identical or Sub tional modifications by one skilled in the art. Thus, examples stantially similar to the native amino acid sequence. For of variations of the sequence set forth in SEQ ID NO:544 example, alanine is encoded in the open reading frame by the include, without limitation, any variation of the sequences as 30 nucleotide codon triplet GCT. Because of the degeneracy of set forth in FIG. 3. Such variations are provided in FIG. 3 in the genetic code, three other nucleotide codon triplets— that a comparison of the amino acid residue (or lack thereof) GCA, GCC, and GCG also code for alanine. Thus, the at a particular position of the sequence set forth in SEQ ID nucleic acid sequence of the open reading frame can be NO:544 with the amino acid residue (or lack thereof) at the changed at this position to any of these three codons without same aligned position of any of the other eleven amino acid 35 affecting the amino acid sequence of the encoded polypeptide sequences of FIG.3 provides a list of specific changes for the or the characteristics of the polypeptide. Based upon the sequence set forth in SEQID NO:544. For example, the “E” degeneracy of the genetic code, nucleic acid variants can be glutamic acid at position 14 of SEQID NO:544 can be sub derived from a nucleic acid sequence disclosed herein using stituted with a “Daspartic acid or “N” asparagine as indi standard DNA mutagenesis techniques as described herein, cated in FIG. 3. It will be appreciated that the sequence set 40 or by synthesis of nucleic acid sequences. Thus, for various forth in SEQID NO:544 can contain any number of variations embodiments the invention encompasses nucleic acid mol as well as any combination of types of variations. It is noted ecules that encode the same polypeptide but vary in nucleic that the amino acid sequences provided in FIG. 3 can be acid sequence by virtue of the degeneracy of the genetic code. polypeptides having carbonic anhydrase activity. The invention also provides an isolated nucleic acid that is As indicated herein, polypeptides having a variant amino 45 at least about 12 bases in length (e.g., at least about 13, 14, 15. acid sequence can retain enzymatic activity. Such polypep 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 100,250, 500, 750, 1000, tides can be produced by manipulating the nucleotide 1500, 2000, 3000, 4000, or 5000 bases in length) and hybrid sequence encoding a polypeptide using standard procedures izes, under hybridization conditions, to the sense or antisense Such as site-directed mutagenesis or various PCR techniques. Strand of a nucleic acid having a sequence listed or otherwise As noted herein, one type of modification includes the Sub 50 disclosed herein. The hybridization conditions can be mod stitution of one or more amino acid residues for amino acid erately or highly stringent hybridization conditions. Also, in residues having a similar chemical and/or biochemical prop Some embodiments the microorganism comprises an endog erty. For example, a polypeptide can have an amino acid enous 3-HP production pathway (which may, in some Such sequence set forth in an amino acid sequence listed or other embodiments, be enhanced), whereas in other embodiments wise disclosed herein comprising one or more conservative 55 the microorganism does not comprise a 3-HP production Substitutions. pathway, but is provided with one or more nucleic acid More Substantial changes can be obtained by selecting sequences encoding polypeptides having enzymatic activity Substitutions that are less conservative, and/or in areas of the or activities to complete a pathway, described herein, result sequence that may be more critical, for example selecting ing in production of 3-HP. In some embodiments, the particu residues that differ more significantly in their effect on main 60 lar sequences disclosed herein, or conservatively modified taining: (a) the structure of the polypeptide backbone in the variants thereof, are provided to a selected microorganism, area of the Substitution, for example, as a sheet or helical Such as selected from one or more of the species and groups conformation; (b) the charge or hydrophobicity of the of species or other taxonomic groups listed herein. polypeptide at the target site; or (c) the bulk of the side chain. VI. Redirecting Malonyl-CoA from Fatty Acid Synthesis The Substitutions that in general are expected to produce the 65 to 3-HP greatest changes in polypeptide function are those in which: Compositions of the present invention, such as genetically (a) a hydrophilic residue, e.g., serine or threonine, is Substi modified microorganisms, comprise a production pathway US 8,883,464 B2 31 32 for a chemical product in which malonyl-CoA is a Substrate, place of the native enoyl-ACP reductase, so that an elevated and may also comprise one or more genetic modifications to culture temperature results in reduced enzymatic activity, reduce the activity of enzymes encoded by one or more of the which then results in a shifting utilization of malonyl-CoA to fatty acid synthetase system genes. The compositions may be production of a desired chemical product. At Such elevated used in the methods and systems of the present invention. temperature the enzyme is considered non-permissive, as is Regarding microbial fermentation of a number of chemical the temperature. One such sequence is a mutant temperature products in many microorganisms of commercial fermenta sensitive fabl (fabI) of E. coli, SEQID NO:769 for DNA, tion interest, malonyl-CoA is a metabolic intermediate that, SEQID NO:770 for protein. under normal growth conditions, is converted to fatty acids It is appreciated that nucleic acid and amino acid sequences and derivatives thereof. Such as phospholipids, that are then 10 for enoyl-ACP reductase in species other than E. coli are used in cell membranes and for other key cellular functions. readily obtained by conducting homology searches in known For example, in Escherichia coli, the fatty acid synthase genomics databases, such as BLASTN and BLASTP. system is a type II or dissociated fatty acid synthase system. Approaches to obtaining homologues in other species and In this system the enzymes of fatty acid production pathway functional equivalent sequences are described herein. are encoded by distinct genes, and, common for many critical 15 Accordingly, it is appreciated that the present invention may metabolic pathways, is well-regulated, including by down be practiced by one skilled in the art for many microorganism stream products inhibiting upstream enzymes. species of commercial interest. In various microorganisms conversion of the metabolic Other approaches than a temperature-sensitive enoyl-ACP intermediate malonyl-CoA to fatty acids via a fatty acid syn reductase may be employed as known to those skilled in the thesis system (i.e., pathway or complex) is the only or the art, Such as, but not limited to, replacing a native enoyl-ACP major use of malonyl-CoA. It has been determined that when or enoyl-coA reductase with a nucleic acid sequence that a production pathway to an alternative chemical product includes an inducible promoter for this enzyme, so that an exists in a microorganism, reducing Such conversion of malo initial induction may be followed by no induction, thereby nyl-CoA to fatty acids can improve metrics for production of decreasing enoyl-ACP or enoyl-coA reductase enzymatic that alternative chemical product (e.g., 3-HP). For example, 25 activity after a selected cell density is attained. in many microorganism cells the fatty acid synthase system In Some aspects, compositions, methods and systems of the comprises polypeptides that have the following enzymatic present invention shift utilization of malonyl-CoA in a activities: malonyl-CoA-acyl carrier protein (ACP) transacy genetic modified microorganism, which comprises at least lase; B-ketoacyl-ACP synthase; B-ketoacyl-ACP reductase: one enzyme of the fatty acid synthase system, such as enoyl B-hydroxyacyl-ACP dehydratase: 3-hydroxyacyl-(acp) 30 acyl carrier protein reductase (enoyl-ACP reductase) or dehydratase; and enoyl-acyl carrier protein reductase (enoyl enoyl-coenzyme A reductase (enoyl-coA reductase). B-ke ACP reductase). In various embodiments nucleic acid toacyl-ACP synthase or B-ketoacyl-coA synthase malonyl sequences that encode temperature-sensitive forms of these CoA-ACP and may further comprise at least one genetic polypeptides may be introduced in place of the native modification of nucleic acid sequence encoding carbonic enzymes, and when Such genetically modified microorgan 35 anhydrase to increase bicarbonate levels in the microorgan isms are cultured at elevated temperatures (at which these ism cell and/or a Supplementation of its culture medium with thermolabile polypeptides become inactivated, partially or bicarbonate and/or carbonate, and may further comprise one completely, due to alterations in protein structure or complete or more genetic modifications to increase enzymatic activity denaturation), there is observed an increase in a product Such of one or more of acetyl-CoA carboxylase and NADPH as 3-HP. In other embodiments other types of genetic modi 40 dependent transhydrogenase. More generally, addition of car fications may be made to otherwise modulate. Such as lower, bonate and/or bicarbonate may be used to increase bicarbon enzymatic activities of one or more of these polypeptides. In ate levels in a fermentation broth. various embodiments a result of such genetic modifications is In some aspects, the present invention comprises a geneti to shift malonyl-CoA utilization so that there is a reduced cally modified microorganism that comprises at least one conversion of malonyl-CoA to fatty acids, overall biomass, 45 genetic modification that provides, completes, or enhances a and proportionally greater conversion of carbon source to a 3-HP production pathway effective to convert malonyl-CoA chemical product such as 3-HP. In various embodiments, the to 3-HP, and further comprises a genetic modification of specific productivity for the microbially produced chemical carbonic anhydrase to increase bicarbonate levels in the product is unexpectedly high. Also, additional genetic modi microorganism cell and/or a Supplementation of its culture fications, such as to increase malonyl-CoA production, may 50 medium with bicarbonate and/or carbonate, and may further be made for certain embodiments. comprise one or more genetic modifications to increase enzy One enzyme, enoyl(acyl carrier protein) reductase (ECNo. matic activity of one or more of acetyl-CoA carboxylase and 1.3.1.9, also referred to as enoyl-ACP reductase) is a key NADPH-dependent transhydrogenase. Related methods and enzyme for fatty acid biosynthesis from malonyl-CoA. In systems utilize Such genetically modified microorganism. Escherichia coli this enzyme, Fab, is encoded by the gene 55 In some aspects, the present invention comprises a geneti fabl (See “Enoyl-Acyl Carrier Protein (fabI) Plays a Deter cally modified microorganism that comprises at least one minant Role in Completing Cycles of Fatty Acid Elongation genetic modification that provides, completes, or enhances a in Escherichia coli,” Richard J. Heath and Charles O. Rock, J. 3-HP production pathway effective to convert malonyl-CoA Biol. Chem. 270:44, pp. 26538-26543 (1995), incorporated to 3-HP, and further comprises a genetic modification of at by reference for its discussion of fab and the fatty acid 60 least one enzyme of the fatty acid synthase system, such as synthase system). enoyl-acyl carrier protein reductase (enoyl-ACP reductase) The present invention may utilize a microorganism that is or enoyl-coenzyme A reductase (enoyl-coA reductase), B-ke provided with a nucleic acid sequence (polynucleotide) that toacyl-ACP synthase or B-ketoacyl-coA synthase, malonyl encodes a polypeptide having enoyl-ACP reductase enzy CoA-ACP and may further comprise a genetic modification matic activity that may be modulated during a fermentation 65 of carbonic anhydrase to increase bicarbonate levels in the event. For example, a nucleic acid sequence encoding a tem microorganism cell and/or a Supplementation of its culture perature-sensitive enoyl-ACP reductase may be provided in medium with bicarbonate and/or carbonate, and may further US 8,883,464 B2 33 34 comprise one or more genetic modifications to increase enzy Without being bound to a particular theory, it is believed matic activity of one or more of acetyl-CoA carboxylase and that reducing the enzymatic activity of enoyl-ACP reductase NADPH-dependent transhydrogenase. Related methods and (and/or of other enzymes of the fatty acid synthase system) in systems utilize Such genetically modified microorganism. a microorganism leads to an accumulation and/or shunting of In various embodiments the present invention is directed to 5 malonyl-CoA, a metabolic intermediate upstream of the a method of making a chemical product comprising: provid enzyme, and Such malonyl-CoA may then be converted to a ing a selected cell density of a genetically modified microor chemical product for which the microorganism cell com ganism population in a vessel, wherein the genetically modi prises a metabolic pathway that utilizes malonyl-CoA. In fied microorganism comprises a production pathway for certain compositions, methods and systems of the present production of a chemical product from malonyl-CoA, and 10 invention the reduction of enzymatic activity of enoyl-ACP reducing enzymatic activity of at least one enzyme of the reductase (or, more generally, of the fatty acid synthase sys genetically modified microorganism’s fatty acid synthase tem) is made to occur after a sufficient cell density of a pathway. genetically modified microorganism is attained. This bi-pha In various embodiments, reducing the enzymatic activity sic culture approach balances a desired quantity of catalyst, in of an enoyl-ACP reductase in a microorganism host cell 15 the cell biomass which Supports a particular production rate, results in production of 3-HP at elevated specific and volu with yield, which may be partly attributed to having less metric productivity. In still other embodiments, reducing the carbon be directed to cell mass after the enoyl-ACP reductase enzymatic activity of an enoyl-CoA reductase in a microor activity (and/or activity of other enzymes of the fatty acid ganism host cell results in production of 3-HP at elevated 20 synthase system) is/are reduced. This results in a shifting net specific and Volumetric productivity. utilization of malonyl-CoA, thus providing for greater carbon Another approach to genetic modification to reduce enzy flux to a desired chemical product. matic activity of these enzymes is to provide an inducible In various embodiments of the present invention the spe promoter that promotes one Such enzyme, such as the enoyl cific productivity is elevated and this results in overall rapid ACP reductase gene (e.g., fab in E. coli). In Such example 25 and efficient microbial fermentation methods and systems. In this promoter may be induced (Such as with isopropyl-L-D- various embodiments the volumetric productivity also is sub thiogalactopyranoiside (IPTG)) during a first phase of a stantially elevated. method herein, and after the IPTG is exhausted, removed or In various embodiments a genetically modified microor diluted out the second step, of reducing enoyl-ACP reductase ganism comprises a metabolic pathway that includes conver enzymatic activity, may begin. Other approaches may be 30 sion of malonyl-CoA to a desired chemical product, 3-hy applied to control enzyme expression and activity Such as are droxypropionic acid (3-HP). This is viewed as quite described herein and/or known to those skilled in the art. advantageous for commercial 3-HP production economics While enoyl-CoA reductase is considered an important and is viewed as an advance having clear economic benefit. enzyme of the fatty acid synthase system, genetic modifica Other chemical products also are disclosed herein. tions may be made to any combination of the polynucleotides 35 The improvements in both specific and volumetric produc (nucleic acid sequences) encoding the polypeptides exhibit tivity parameters are unexpected and advance the art. ing the enzymatic activities of this system, such as are listed The reduction of enoyl-ACP reductase activity and/or of herein. For example, FabB. B-ketoacyl-acyl carrier protein other enzymes of the fatty acid synthase system may be synthase I, is an enzyme in E. coli that is essential for growth achieved in a number of ways, as is discussed herein. and the biosynthesis of both saturated and unsaturated fatty 40 By “means for modulating the conversion of malonyl acids. Inactivation of FabB results in the inhibition of fatty CoA to fatty acyl-ACP or fatty acyl-coA molecules, and to acid elongation and diminished cell growth as well as elimi fatty acid molecules, is meant any one of the following: 1) nating a futile cycle that recycles the malonate moiety of providing in a microorganism cell at least one polynucleotide malonyl-ACP back to acetyl-CoA. FabF. B-ketoacyl-acyl car that encodes at least one polypeptide having activity of one of rier protein synthase II, is required for the synthesis of Satu- 45 the fatty acid synthase system enzymes (such as recited rated fatty acids and the control membrane fluidity in cells. herein), wherein the polypeptide so encoded has (Such as by Both enzymes are inhibited by cerulenin. mutation and/or promoter Substitution, etc., to lower enzy It is reported that overexpression of FabF results in dimin matic activity), or may be modulated to have (such as by ished fatty acid biosynthesis. It is proposed that FabF out temperature sensitivity, inducible promoter, etc.) a reduced competes FabB for association with Fab), malonyl-CoA: 50 enzymatic activity; 2) providing to a vessel comprising a ACP transacylase. The association of FabB with Fab) is microorganism cell or population an inhibitor that inhibits required for the condensation reaction that initiates fatty acid enzymatic activity of one or more of the fatty acid synthase elongation. (See Microbiological Reviews, September 1993, system enzymes (such as recited herein), at a dosage effective p. 522-542 Vol. 57, No. 3; K. Magnuson et al., “Regulation of to reduce enzymatic activity of one or more of these enzymes. Fatty Acid Biosynthesis in Escherichia coli. American Soci- 55 These means may be provided in combination with one ety for Microbiology: W. Zha et al., “Improving cellular another. When a means for modulating involves a conversion, malonyl-CoA level in Escherichia coli via metabolic engi during a fermentation event, from a higher to a lower activity neering.” Metabolic Engineering 11 (2009) 192-198). An of the fatty acid synthetase system, Such as by increasing alternative to genetic modification to reduce Such fatty acid temperature of a culture vessel comprising a population of synthase enzymes is to provide into a culture system a Suit- 60 genetically modified microorganism comprising a tempera able inhibitor of one or more such enzymes. This approach ture-sensitive fatty acid synthetase system polypeptide (e.g., may be practiced independently or in combination with the enoyl-ACP reductase), or by adding an inhibitor, there are genetic modification approach Inhibitors, such as cerulenin, conceived two modes—one during which there is higher thiolactomycin, and triclosan (this list not limiting) or genetic activity, and a second during which there is lower activity, of modifications directed to reduce activity of enzymes encoded 65 Such fatty acid synthetase system. During the lower activity by one or more of the fatty acid synthetase system genes may mode, a shift to greater utilization of malonyl-CoA to a be employed, singly or in combination. selected chemical product may proceed. US 8,883,464 B2 35 36 Once the modulation is in effect to decrease the noted A mono-functional malonyl-CoA reductase in combina enzymatic activity(ies), each respective enzymatic activity so tion with a 3-HP dehydrogenase. By mono-functional is modulated may be reduced by at least 10, at least 20, at least meant that the malonyl-CoA reductase catalyzes the conver 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent compared with the activity of the native, sion of malonyl-CoA to malonate semialdehyde. non-modulated enzymatic activity (such as in a cell or iso 5 Any of the above polypeptides may be NADH- or lated). Similarly, the conversion of malonyl-CoA to fatty NADPH-dependent, and methods known in the art may be acyl-ACP or fatty acyl-coA molecules may be reduced by at used to convert a particular enzyme to be either form. More least 10, at least 20, at least 30, at least 40, at least 50, at least particularly, as noted in WO 2002/042418, “any method can 60, at least 70, at least 80, or at least 90 percent compared with be used to convert a polypeptide that uses NADPH as a Such conversion in a non-modulated cell or other system. 10 cofactor into a polypeptide that uses NADH as a cofactor such Likewise, the conversion of malonyl-CoA to fatty acid mol as those described by others (Eppink et al., J. Mol. Biol. 292 ecules may be reduced by at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at (1): 87-96 (1999), Hall and Tomsett, Microbiology, 146 (Pt least 90 percent compared with Such conversion in a non 6): 1399-406 (2000), and Dohr et al., Proc. Natl. Acad. Sci., modulated cell or other system. 15 98 (1): 81-86 (2001)).” VII. Production Pathway from Malonyl-CoA to 3-HP Without being limiting, a bi-functional malonyl-CoA In various embodiments the compositions, methods and reductase may be selected from the malonyl-CoA reductase systems of the present invention involve inclusion of a meta of Chloroflexus aurantiacus (such as from ATCC 29365) and bolic production pathway that converts malonyl-CoA to a other sequences. Also without being limiting, a mono-func chemical product of interest. tional malonyl-CoA reductase may be selected from the As one example, 3-HP is selected as the chemical product malonyl-CoA reductase of Sulfolobus tokodaii (SEQ ID of interest. NO:826). As to the malonyl-CoA reductase of C. aurantia Further as to specific sequences for 3-HP production path cus, that sequence and other species’ sequences may also be way, malonyl-CoA reductase (mcr) from C. aurantiacus was bi-functional as to this enzymatic activity. gene synthesized and codon optimized by the services of 25 When a mono-functional malonyl-CoA reductase is pro DNA 2.0. The FASTA sequence is shown in SEQID NO:783 vided in a microorganism cell, 3-HP dehydrogenase enzy (gil42561982gb|AAS20429.11 malonyl-CoA reductase matic activity also may be provided to convert malonate semi (Chloroflexus aurantiacus)). aldehyde to 3-HP. As shown in the examples, a mono Mcr has very few sequence homologs in the NCBI data functional malonyl-CoA reductase may be obtained by base. Blast searches finds 8 different sequences when search 30 ing over the entire protein. Hence development of a pile-up truncation of a bi-functional mono-functional malonyl-CoA, sequences comparison is expected to yield limited informa and combined in a strain with an enzyme that converts mal tion. However, embodiments of the present invention none onate semialdehyde to 3-HP. theless may comprise any of these eight sequences, shown Also, it is noted that another malonyl-CoA reductase is herein and identified as SEQID NOs:784 to 791, which are 35 known in Metallosphaera sedula (Msed 709, identified as expected to be but are not yet confirmed to be bi-functional as malonyl-CoA reductase/Succinyl-CoA reductase). to this enzymatic activity. Other embodiments may comprise By providing nucleic acid sequences that encode polypep mutated and other variant forms of any of SEQID NOS:784 to tides having the above enzymatic activities, a genetically 791, as well as polynucleotides (including variant forms with modified microorganism may comprise an effective 3-HP conservative and other Substitutions). Such as those intro 40 pathway to convert malonyl-CoA to 3-HP in accordance with duced into a selected microorganism to provide or increase the embodiments of the present invention. 3-HP production therein. Other 3-HP pathways, such as those comprising an ami The portion of a CLUSTAL 2.0.11 multiple sequence notransferase (see, e.g., WO 2010/01 1874, published Jan. 28, alignment identifies these eight sequences with respective 2010), may also be provided in embodiments of a genetically SEQ ID NOs: 783-791, as shown in the following table. modified microorganism of the present invention. TABLE 2 Reference Nos. Seq ID No. Genus Species gi42561982gb|AAS20429.1 783 Chloroflexus aurantiacus gil 163848.165 refYP 001636209 784 Chloroflexus aurantiacus J-10-fl gi219848167 refYP 002462600 785 Chloroflexus aggregains DSM 94.85 gil 156742880 refYP 001433009 786 Roseiflexus castenholzii DSM 13941 gil 148657307 refYP 001277512 787 Roseiflexus sp. RS-1 gi85708113 refZP 01039179.1 788 Erythrobacter sp. NAP1 gi254282228 |ref ZP 0495.7196.1 789 gamma proteobacterium NOR51-B gi254513883|ref ZP 05125944.1 790 gamma proteobacterium NOR5-3 gil 1195.04313|ref ZP 01626393.1 791 3 marine gamma proteobacterium HTCC208

Malonyl-CoA may be converted to 3-HP in a microorgan Incorporated into this section, the present invention pro ism that comprises one or more of the following: 60 vides for elevated specific and volumetric productivity met rics as to production of a selected chemical product, Such as A bi-functional malonyl-CoA reductase, such as may be 3-hydroxypropionic acid (3-HP). In various embodiments, obtained from Chloroflexus aurantiacus and other microor production of a chemical product, such as 3-HP, is not linked ganism species. By bi-functional in this regard is meant that to growth. the malonyl-CoA reductase catalyzes both the conversion of 65 In various embodiments, production of 3-HP, or alterna malonyl-CoA to malonate semialdehyde, and of malonate tively one of its downstream products such as described semialdehyde to 3-HP. herein, may reach at least 1, at least 2, at least 5, at least 10, at US 8,883,464 B2 37 38 least 20, at least 30, at least 40, and at least 50 g/liter titer, such In various aspects and embodiments of the present inven as by using one of the methods disclosed herein. tion, there is a resulting Substantial increase in microorganism As may be realized by appreciation of the advances dis specific productivity that advances the fermentation art and closed herein as they relate to commercial fermentations of commercial economic feasibility of microbial chemical pro selected chemical products, embodiments of the present 5 duction, such as of 3-HP (but not limited thereto). invention may be combined with other genetic modifications Stated in another manner, in various embodiments the spe and/or method or system modulations so as to obtain a micro cific productivity exceeds (is at least) 0.01 g chemical prod organism (and corresponding method) effective to produce at uct/g DCW-hr, exceeds (is at least) 0.05 g chemical product/g least 10, at least 20, at least 30, at least 40, at least 45, at least DCW-hr, exceeds (is at least) 0.10 g chemical product/g 10 DCW-hr, exceeds (is at least) 0.15 g chemical product/g 50, at least 80, at least 100, or at least 120 grams of a chemical DCW-hr, exceeds (is at least) 0.20 g chemical product/g product, Such as 3-HP. per liter of final (e.g., spent) fermen DCW-hr, exceeds (is at least) 0.25 g chemical product/g tation broth while achieving this with specific and/or volu DCW-hr, exceeds (is at least) 0.30 g chemical product/g metric productivity rates as disclosed herein. DCW-hr, exceeds (is at least) 0.35 g chemical product/g In some embodiments a microbial chemical production 15 DCW-hr, exceeds (is at least) 0.40 g chemical product/g event (i.e., a fermentation event using a cultured population of DCW-hr, exceeds (is at least) 0.45 g chemical product/g a microorganism) proceeds using a genetically modified DCW-hr, exceeds (is at least) 0.50 g chemical product/g microorganism as described herein, wherein the specific pro DCW-hr, exceeds (is at least) 0.60 g chemical product/g ductivity is between 0.01 and 0.60 grams of 3-HP produced DCW-hr. per gram of microorganism cell on a dry weight basis per hour More generally, based on various combinations of the (g 3-HP/g DCW-hr). In various embodiments the specific genetic modifications described herein, optionally in combi productivity is greater than 0.01, greater than 0.05, greater nation with Supplementations described herein, specific pro than 0.10, greater than 0.15, greater than 0.20, greater than ductivity values for 3-HP, and for other chemical products 0.25, greater than 0.30, greater than 0.35, greater than 0.40, described herein, may exceed 0.01 g chemical product/g greater than 0.45, or greater than 0.50 g 3-HP/g DCW-hr. 25 DCW-hr, may exceed 0.05 g chemical product/g DCW-hr, Specific productivity may be assessed over a 2, 4, 6, 8, 12 or may exceed 0.10g chemical product/g DCW-hr, may exceed 24 hour period in a particular microbial chemical production 0.15 g chemical product/g DCW-hr, may exceed 0.20 g event. More particularly, the specific productivity for 3-HP or chemical product/g DCW-hr, may exceed 0.25 g chemical other chemical product is between 0.05 and 0.10, 0.10 and product/g DCW-hr, may exceed 0.30 g chemical product/g 0.15, 0.15 and 0.20, 0.20 and 0.25, 0.25 and 0.30, 0.30 and 30 DCW-hr, may exceed 0.35 g chemical product/g DCW-hr, 0.35, 0.35 and 0.40, 0.40 and 0.45, or 0.45 and 0.50 g3-HP/g may exceed 0.40 g chemical product/g DCW-hr, may exceed DCW-hr., 0.50 and 0.55, or 0.55 and 0.60 g3-HP/g DCW-hr. 0.45 g chemical product?gDCW-hr, and may exceed 0.50g or Various embodiments comprise culture systems demonstrat 0.60 chemical product/g DCW-hr. Such specific productivity ing Such productivity. may be assessed over a 2, 4, 6, 8, 12 or 24 hour period in a Also, in various embodiments of the present invention the 35 particular microbial chemical production event. volumetric productivity achieved may be 0.25 g 3-HP (or The improvements achieved by embodiments of the other chemical product) per liter per hour (g (chemical prod present invention may be determined by percentage increase uct)/L-hr), may be greater than 0.25 g 3-HP (or other chemi in specific productivity, or by percentage increase in Volumet cal product)/L-hr, may be greater than 0.50 g 3-HP (or other ric productivity, compared with an appropriate control micro chemical product)./L-hr, may be greater than 1.0 g 3-HP (or 40 organism lacking the particular genetic modification combi other chemical product)/L-hr, may be greater than 1.50 g nations taught herein (with or without the Supplements taught 3-HP (or other chemical product)/L-hr, may be greater than herein, added to a vessel comprising the microorganism 2.0 g 3-HP (or other chemical product)/L-hr, may be greater population). For particular embodiments and groups thereof, than 2.50 g 3-HP (or other chemical product)/L-hr, may be Such specific productivity and/or Volumetric productivity greater than 3.0 g 3-HP (or other chemical product)/L-hr, may 45 improvements is/are at least 10, at least 20, at least 30, at least be greater than 3.50 g3-HP (or other chemical product)/L-hr, 40, at least 50, at least 100, at least 200, at least 300, at least may be greater than 4.0 g 3-HP (or other chemical product)/ 400, and at least 500 percent over the respective specific L-hr, may be greater than 4.50 g 3-HP (or other chemical productivity and/or Volumetric productivity of such appropri product)/L-hr, may be greater than 5.0 g 3-HP (or other ate control microorganism. chemical product)/L-hr, may be greater than 5.50 g 3-HP (or 50 The specific methods and teachings of the specification, other chemical product)/L-hr, may be greater than 6.0 g 3-HP and/or cited references that are incorporated by reference, (or other chemical product)/L-hr, may be greater than 6.50 g may be incorporated into the examples. Also, production of 3-HP (or other chemical product)/L-hr, may be greater than 3-HP, or one of its downstream products such as described 7.0 g 3-HP (or other chemical product)/L-hr, may be greater herein, may reach at least 1, at least 2, at least 5, at least 10, at than 7.50 g 3-HP (or other chemical product)/L-hr, may be 55 least 20, at least 30, at least 40, and at least 50 g/liter titer in greater than 8.0 g 3-HP (or other chemical product)/L-hr, may various embodiments. be greater than 8.50 g3-HP (or other chemical product)/L-hr, The metrics may be applicable to any of the compositions, may be greater than 9.0 g 3-HP (or other chemical product)/ e.g., genetically modified microorganisms, methods, e.g., of L-hr, may be greater than 9.50 g 3-HP (or other chemical producing 3-HP or other chemical products, and systems, product)/L-hr, or may be greater than 10.0 g 3-HP (or other 60 e.g., fermentation systems utilizing the genetically modified chemical product)/L-hr. microorganisms and/or methods disclosed herein. In some embodiments, specific productivity as measured It is appreciated that iterative improvements using the strat over a 24-hour fermentation (culture) period may be greater egies and methods provided herein, and based on the discov than 0.01, 0.05, 0.10, 0.20, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, eries of the interrelationships of the pathways and pathway 8.0, 9.0, 10.0, 11.0 or 12.0 grams of chemical product per 65 portions, may lead to even greater 3-HP production and tol gram DCW of microorganisms (based on the final DCW at erance and more elevated 3-HP titers at the conclusion of a the end of the 24-hour period). 3-HP bio-production event. US 8,883,464 B2 39 40 Any number of strategies may lead to development of a (“3-HP) production, wherein the increased level of 3-HP suitable modified enzyme suitable for use in a 3-HP produc production is greater than the level of 3-HP production in the tion pathway. With regard to malonyl-CoA-reductase, one wild-type microorganism, and at least one genetic modifica may utilize or modify an enzyme such as encoded by the tion of a metabolic complex identified herein as the 3-HP sequences in the table immediately above, to achieve a Suit Toleragenic Complex (3HPTGC). Under certain condi able level of 3-HP production capability in a microorganism tions, such as culture in minimal media, the 3HPTGC genetic strain. modification(s) allow the genetically modified microorgan VIII. Increasing Tolerance to 3-HP ism to produce 3-HP under specific culture conditions such A complex comprising all or portions of a number of inter that 3-HP may accumulate to a relatively higher concentra related metabolic pathways has been identified, wherein 10 tion without the toxic effects observed in unmodified micro genetic modification to increase enzymatic activities of organisms. The at least one genetic modification of a 3-HP enzymes of such complex, named the 3-HP Toleragenic Com production pathway may be to improve 3-HP accumulation plex (3HPTGC), are demonstrated to increase microorgan and/or production of a 3-HP production pathway found in the ism tolerance to exposure to 3-HP. The 3HPTGC is described wild-type microorganism, or may be to provide Sufficient in WO 2010/01 1874, published Jan. 28, 2010, which is incor 15 enzymatic conversions in a microorganism that normally porated in the present application for its teachings of the does not synthesize 3-HP so that 3-HP is thus bio-produced. 3HPTGC and combinations of genetic modifications related Methods of making such genetically modified microorgan to 3HP production and tolerance based on the 3HPTGC and isms also are described and are part of this aspect of the groups therein. invention. As described and detailed herein, the present invention Another aspect of the invention relates to a genetically broadly relates to alterations, using genetic modifications, modified microorganism comprising at least one genetic and/or medium modulations (e.g., additions of enzymatic con modification from two or more of the chorismate, threonine/ version products or other specific chemicals), to achieve homocysteine, polyamine synthesis, lysine synthesis, and desired results in microbe-based industrial bio-production nucleotide synthesis portions of the 3HPTGC. Non-limiting methods, systems and compositions. As to the tolerance 25 examples of multiple combinations exemplify the advantages aspects, this invention flows from the discovery of the unex of this aspect of the invention. Additional genetic modifica pected importance of the 3HPTPC which comprises certain tions pertain to other portions of the 3HPTGC. Capability to metabolic pathway portions comprising enzymes whose bio-produce 3-HP may be added to some genetically modi increased activity (based on increasing copy numbers of fied microorganisms by appropriate genetic modification. nucleic acid sequences that encode there) correlates with 30 Methods of identifying genetic modifications to provide a increased tolerance of a microorganism to 3-HP. microorganism achieving an increased 3-HP tolerance, and Actual data and/or prophetic examples directed to alter microorganisms made by such methods, relate to this aspect ations of the 3HPTGC are provided herein. These examples of the invention. are intended to demonstrate the breadth of applicability Another aspect of the invention relates to a genetically (based on the large number of genomic elements related to the 35 modified microorganism that is able to produce 3-hydrox 3HPTGC that demonstrate increased 3-HP tolerance) and ypropionic acid (3-HP), comprising at least one genetic Some specific approaches to achieve increased tolerance to modification to the 3HPTGC that increases enzymatic con 3-HP. Approaches may be combined to achieve additive or version at one or more enzymatic conversion steps of the synergistic improvements in 3-HPtolerance, and may include 3HPTGC for the microorganism, and wherein the at least one alterations that are genetic or non-genetic (e.g., relating to 40 genetic modification increases 3-HP tolerance of the geneti system supplementation with particular chemicals, or general cally modified microorganism above the 3-HPtolerance of a alterations to the industrial system). In addition, specific pro control microorganism lacking the genetic modification. duction strategies are disclosed and exemplified. Methods of making such genetically modified microorgan Thus, in addition to the above-described genetic modifica isms also are described and are part of this aspect of the tions, directed to providing a 3-HP production pathway and to 45 invention. providing a nucleic acid sequence comprising and/or control Another aspect of the invention relates to a genetically ling a gene encoding an enoyl-ACP reductase that allows for modified microorganism comprising various core sets of spe control of enzymatic activity of the latter enzyme, and/or as cific genetic modification(s) of the 3HPTGC. In various described herein other modifications of the fatty acid syn embodiments this aspect may additionally comprise at least thetase system, in various embodiments one or more genetic 50 one genetic modification from one or more or two or more of modifications may be made to the genetically modified the chorismate, threonine/homocysteine, polyamine synthe microorganism to increase its tolerance to 3-HP (or other sis, lysine synthesis, and nucleotide synthesis portions of the chemical products). 3HPTGC. Methods of making such genetically modified Accordingly, in some embodiments of the present inven microorganisms also are described and are part of this aspect tion, a genetically modified microorganism may comprise at 55 of the invention. least one genetic modification to provide, complete, or Further, the invention includes methods of use to improve enhance one or more 3-HP production pathways, at least one a microorganisms tolerance to 3-HP, which may be in a genetic modification to provide enoyl-ACP reductase enzy microorganism having 3-HP production capability (whether matic activity and/or other modifications of the fatty acid the latter is naturally occurring, enhanced and/or introduced synthetase system that can be controlled so as to reduce Such 60 by genetic modification). activity at a desired cell density, and at least one genetic Also, another aspect of the invention is directed to provid modification of the 3HPTGC, or one, two, or three or more ing one or more Supplements, which are Substrates (i.e., reac groups thereof, to increase tolerance of the genetically modi tants) and/or products of the 3HPTGC (collectively herein fied microorganism to 3-HP. “products' noting that substrates of all but the initial conver Accordingly, one aspect of the invention relates to a geneti 65 sion steps are also products of the 3HPTGC), to a culture of a cally modified microorganism comprising at least one genetic microorganism to increase the effective tolerance of that modification effective to increase 3-hydroxypropionic acid microorganism to 3-HP. US 8,883,464 B2 41 42 Another aspect of the invention regards the genetic modi for genetic modification(s) to increase 3-HP tolerance in a fication to introduce a genetic element that encodes a short selected microorganism species. polypeptide identified herein as IroK. The introduction of As indicated, in various embodiments the combinations of genetic elements encoding this short polypeptide has been genetic modifications as described in this section are prac demonstrated to improve 3-HP tolerance in E. coli under ticed in combination with aspects of the invention pertaining microaerobic conditions. This genetic modification may be to modulation of the fatty acid synthase system. combined with other genetic modifications and/or Supple VIIIA. SCALES Technique ment additions of the invention. As described in WO 2010/01 1874, published Jan. 28, As to methods of making 3-HP in accordance with the 2010, to obtain genetic information, initial 3-HP-related fit teachings of this invention, and to genetically modified 10 ness data was obtained by evaluation offitness of clones from microorganisms that make 3-HP, one or more genetic modi a genomic-library population using the SCALES technique. fications may be provided to a microorganism to increase These clones were grown in a selective environment imposed tolerance to 3-HP. That is, SEQID NOs:001 to 189 are incor by elevated concentrations of 3-HP shown to be a reliable test porated into this section, SEQ ID NOS:190 to 603 are pro of 3-HP tolerance. vided as nucleic acid sequences (gene, DNA) and encoded 15 amino acid sequences (proteins) of the E. coli 3HPTGC, and More particularly, to obtain data potentially useful to iden SEQ ID NOS:604 to 766 are provided as sequences of the tify genetic elements relevant to increased 3-HPtolerance, an nucleic acid sequences of the Saccharomyces cerevisiae initial population of five representative E. coli K12 genomic 3HPTG.C. libraries was produced by methods known to those skilled in Moreover, a particular genetic modification to increase the art. The five libraries respectively comprised 500, 1000, expression of carbonic anhydrase (for example, E. coli's 2000, 4000, 8000 base pair (“bp') inserts of E. coli K12 cynT SEQ ID NO:337 for DNA and SEQ ID NO:544 for genetic material. Each of these libraries, essentially compris protein sequences), may act in a dual function manner to ing the entire E. coli K12 genome, was respectively trans advantageously improve both 3-HP production and 3-HPtol formed into MACH1TM-T1(R) E. coli cells and cultured to erance. This is particularly the case when malonyl-CoA 25 mid-exponential phase corresponding to microaerobic con reductase is provided for 3-HP production. FIG. 1 depicts a ditions (ODoo-0.2). Batch transfer times were variable and production pathway from malonyl-CoA to 3-HP comprising were adjusted as needed to avoid a nutrient limited selection a bi-functional malonyl-CoA reductase, and other enzymatic environment (i.e., to avoid the cultures from entering station conversions and pathways described herein. Carbonic anhy ary phase). Although not meant to be limiting as to alternative drase is not meant to be limiting. For instance, in E. coli a 30 approaches, selection in the presence of 3-HP was carried out carbonic anhydrase 2 is known, variously designated as can over 8 serial transfer batches with a decreasing gradient of andyadE, and use of genetic modifications in embodiments of 3-HP over 60 hours. More particularly, the 3-HP concentra the present invention may use this or other genes and their tions were 20 g3-HP/L for serial batches 1 and 2, 15 g3-HP/L encoded enzymes. The sequences for can are provided as for serial batches 3 and 4, 10 g3-HP/L for serial batches 5 and SEQ ID NO: 767 (EG12319 can “carbonic anhydrase 2 35 6, and 5 g3-HP/L for serial batches 7 and 8. For serial batches monomer' (complement (142670.142008)) Escherichia coli 7 and 8 the culture media was replaced as the culture K-12 substr. MG 1655) and SEQ ID NO: 768 (EG12319 approached Stationary phase to avoid nutrient limitations. MONOMER carbonic anhydrase 2 monomer (complement Samples were taken during and at the culmination of each (142670.142008)) Escherichia coli K-12 substr. MG1655). batch in the selection, and were Subjected to microarray Also, it is appreciated that genetic modifications to 40 analysis that identified signal strengths. The individual stan increase 3-HP tolerance may be further classified by genetic dard laboratory methods for preparing libraries, transforma modifications made along particular respective portions of tion of cell cultures, and other standard laboratory methods the 3HPTGC. For example, genetic modifications may be used for the SCALES technique prior to array and data analy made to polynucleotides that encode polypeptides that cata ses are well-known in the art, such as Supported by methods lyze enzymatic reactions along specific portions of the of the 45 taught in Sambrook and Russell, Molecular Cloning: A Labo 3HPTGC and so are expected to increase production of ratory Manual. Third Edition 2001 (volumes 1-3), Cold respectively, aromatic amino acids (tyr and phe), tryptophan Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (trp), ubiquinone-8, menaquinone, enterobactin, tetrahydro (hereinafter, Sambrook and Russell, 2001). Aspects of indi folate (see respective enzymatic conversions of Group A vidual methods also are discussed in greater detail in the sheet (and inputs thereto)), one or more of the polar 50 Examples and in the SCALES technique patent applications, uncharged amino acids (gly, ser, cys, homocysteine), isoleu U.S. Patent Publication No. 2006/0084.098A1, filed Sep. 20, cine, methionine (see respective enzymatic conversions of 2005, entitled: “Mixed-Library Parallel Gene Mapping Group B sheet (and inputs thereto)), glutamine, arginine, Quantitation Microarray Technique for Genome Wide Iden putrescine, spermidine, aminopropylcadaverine (see see tification of Trait Conferring Genes' (hereinafter, the respective enzymatic conversions of Group C sheet (and 55 “SCALES Technique'), which is incorporated herein by ref inputs thereto)), cadaverine (see respective enzymatic con erence for teaching additional details of this technique. versions of Group D sheet (and input thereto)), inosine-5- Microarray technology also is well-known in the art (see, phosphate, Xanthosine-5-phosphate, adenylo-Succinate, oro e.g. ). To obtain data of which tidine-5'-phosphate, and any of the mono-, di-, and tri clones were more prevalent at different exposure periods to phosphate nucleosides (i.e., adenosine, guanosine, cytosine, 60 3-HP, Affymetrix E. coli Antisense Gene Chip arrays (Af uridine) obtainable there from (see respective enzymatic con fymetrix, Santa Clara, Calif.) were handled and scanned versions of Group E sheet (and input thereto)), glutamate, according to the E. coli expression protocol from Affymetrix Succinate. Succinate semialdehyde, oxaloacetate, and aspar producing affymetrix.cel files. A strong microarray signal tate (see respective enzymatic conversions of Group F sheet, after a given exposure to 3-HP indicates that the genetic including reactions shown along dashed lines). Such that 65 sequence introduced by the plasmid comprising this genetic 3-HP tolerance thereby increases as a result of such genetic sequence confers 3-HP tolerance. These clones can be iden modification(s). Any portion or Sub-portion may be selected tified by numerous microarray analyses known in the art. US 8,883,464 B2 43 44 Also, for the purposes of incorporation by reference as TABLE 3-continued applied in the United States, “A genomics approach to improve the analysis and design of Strain selections. T. E. SCALES Fitness Data Warnecke et al., Metabolic Engineering 10 (2008)154-165, is Cumulative Cumulative Cumulative incorporated by reference herein for its additional specific Gene Fitness Gene Fitness Gene Fitness teachings that demonstrate that SCALEs fitness data corre bur) 1.32 sdaB 122 trpE 2.85 lates with and can be used as a Surrogate of increased toler burE 1.82 serA 3.11 tynA 2.36 ance to 3-HP. This conclusion is based on the standard use of burF 2.04 SerB 2.46 tyra 9.1 burEH 1.66 serC 2.15 tyrB 1.49 a receiver operator characteristic curve (ROC) curve. ROC 10 burK 2.65 speA 2.09 ubiA 1.51 analysis is routinely used in the medical diagnostic field to l 4.83 speB 1.66 ubiB 2.09 evaluate the correlation for a diagnostic test to the actual bury 3.13 spec 1.52 ubiC 2.4 presence or absence of a disease. Currently diagnostic tests burn 2.94 speD 3.43 ubiD O.91 used through the world in medical applications that perform burT 3.73 talA 1.24 ubiF 1.02 well in a ROC analysis are routinely used to identify the buuE 1.53 talB 4.78 ubiF 1.78 15 pyrB 6.36 tdcB 1.87 ubiG 3.17 absence or presence of a disease. This analysis was adapted to pyrC 14.48 tdcD 1.64 ubi 5.35 evaluate the sensitivity and specificity of different microbial pyrD 2.26 tdcE 1.16 ubiX 1.72 pyrE 1.03 tdh 1.38 ydcW O.89 growth based selections resulting in fitness values as reliable pyrF 1.38 tktA 1.89 ydi B O.87 tests for 3-HP tolerance. In particular a growth based selec pyrC 2.23 tktB 1.21 ygG 2.51 tion using serial batch cultures with decreasing levels of 3-HP pyrH 1.78 trp A 2.45 yneIsad 4.18 was identified as a sensitive and specific test for 3-HP toler pyrI O.83 trpB 1.93 ance. As a result clones in this selection with a fitness metric rpe 2.06 greater than a cutoff of 0 are identified as clones conferring tolerance to 3-HP. VIIIB. Analysis of the SCALES Technique The following table lists some of the genes (introduced by 25 Also as described in WO 2010/01 1874, published Jan. 28, vectors of the libraries) that were shown to have elevated 2010, analysis of the 3-HPtolerance SCALEs data has led to fitness values, shown herein to confer tolerance to 3-HP. an understanding of interrelationships among various identi fied pathways and portions thereof. It is noted that the TABLE 3 3HPTGC, in its entirety, was deduced from interrelationships SCALES Fitness Data 30 between genes having elevated fitness values. Not every enzyme of the 3HPTGC was shown in the SCALES data to Cumulative Cumulative Cumulative have positive fitness values. This may be attributed to certain Gene Fitness Gene Fitness Gene Fitness deficiencies in the commercial arrays used to obtain that aceE 11.2 cysM 26.63 ilvC 2.61 SCALES data. Accordingly, some members of the E. coli aceF 8.39 (O 6.98 ilv) 1.6 35 ackA 2.36 entA 1.58 ilw O.94 3HPTGC not so derived from the SCALES genetic element acna 3.58 entB O.93 vEH 1.18 data were deduced to fill in the 3HPTGC. However, it is noted acnB 3.18 entC 1.26 ilw 1.77 that most of the enzymes in the 3HPTGC do have positive adhE 3.68 ent) 1 ilwM 1.02 fitness values, and the overall fitness data incombination with adiA 1.95 ent 1.03 wN 1.53 the Supplements and genetic modifications data, provided adk 2.18 entF 1.03 kb 3.11 40 aldA 1.83 fbaA 2.87 italE 1.14 herein, prove the validity of the deduction and the overall argA 3.94 fbaB 2.28 lysC 1.97 significance of the 3HPTGC being related to 3-HPtolerance. argB 8.94 foLA 15.07 may 2.58 As described herein, the 3HPTGC is divided into an “upper argC 4.02 follB 0.57 menA 3.2 section' comprising the glycolysis pathway, the tricarboxylic arg) 2.87 folC 72 menB O.86 argE 2.15 fol) 8.54 menC O.92 acid cycle, the glyoxylate pathway, and a portion of the pen argF 2.04 foE O8 menD 2.33 45 tose phosphate pathway, and a “lower section' comprising all argG 2.62 folk 73 menE 3.06 or portions of the chorismate Super-pathway, the carbamoyl argh 8.06 foP 2.45 menF 3.09 phosphate to carbamate pathway, the threonine/homocys arg 4.06 fumA 3.84 metA 1.56 aroA 2.31 fumB 2.51 metB 1.83 teine Super-pathway, the nucleotide synthesis pathway, and aroB 8.68 fumC 86 metC 6.08 the polyamine synthesis pathway. aroC 95 gabl) .83 metE 2.46 50 In various embodiments microorganisms are genetically aroD .93 gabT 41 metH 2.44 aroE 8.44 gap A 3.03 metK 3.35 modified to affect one or more enzymatic activities of the aroF 6.24 gcvH 5.9 le 2.97 3HPTGC so that an elevated tolerance to 3-HP may be aroG 2.26 gcvP 7.91 mhpF .44 achieved, such as in industrial systems comprising microbial aroH 61 gcvT .78 ilk 66 3-HP biosynthetic activity. Also, genetic modifications may aroK 4 gdh A 2.84 nrdA 2.01 55 8O 63 gldA 2.08 nrdB 81 be made to provide and/or improve one or more 3-HP bio 8S 2.96 glk 17 nrdD 2.79 synthesis pathways in microorganisms comprising one or aspC 2.82 glnA 34 nrdE 91 more genetic modifications for the 3-HP toleragenic com astC 2.29 gltA 6.37 nirdE 25 plex, thus providing for increased 3-HP production. These car.A O.89 glyA S.O6 pabA 2.33 carE 17 gmk 86 pabB 92 latter recombinant microorganisms may be referred to as cynS 4.83 gnd 69 thrA 2.79 60 3-HP-syntha-toleragenic recombinant microorganisms cysE 19 gpmA 2.01 thrB O.96 (3HPSATG' recombinant microorganisms). cysK 2.41 guaA 3.65 thrC S1 The 3HPTGC for E. coli is disclosed in FIG.9A, sheets 1-7 pabC .75 guaB 2.63 phe A 6.7 pfkA .78 ilvA 12.21 pta 2.7 (a guide for positioning these sheets to view the entire pflB 2.83 ilwB 2.7 purA S.1 depicted 3HPTGC is provided in sheet 1 of FIG.9A). As may purB 3.65 rpiA 1.85 trpC S6 65 be observed in FIG.9, sheets 1-7, the 3HPTGC comprises all purc .78 sdaA 1.62 trpD 2.48 or various indicated portions of the following: the chorismate Super-pathway, the carbamoyl-phosphate to carbamate path US 8,883,464 B2 45 46 way, the threonine/homocysteine Super-pathway; a portion of both cynT and cynS. Additional control and other genetic the pentose phosphate pathway; the nucleotide synthesis elements may also be in Such nucleic acid sequences, which pathway; the glycolysis/tricarboxylic acid cycle? glyoxylate may be collectively referred to as “genetic elements' when bypass Super-pathway; and the polyamine synthesis pathway. added in a genetic modification, and which is intended to include a genetic modification that adds a single gene. It is noted that the chorismate pathway and the threonine However, similarly functioning genes are readily found in pathway are identified as Super-pathways since they respec different species and strains, encoding enzymes having the tively encompass a number of Smaller known pathways. same function as shown in FIG. 9A, sheets 1-7, and such However, the entire3HPTGC comprises these as well as other genes, and the 3HPTGCs of such other species and strains pathways, or portions thereof, that normally are not associ may be utilized in the practice of the invention. This can be ated with either the chorismate super-pathway or the threo 10 achieved by the following methods, which are not meant to be nine/homocysteine Super-pathway. limiting. More particularly, FIG.9A, comprising sheets 1-7, is sub For the set of genes within the 3HPTGC of E. coli, protein divided into the lower section, which is further subdivided sequences were obtained from NCBI. To identify similarly into Groups A-E and the upper section, identified simply as functioning genes in S. cerevisiae, a pathway comparison tool Group F. The lower section groups are identified as follows: 15 at was utilized using the genes identi Group A, or "chorismate comprising the indicated, major fied in the E. coli 3HPTGC. For B. subtilis, this annotated portion of the chorismate super-pathway (sheet 3); Group B, approach was used in part, and enzymes or pathway portions or “threonine/homocysteine, comprising the indicated por not obtained by that approach were obtained by a homology tion of the threonine/homocysteine pathway (sheet 7); Group comparison approach. For the homology approach, a local C, or “polyamine synthesis, comprising the indicated por blast () (blastp) compari tion of the polyamine pathway, which includes arginine Syn son using the selected set of E. coli proteins and Bacillus thesis steps and also the carbamoyl-phosphate to carbamate protein sequence (4096 sequences) was performed using dif pathway (sheet 5); Group D, or “lysine synthesis,” compris ferent thresholds (, and alternatively at >. As is obtained by the comparison. Not all of the homologous known to one skilled in the art, some genes may be found on sequences in C. necator are expected to encode a desired a chromosome within an operon, under the control of a single enzyme Suitable for an enzymatic conversion step of the promoter, or by other interrelationships. When a nucleic acid 3HPTGC for C. necator. However, through one or more of a sequence herein is referred to as a combination, Such as 40 combination of selection of genetic elements known to sucCD or cynTS, by this is meant that the nucleic acid encode desired enzymatic reactions, the most relevant genetic sequence comprises, respectively, both SucC and SucD, and elements are selected for the 3HPTGC for this species. TABLE 4 Homology Relationships for Genetic Elements of C. necator

E. coi C. necator Gene E. coli enzyme Gene C. necator C. necator Gene Symbol E. coli enzyme product substrate Symbol E-value Product

80te Pyruvate acetyl-coA aceE O pyruvate enydrogenase subunit E1 80te Pyruvate acetyl-coA aceE O pyruvate enydrogenase subunit E1 80te Pyruvate acetyl-coA aceE O 2-oxoacid enydrogenase subunit E1 acef gil 16128108 refNP 414 pyruvate pdhB 2.00E-102 dihydrolipoamide 657.1 acetyltransferase acef gil 16128108 refNP 414 pyruvate pdhB 2.00E-25 dihydrolipoamide 657.1 acetyltransferase acef Pyruvate acetyl-coA pdhB 2.00E-22 dihydrolipoamide acetyltransferase acef Pyruvate acetyl-coA pdhB 1.00E-10 dihydrolipoamide acetyltransferase US 8,883,464 B2 47 48 TABLE 4-continued Honology Relationships for Genetic Elements of C. recator E. coi C. necator Gene E. coli enzyme Gene C. necator C. necator Gene Symbol E. coli enzyme product substrate Symbol E-value Product 800 Pyruvate acetyl-coA pdhL 6.OOE-11 rolipoamide rogenase (E3) C mponent of pyruvate |rogenase 800 Pyruvate acetyl-coA pdhL 2.OOE-09 nyny rolipoamide hydrogenase (E3) ponent of pyruvate hydrogenase 800 Pyruvate acetyl-coA pdhL 8.OOE-08 ny rolipoamide hydrogenase (E3) CC ponent of pyruvate hydrogenase 800 Pyruvate acetyl-coA OdhB 9.OOE-36 hydrolipoamide etyltransferase 800 Pyruvate acetyl-coA bkdB 1.OOE-30 8 ched-chain alpha aci hydrogenase bunit E2 800 Pyruvate acetyl-coA bkdB 1.OOE-07 hed-chain alpha e O aci |rogenase bl l it E2 800 Pyruvate acetyl-coA bkdB 2.OOE-07 hed-chain alpha o acid hydrogenase subunit E2 808 gil 16129237 ref INP 415 citrate euC1 2.OOE-19 isopropylmalate 792.1 isomerase large Subunit 808 gil 16129237 ref INP 415 citrate euC2 7.OOE-22 isopropylmalate 792.1 isomerase large Subunit 808 gil 16129237 refNP 415 citrate acnM O aconitate hy ratase 792.1 808 gil 16129237| ref INP 415 citrate leuC3 6.OOE-2O isopropylmalate 792.1 isomerase large Subunit 808 Citrate cis-aconitate acna O aconitate hy ratase 808 Citrate cis-aconitate leuc4 6.OOE-14 3-isopropylmalate dehydratase arge Subunit 808 Citrate cis-aconitate leuCS 1.OOE-12 isopropylmalate isomerase large Subunit ytic gil 16132212|refNP 418 3-phospho- pgam2 3.OOE-2S phosphoglycerate 812.1 glycerate mutase 2 protein ytic 3-phosphoglycerate 2-phospho- pgam2 3.OOE-2S phosphoglycerate glycerate mutase 2 protein Zwf gil 16129805 refNP 416 glucose-6- Zwf1 2.00E-132 glucose-6-phosphate 366.1 phosphate 1-dehydrogenase Zwf glucose-6-phosphate glucono- Zwf2 7.00E-126 glucose-6-phosphate lactone-6- 1-dehydrogenase phosphate Zwf glucose-6-phosphate glucono- Zwf3 8.00E-130 glucose-6-phosphate lactone-6- 1-dehydrogenase phosphate

FIG.9B, sheets 1-7, shows the 3HPTGC for Bacillus sub- 5 5 or both of the above approaches may be employed to identify tilis, FIG. 9C, sheets 1-7, shows the 3HPTGC for the yeast relevant genes and enzymes in a selected microorganism Saccharomyces cerevisiae and FIG.9D, sheets 1-7, shows the species (for which its genomic sequence is known or has been 3HPTGC for Cupriavidus necator. Enzyme names for the obtained), evaluate the relative improvements in 3-HP toler latter are shown, along with an indication of the quantity of 60 ance of selected genetic modifications of such homologously homologous sequences meeting the criterion of having an matched and identified genes, and thereby produce a recom E-value less than E' when compared against an E. coli binant selected microorganism comprising improved toler enzyme known to catalyze a desired 3HPTGC enzymatic ance to 3-HP. conversion step. Additionally, it is appreciated that alternative pathways in Based on either of the above approaches, and the present 65 various microorganisms may yield products of the 3HPTGC, existence of or relative ease and low cost of obtaining the increased production or presence of which are demon genomic information of a given microorganism species, one strated herein to result in increased 3-HP tolerance. For US 8,883,464 B2 49 50 example, in yeast species there are alternative pathways to translational, and post-translational modifications that result lysine, a product within Group D. Accordingly, alterations of in a change of enzyme activity and/or overall enzymatic con Such alternative pathways are within the scope of the inven version rate under selected and/or identified culture condi tion for Such microorganism species otherwise falling within tions, and/or to provision of additional nucleic acid sequences the scope of the relevant claim(s). Thus, in various embodi (as provided in some of the Examples) so as to increase copy ments the invention is not limited to the specific pathways number and/or mutants of an enzyme of the 3HPTGC.. depicted in FIGS. 9A-D. That is, various pathways, and Specific methodologies and approaches to achieve Such enzymes thereof, that yield the products shown in FIGS. genetic modification are well known to one skilled in the art, 9A-D may be considered within the scope of the invention. and include, but are not limited to: increasing expression of an It is noted that when two or more genes are shown for a 10 endogenous genetic element; decreasing functionality of a particular enzymatic conversion step, these may be compo repressor gene; introducing a heterologous genetic element; nents of a single multi-enzyme complex, or may represent increasing copy number of a nucleic acid sequence encoding alternative enzymes that have different control factors that a polypeptide catalyzing an enzymatic conversion step of the control them, or are induced differently. Also, as is clear to 3HPTGC; mutating a genetic element to provide a mutated one skilled in the art, the major reactants (i.e., Substrates) and 15 protein to increase specific enzymatic activity; over-express products are shown for the enzymatic conversion steps. This ing; under-expressing: over-expressing a chaperone; knock is to minimize details on an already-crowded figure. For ing out a protease; altering or modifying feedback inhibition; example, electron carriers and energy transfer molecules, providing an enzyme variant comprising one or more of an such as NAD(P)(H) and ADP/ATP. are not shown, and these impaired binding site for a repressor and/or competitive (and other Small-molecule reactants not shown in the inhibitor, knocking out a repressor gene; evolution, selection 3HPTGC figures) are not considered “products of the and/or other approaches to improve mRNA stability. Random 3HPTGC as that term is used herein. Also, for at least two mutagenesis may be practiced to provide genetic modifica steps (dihydroneopterin phosphate to 7,8-dihydro-D-neop tions of the 3HPTGC that may fall into any of these or other terin and 1,4-dihydroxy-2-naphthoyl-CoA to 1,4-dihydroxy stated approaches. The genetic modifications further broadly 2-naphthoate) no enzyme is shown because no enzyme has 25 fall into additions (including insertions), deletions (such as by been known to be identified for this step at the time offiling. a mutation) and Substitutions of one or more nucleic acids in Accordingly, in some embodiments the 3HPTGC is under a nucleic acid of interest. In various embodiments a genetic stood and/or taken to exclude enzymes, nucleic acid modification results in improved enzymatic specific activity sequences, and the like, for these steps. Also, as discussed and/or turnover number of an enzyme. Without being limited, herein, also included within the scope of the invention are 30 changes may be measured by one or more of the following: nucleic acid sequence variants encoding identified enzymatic KM, Keat; and Karaia. functional variants of any of the enzymes of the 3HPTGC or Such genetic modifications overall are directed to increase a related complex or portion thereofas set forth herein, and enzymatic conversion at least one enzymatic conversion step their use in constructs, methods, and systems claimed herein. of the 3HPTGC so as to increase 3-HPtolerance of a micro Some fitness data provided in Table 3 is not represented in 35 organism so modified. Also, the enzymatic conversion steps the figures of the 3HPTGC but nonetheless is considered to shown in FIGS. 9A-D may be catalyzed by enzymes that are Support genetic modification(s) and/or Supplementation to readily identified by one skilled in the art, such as by search improve 3-HPtolerance. For example, the relatively elevated ing for the enzyme name corresponding to the gene name at a fitness scores forgcvH.gcVP and gcVT, related to the glycine particular enzymatic conversion step in FIGS.9A-D, and then cleavage system. These enzymes are involved in the glycine/ 40 identifying enzymes, such as in other species, having the 5,10-methylene-tetrahydrofolate (“5,10 mTHF) conversion same name and function. The latter would be able to convert pathway, depicted in FIG. 10. In the direction shown in FIG. the respective reactant(s) to the respective product(s) for that 10, the three enzymatically catalyzed reactions result in enzymatic conversion step. Public database sites, such as decarboxylation of glycine (a 3HPTGC product, see FIG.9A, , , sheet 4), production of 5,10-methylene-THF from tet 45 , and

Further as to Supplements, as to Group C regarding Supplementation and/or genetic modification(s), may be polyamine synthesis, the results of the examples demonstrate 35 effective to increase the intracellular concentration of one or that 3-HP tolerance of E. coli was increased by adding the more 3HPTGC products in a microorganism and/or in the polyamines putrescine, spermidine and cadaverine to the media in which Such microorganism is cultured. media. Minimum inhibitory concentrations (MICs) for E. coli Taken together, the fitness data and Subsequently obtained K12 in control and supplemented media were as follows: in data from the examples related to genetic modifications and/ M9 minimal media supplemented with putrescine 40 g/L, in 40 or supplements pertaining to the 3HPTGC support a concept M9 minimal media supplemented with spermidine 40 g/L, in of a functional relationship between such alterations to M9 minimal media supplemented with cadaverine 30 g/L. increase enzymatic conversion along the pathways of the Minimum inhibitory concentrations (MICs) for added 3HPTGC and the resulting functional increase in 3-HPtoler sodium bicarbonate in M9 minimal media was 30 g/L. The ance in a microorganism cell or culture system. This is Minimum inhibitory concentrations (MICs) for E. coli K12 in 45 observable for the 3HPTGC as a whole and also within and 100 g/L stock solution 3-HP was 20 g/L. among its defined groups. Further, in view of the increase over the control MIC with Further, tables 47, 48, 50, 52, 53, and 56, incorporated into Sodium bicarbonate Supplementation, other alteration, Such this section, provide non-limiting examples Supplements as regulation and/or genetic modification of carbonic anhy additions, genetic modifications, and combinations of drase, such as providing a heterologous nucleic acid sequence 50 Supplements additions and genetic modifications. Additional to a cell of interest, where that nucleic acid sequence encodes Supplementations, genetic modifications, and combinations a polypeptide possessing carbonic anhydrase activity are con thereof, may be made in view of these examples and the sidered of value to increase tolerance to 3-HP (such as in described methods of identifying genetic modifications combination with other alterations of the 3HPTGC). Simi toward achieving an elevated tolerance to 3-HP in a microor larly, and as Supported by other data provided herein, alter 55 ganism of interest. Particular combinations may involve only ations of the enzymatic activities, such as by genetic modifi the 3HPTGC lower section, including combinations involv cation(s) of enzyme(s) along the 3HPTGC pathway portions ing two or more, three or more, or four or more, of the five that lead to arginine, putrescine, cadaverine and spermidine, groups therein (each involving Supplement additions and/or are considered of value to increase tolerance to 3-HP (such as genetic modification), any of these in various embodiments in combination with other alterations of the 3HPTGC). 60 also comprising one or more genetic modifications or Supple It is appreciated that the results of Supplementations evalu ment additions regarding the 3HPTGC uppersection. Subject ations provide evidence of the utility of direct supplementa matter in the Examples is incorporated into this section to the tion into a culture media, and also of improving 3-HP toler extent not already present. ance by a genetic modification route. Such as is provided in Based on these results, it is appreciated that in various Some examples herein. It is appreciated that increasing the 65 embodiments of the invention, whether methods or compo concentration of a product of a 3HPTGC enzymatic conver sitions, as a result of genetic modification and/or Supplemen sion step. Such as by a genetic modification, whether by tation of reactants of the 3HPTGC, the alteration(s) directed US 8,883,464 B2 55 56 to the 3HPTGC are effective to increase 3-HPtolerance by at the examples are meant to be exemplary and not limiting. least 5 percent, at least 10 percent, at least 20 percent, at least Genetic manipulations may be made to achieve a desired 30 percent, or at least 50 percent above a 3-HPtolerance of a alteration in overall enzyme function, Such as by reduction of control microorganism, lacking said at least one 3HPTGC feedback inhibition and other facets of control, including genetic modification. alterations in DNA transcriptional and RNA translational As is appreciated by the examples, any of the genetically control mechanisms, improved mRNA stability, as well as modified microorganisms of the invention may be provided in use of plasmids having an effective copy number and promot a culture system and utilized, such as for the production of ers to achieve an effective level of improvement. Such genetic 3-HP. In some embodiments, one or more supplements (that modifications may be chosen and/or selected for to achieve a are products of the 3HPTGC enzymatic conversion steps) are 10 higher flux rate through certain basic pathways within the provided to a culture system to further increase overall 3-HP 3HPTGC and so may affect general cellular metabolism in tolerance in Such culture system. fundamental and/or major ways. Accordingly, in certain alter Increased tolerance to 3-HP, whether of a microorganism natives genetic modifications are made more selectively, to or a culture system, may be assessed by any method or other parts of the 3HPTG.C. approach known to those skilled in the art, including but not 15 Further, based on analysis of location and properties of limited to those described herein. committed steps, feedback inhibition, and other factors and The genetic modification of the 3HPTGC upper portion constraints, in various embodiments at least one genetic may involve any of the enzymatic conversion steps. One, modification is made to increase overall enzymatic conver non-limiting example regards the tricarboxylic acid cycle. It sion for one of the following enzymes of the 3HPTGC: 2-de is known that the presence and activity of the enzyme citrate hydro-3-deoxyphosphoheptonate aldolase (e.g., aroF, aroG, synthase (E.C. 2.3.3.1 (previously 4.1.3.7)), which catalyzes aroH); cyanase (e.g., cynS); carbonic anhydrase (e.g., cynT); the first step in that cycle, controls the rate of the overall cycle cysteine synthase B (e.g., cysM); threonine deaminase (e.g., (i.e., is a rate-limiter). Accordingly, genetic modification of a ilvA); ornithine decarboxylase (e.g., spec, speF); adenosyl microorganism, Such as to increase copy numbers and/or methionine decarboxylase (e.g., spel)); and spermidine Syn specific activity, and/or other related characteristics (such as 25 thase (e.g., speE). Genetic modifications may include lower effect of a feedback inhibitor or other control mol increasing copy numbers of the nucleic acid sequences ecule), may include a modification of citrase synthase. Ways encoding these enzymes, and providing modified nucleic acid to effectuate Such change for citrate synthase may utilize any sequences that have reduced or eliminated feedback inhibi number of laboratory techniques, such as are known in the art, tion, control by regulators, increased affinity for Substrate, including approaches described herein for other enzymatic 30 and other modifications. Thus, one aspect of the invention is conversion steps of the 3HPTGC. Further, several commonly to genetically modify one or more of these enzymes in a known techniques are described in U.S. Pat. Nos. 6,110,714 manner to increase enzymatic conversion at one or more and 7.247,459, both assigned to Ajinomoto Co., Inc., both of 3HPTGC enzymatic conversion steps so as to increase flux which are herewith incorporated by reference for their respec and/or otherwise modify reaction flows through the 3HPTGC tive teachings about amplifying citrate synthase activity (spe 35 so that 3-HP tolerance is increased. In addition to the cifically, cols. 3 and 4, and Examples 3 and 4, of U.S. Pat. No. examples which pertain to genetic modifications regarding 6,110,714, and cols. 11 and 12 (specifically Examples (1) and aroH and cyanase (with carbonic anhydrase), respectively, (2)) of U.S. Pat. No. 7,247,459). the following examples are provided. It is noted that in E. coli In various embodiments E. coli strains are provided that a second carbonic anhydrase enzyme is known. This is iden comprise selected gene deletions directed to increase enzy 40 tified variously as Can and yadf. matic conversion in the 3HPTGC and accordingly increase Also, it is appreciated that various embodiments of the microorganism tolerance to 3-HP. For example, the following invention may comprise genetic modifications of the genes, which are associated with repression of pathways in 3HPTGC (as may be provided in a microorganism, as the indicated 3HPTGC Groups, may be deleted: Group described herein), and/or Supplements thereof, excluding any A tyrR, trpR: Group B—metJ; Group C purR; Group 45 one or more designated enzymatic conversion steps, product D-lysR; Group E nrdR. There are for E. coli and it is additions, and/or specific enzymes. For example, an embodi known and determinable by one skilled in the art to identify ment of the invention may comprise genetic modifications of and genetically modify equivalent repressor genes in this and the 3HPTGC in a microorganism, however excluding those of other species. Group A, or of Groups A and B, or of a defined one or more A disruption of gene function may also be effectuated, in 50 members of the 3HPTGC (which may be any subset of the which the normal encoding of a functional enzyme by a 3HPTGC members). nucleic acid sequence has been altered so that the production For example, without being limiting, a modified 3HPTGC of the functional enzyme in a microorganism cell has been may comprise all members of the 3HPTGC as depicted herein reduced or eliminated. A disruption may broadly include a except the degradative form of arginine decarboxylase (adiA, gene deletion, and also includes, but is not limited to gene 55 which is known to be induced in rich medium at low pH under modification (e.g., introduction of stop codons, frame shift anaerobic conditions in the presence of excess Substrate), or mutations, introduction or removal of portions of the gene, other Subsets excluding Such degradative arginine decarboxy introduction of a degradation signal), affecting mRNA tran lase and other selected enzyme steps. Other modified Scription levels and/or stability, and altering the promoter or 3HPTGC complexes may also be practiced in various repressor upstream of the gene encoding the polypeptide. In 60 embodiments. Based on the noted induction of adiA, the use Some embodiments, a gene disruption is taken to mean any of the degradative form of arginine decarboxylase is not be genetic modification to the DNA, mRNA encoded from the considered within the scope of the 3HPTGC for 3-HP toler DNA, and the amino acid sequence that results in at least a 50 ance improvement as practiced under aerobic conditions. percent reduction of enzyme function of the encoded gene in Moreover, various non-limiting aspects of the invention the microorganism cell. 65 may include, but are not limited to: Further, as to the full scope of the invention and for various A genetically modified (recombinant) microorganism embodiments, it is recognized that the above discussion and comprising a nucleic acid sequence that encodes a polypep