Granted Patent
B2
US 12,662,679 · App. 18/344,219 · Granted Jun 23, 2026
Production of fatty acid derivatives
Inventors:
Derek L. Greenfield (South San Francisco, CA); Andreas W. Schirmer (Hayward, CA); Elizabeth J. Clarke (South San Francisco, CA); Eli S. Groban (South San Francisco, CA); Bernardo M. Da Costa (South San Francisco, CA); Zhihao Hu (Zhajalgang, CN); Kevin Holden (South San Francisco, CA); Noah Helman (South San Francisco, CA)
Assignee:
Genomatica, Inc.
C12N15/70C12N9/0006C12N9/001C12N9/1029C12N9/1288C12N9/88C12N9/90C12N9/93C12P7/04C12P7/6409C12P7/6436C12Y101/011C12Y103/01009C12Y103/0101C12Y203/01039C12Y203/01041C12Y203/01179C12Y203/0118C12Y207/08007C12Y402/01059C12Y503/03014C12Y604/01002
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Abstract
The disclosure relates to recombinant host cells including strain modifications effective to improve titer, yield and/or productivity of fatty acid derivatives. The disclosure further relates to cell cultures including the recombinant host cells for the fermentative production of fatty acid derivatives and compositions thereof.
Claims (14)
1 . A recombinant bacterial host cell expressing a polypeptide having 3-hydroxy-acyl-[acp] dehydratase activity;
wherein the polypeptide having 3-hydroxy-acyl-[acp] dehydratase activity is FabA;
wherein the recombinant bacterial host cell expresses one or more additional polypeptides, and the one or more additional polypeptides are FabV, FabH, FabD, FabG, and FabF; and
wherein the recombinant bacterial host cell produces a fatty acid derivative composition at a higher titer, yield, or productivity than a corresponding wild-type host cell when cultured in a medium containing a carbon source.
2 . The recombinant bacterial host cell of claim 1 ,
wherein the polypeptide with 3-hydroxy-acyl-[acp] dehydratase activity is FabA from Salmonella typhimurium ; and
wherein the one or more additional polypeptides are FabV from Vibrio cholerae ; FabH from Salmonella typhimurium, Acinetobacter sp. ADP1, Marinobacter aquaeoli VT8, or Rhodococcus opacus B4; FabD from Salmonella typhimurium ; FabG from Salmonella typhimurium ; and FabF from Clostridium acetobutylicum.
3 . The recombinant bacterial host cell of claim 1 , wherein the polypeptide with 3-hydroxy-acyl-[acp] dehydratase activity and the one or more additional polypeptides are (1) heterologous, or (2) are native and are overexpressed.
4 . The recombinant bacterial host cell of claim 1 , further comprising a genetically engineered polynucleotide sequence encoding an acyl carrier protein (ACP), wherein the recombinant bacterial host cell produces a fatty acid derivative composition at a higher titer, yield, or productivity than a corresponding wild type host cell when cultured in a medium containing a carbon source under conditions effective to express said ACP.
5 . The recombinant bacterial host cell of claim 4 , wherein the ACP is derived from the same organism as a terminal pathway enzyme expressed in the recombinant bacterial host cell, and wherein the terminal pathway enzyme cleaves any acyl-ACP species that is part of a fatty acid biosynthetic pathway.
6 . The recombinant bacterial host cell of claim 1 , wherein the recombinant bacterial host cell further comprises a recombinant polynucleotide encoding a polypeptide that has phosphopantetheinyl transferase activity.
7 . The recombinant bacterial host cell of claim 6 , wherein the polypeptide that has phosphopantetheinyl transferase activity is encoded by an sfp gene.
8 . The recombinant bacterial host cell of claim 1 , wherein the recombinant bacterial host cell further comprises a recombinant polynucleotide encoding a polypeptide that has acetyl-CoA carboxylase activity.
9 . The recombinant bacterial host cell of claim 8 , further comprising a recombinant polynucleotide encoding a polypeptide that has phosphopantetheinyl transferase activity.
Assignments (5)
SECURITY INTEREST
Recorded Feb 10, 2026
From: GENOMATICA, INC.
To: AGAIN BIO APS
Reel/Frame 074708/0001 →
SECURITY INTEREST
Recorded Dec 9, 2025
From: GENOMATICA, INC.
To: NOVO HOLDINGS A/S, AS COLLATERAL AGENT
Reel/Frame 073915/0027 →
SECURITY INTEREST
Recorded Jun 2, 2025
From: GENOMATICA, INC.
To: OXFORD FINANCE LLC
Reel/Frame 071471/0770 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jan 16, 2024
From: GREENFIELD, DEREK L.; CLARKE, ELIZABETH J.; GROBAN, ELI S.; HU, ZHIHAO; HELMAN, NOAH; SCHIRMER, ANDREAS W.; DA COSTA, BERNARDO M.; HOLDEN, KEVIN
To: REG LIFE SCIENCES, LLC.
Reel/Frame 066134/0766 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jan 16, 2024
From: REG LIFE SCIENCES, LLC.
To: GENOMATICA, INC.
Reel/Frame 066338/0028 →
Continuity (5)
Division
14390378
Provisional Application
61619324
· Apr 2, 2012
References Cited (158)
US 5000000A
· Ingram et al.
· 1991
[cited by applicant]
US 5028539A
· Ingram et al.
· 1991
[cited by applicant]
US 5424202A
· Ingram et al.
· 1995
[cited by applicant]
US 5482846A
· Ingram et al.
· 1996
[cited by applicant]
US 5602030A
· Ingram et al.
· 1997
[cited by applicant]
US 5939250A
· Short
· 1999
[cited by applicant]
US 5965408A
· Short
· 1999
[cited by applicant]
US 7192735B2
· Lambalot et al.
· 2007
[cited by applicant]
US 7897369B2
· Schmidt-Dannert et al.
· 2011
[cited by applicant]
US 8097439B2
· Alibhai et al.
· 2012
[cited by applicant]
US 20090140696A1
· Okuto
· 2009
[cited by applicant]
US 20100242345A1
· Keasling et al.
· 2010
[cited by applicant]
US 20100274033A1
· Sanchez-Riera et al.
· 2010
[cited by applicant]
US 20110111458A1
· Masuda et al.
· 2011
[cited by applicant]
US 20110151526A1
· Saunders et al.
· 2011
[cited by applicant]
US 20110162259A1
· Gaertner
· 2011
[cited by applicant]
US 20150064782A1
· Greenfield et al.
· 2015
[cited by applicant]
US 20160002681A1
· Simpson
· 2016
[cited by examiner]
US 20210348173A1
· Greenfiled et al.
· 2021
[cited by applicant]
CN 1295130A
· 2001
[cited by applicant]
CN 101490241A
· 2009
[cited by applicant]
EP 2194119A2
· 2010
[cited by examiner]
JP 2010507369A
· 2010
[cited by applicant]
JP 2010526805A
· 2010
[cited by applicant]
JP 2012504963A
· 2012
[cited by applicant]
WO 9116427A1
· 1991
[cited by applicant]
WO 2007136762A2
· 2007
[cited by applicant]
WO 2008130437A2
· 2008
[cited by applicant]
WO WO2008119082A2
· 2008
[cited by examiner]
WO 2008147781A2
· 2008
[cited by applicant]
WO 2009002480A2
· 2008
[cited by applicant]
WO 2009085278A1
· 2009
[cited by applicant]
WO 2009140695A1
· 2009
[cited by applicant]
WO 2010042664A1
· 2010
[cited by applicant]
WO 2010062480A2
· 2010
[cited by applicant]
WO 2010075483A2
· 2010
[cited by applicant]
WO 2010127318A2
· 2010
[cited by applicant]
WO 2011038134A1
· 2011
[cited by applicant]
WO 2012009660A2
· 2012
[cited by applicant]
WO 2012019175A2
· 2012
[cited by applicant]
WO 2013019647A1
· 2013
[cited by applicant]
WO 2013152051A2
· 2013
[cited by applicant]
WO 2013152052A2
· 2013
[cited by applicant]
GenBank, Accession No. BAA16180, 2008, www.nlm.nih.gov. (Year: 2008).
[cited by examiner]
GenBank, Accession No. NP_418398.2, 2010, www.nlm.nih.gov. (Year: 2010).
[cited by examiner]
Jeon et al., Development of
[cited by examiner]
Yu et al., In vitro reconstitution and steady-state analysis of the fatty acid synthase from
[cited by examiner]
Massengo-Tiasse et al., Vibrio cholerae FabV Defines a New Class of Enoyl-Acyl Carrier Protein Reductase, J. Biol. Chem. 283, 2008, 1308-16. (Year: 2008).
[cited by examiner]
Cao et al., Increasing unsaturated fatty acid contents in
[cited by examiner]
Feng et al.,
[cited by examiner]
Uniprot, Accession No. P64105, 2005, www.uniprot.org. (Year: 2005).
[cited by examiner]
Amann et al., “Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in
[cited by applicant]
Arkin et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7811-7815.
[cited by applicant]
Arnold, “Protein engineering for unusual environments,” Curr. Opin. Biotech. 4: 450-455 (1993).
[cited by applicant]
Baldari et al., “A novel leader peptide which allows efficient secretion of a fragment of human interleukin 1 beta in
[cited by applicant]
Bergler et al., “Protein EnvM is the NADH-dependent Enoyl-ACP Reductase (Fabl) of
[cited by applicant]
Bergler et al., “The enoyl-[acyl-carrier-protein] reductase (Fabl) of
[cited by applicant]
Bowie et al. “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” (1990) Science, 247:1306-1310.
[cited by applicant]
Caldwell et al., “Randomization of Genes by PCR Mutagenesis,” PCR Methods Applic. 2: 28-33 (1992).
[cited by applicant]
Communication issued on EP Appl. 13715879.6, mailed Jun. 1, 2016.
[cited by applicant]
Communication issued on EP Application 13715879.6, mailed May 12, 2017.
[cited by applicant]
Cronan et al., “FadR, transcriptional co-ordination of metabolic expediency,” Mol. Microbial. 29(4): 937-943 (1998).
[cited by applicant]
Datsenko et al., “One-step Inactivation of Chromosomal Genes in
[cited by applicant]
Davis et al., “Overproduction of Acetyl-CoA Carboxylase Activity Increases the Rete of Fatty Acid Biosynthesis in
[cited by applicant]
Decision of Rejection issued on Japanese Application2 015-504687, mailed Dec. 4, 2017.
[cited by applicant]
Decision of Rejection in JP Patent No. 2018-072000 dated Jan. 8, 2020 (with English translation) (9 pages).
[cited by applicant]
Delegrave et al., “Searching Sequence Space to Engineer Proteins: Exponential Ensemble Mutagenesis,” Biotech. Res, 11: 1548-1552 (1993).
[cited by applicant]
Exam Report issued on Malaysian Appl. PI 2014002773, mailed Sep. 15, 2017.
[cited by applicant]
Examination Report issue on Australian Application 2013243601, mailed Oct. 5, 2017.
[cited by applicant]
Examination Report issued on Indonesian Appl. P-00201406815, mailed Dec. 29, 2017.
[cited by applicant]
Extended European Search Report in EP Patent Application No. 19211601.0 dated May 14, 2020 (9 pages).
[cited by applicant]
Final Office Action on U.S. Appl. No. 14/390,378 mailed Jan. 19, 2017.
[cited by applicant]
First Examination Report on IN Patent Application No. 9107/DELNP/2014 dated Aug. 28, 2019, 7 pages.
[cited by applicant]
Flores et al., “Expression of PEP carboxylase from
[cited by applicant]
Fourth Office Action in CN Patent Application No. 201380026304.9, dated Jan. 5, 2018 (16 pages with English translation).
[cited by applicant]
Gaviglia et al., “Rat Long Chain Acyl-CoA Synthetase 5, but Not 1, 2, 3, or 4, Complements
[cited by applicant]
GenBank, Accession No. 460041.1, 2011, VWVW.ncbi.nlm.nih.gov.
[cited by applicant]
GenBank, Accession No. AAC74178.1, 2010, VWWW.ncbi.nlm.nih.gov.
[cited by applicant]
GenBank, Accession No. BAA16180, 2008, VWVW.ncbi.nlm.nih.gov.
[cited by applicant]
GenBank, Accession No. NP_350156.1, 2010, VWVW.ncbi.nlm.nih.gov.
[cited by applicant]
GenBank, Accession No. NP_416826.1, 2011, www.ncbi.nlm.nih.gov.
[cited by applicant]
GenBank, Accession No. NP_460163.1, 2011, www.ncbi.nlm.nih.gov.
[cited by applicant]
GenBank, Accession No. NP_460164.1, 2011, www.ncbi.nlm.nih.gov.
[cited by applicant]
GenBank, Accession No. NP_460165.1, 2011, www.ncbi.nlm.nih.gov.
[cited by applicant]
GenBank, Accession No. YP_001217283.2, 2010, www.ncbi.nlm.nih.gov.
[cited by applicant]
Goeddel, Gene Expression Technology: Methods in Enzymology, vol. 185, Academic Press, San Diego, Calif. (1990).
[cited by applicant]
Gokarn et al., “Metabolic Analysis of
[cited by applicant]
Grosjean et al., “Preferential codon usage in prokaryotic genes: the optimal codon-anticodon; interaction energy and the selective codon usage in efficiently expressed genes,” Gene. 18: 199-209 (1982).
[cited by applicant]
Handke, P., et al., “Application and engineering of fatty acid biosynthesis in
[cited by applicant]
Heath et al., “Inhibition of beta.-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) by Acyl-Acyl Carrier Protein in
[cited by applicant]
Heath et al., “Regulation of Fatty Acid Elongation and Initiation by Acyl-Acyl Carrier Protein in
[cited by applicant]
Heath, “The Enoly-[acyl-carrier-protein] Reductases Fabl and FabL from Bacillus subtilis,” Journal of Biological Chemistry, vol. 275, No. 51, Sep. 27, 2000, pp. 40128-40133.
[cited by applicant]
International Preliminary Report on Patentability on PCT/US2013/035037, dated Oct. 7, 2014, 13 pages.
[cited by applicant]
International Search Report and Written Opinion on PCT/US2013/035037, mailed Oct. 31, 2013.
[cited by applicant]
Ku et al., “High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants,” Nature Biotechnology, vol. 17, Jan. 1999, pp. 76-80.
[cited by applicant]
Kurjan et al., Struture of a Yeast Pheromone Gene (MFx): A Putative x-Factor precursor Contains Four Tandem Copies of Mature x-Factor, Cell, vol. 30, pp. 933-943 (1982).
[cited by applicant]
Lennen et al., Engineering
[cited by applicant]
Leung et al. “A Journal of Methods in Cell and Molecular Biology,” Technique 1:(1): 11-15 (1989).
[cited by applicant]
Liu et al., “Quantitative analysis and engineering of fatty acid biosynthesis in
[cited by applicant]
Liu et al., Fatty acid production in genetically modified cyanobacteria, Proc. Natl. Acad. Sci. USA, 2011, 108, 6800-6904.
[cited by applicant]
Lu et al., Overproduction of free fatty acids in
[cited by applicant]
Lucklow et al., “High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors,” Virology 170(1): 31-39 (1989).
[cited by applicant]
Maniatis et al., “Regulation of Inducible and Tissue-Specific Gene Expression,” Science 236: 1237-1245 (1987).
[cited by applicant]
Massengo-Tiasse et al., “Vibrio cholerae FabV Defines a New Class of Enoyl-Acyl Carrier Protein Reductase”, J. Biol. Chem. 283(3): 1308-1316 (2008).
[cited by applicant]
Mori et al., A SecE Mutation That Modulates SecY-SecE Translocase Assembly, Identified as a Specific Suppressor of SecY Defects, J. Bacterial., 2003, 185, 948-56.
[cited by applicant]
Needleman and Wunsch, “A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins,” J. Mol. Biol. 48:443-453 (1970).
[cited by applicant]
Non-Final Office Action on U.S. Appl. No. 14/390,378 mailed May 23, 2016.
[cited by applicant]
Notice of Reasons for Refusal in KR 10-2014-7030467 dated Mar. 2, 2020 (5 pages) (with English Translation).
[cited by applicant]
Notice of Reasons for Rejection issued on Japanese application 2015-504687, mailed Jan. 19, 2017, English translation.
[cited by applicant]
Office Action from corresponding JP Application No. 2020081804 dated Aug. 2, 2021.
[cited by applicant]
Office Action from corresponding Korean Application No. 10-2020-7015194 dated Apr. 15, 2021.
[cited by applicant]
Office Action in CA Patent Application No. 2883968 dated Apr. 28, 2020 (3 pages).
[cited by applicant]
Office Action in CO Patent Application No. 14-240.053, dated May 12, 2016, (20 pages) (with English translation).
[cited by applicant]
Office Action in CO Patent Application No. 14-240.053, dated Nov. 11, 2016, (14 pages) (with English translation).
[cited by applicant]
Office Action issued on Chinese Application 201380026304.9, mailed Apr. 1, 2017, English translation only.
[cited by applicant]
Office Action issued on Chinese Application 201380026304.9, mailed Dec. 29, 2015, English translation provided.
[cited by applicant]
Office Action on Application No. MX/a/2014/011905, dated Dec. 3, 2018, 4 pages.
[cited by applicant]
Office Action on CN Application No. 201380026304.9, dated Jan. 5, 2018, 8 pages.
[cited by applicant]
Office Action on CN Application No. 201380026304.9, dated Sep. 9, 2016, 9 pages (with translation).
[cited by applicant]
Office Action on CN Application No. 201380026304.9, dated Oct. 26, 2018, 12 pages (with translation).
[cited by applicant]
Office Action on CO Application 14-240.053, dated May 12, 2016, 10 pages.
[cited by applicant]
Office Action on CO Application No. 14-240.053, dated Nov. 11, 2016, 7 pages.
[cited by applicant]
Office Action on ID Application No. P00201406815, dated Apr. 12, 2018, 3 pages (with translation).
[cited by applicant]
Palmeros et al., “A family of removable cassets designed to obtain antiobiotic-resistance-free genomic modifications of
[cited by applicant]
Peng et al., Metabolic flux analysis for a ppc mutant
[cited by applicant]
Preliminary Office Action in BR Patent Application No. 112014024675.0 dated Sep. 2, 2019, 6 pages (translation).
[cited by applicant]
Quadri, L.E.N., et al., “Characterization of Sfp, a Bacillus subtilis Phosphopantetheinyl Transferase for Peptidyl Carrier Protein Domains in Peptide Synthetases,” Biochemisty, 37: 1585-1595 (1998).
[cited by applicant]
Reidhaar-Olson et al., “Combinatorial Cassette Mutagenesis as a Probe of the Informational Content of Protein Sequences,” Science 241: 53-57 (1988).
[cited by applicant]
Rosenberg, “Multiple Sequence Alignment Accuracy and Evolutionary Distance Estimation,” BMC Bioinformatics 6: 278 (2005).
[cited by applicant]
Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 1989, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
[cited by applicant]
Sanchez, Cesar, et al., “Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin,” Chemistry & Bi…
[cited by applicant]
Schultz et al., “Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus,” Gene 54: 113-123 (1987).
[cited by applicant]
Second Office Action in CA Patent Application No. 2,883,968 dated Apr. 23, 2019, 3 pages.
[cited by applicant]
Second Office Action in MX Patent Application No. MX/a/2014/011905, dated Dec. 3, 2018 (with English language comments; no Office Action translation available) (8 pages).
[cited by applicant]
Smith et al., “Production of Human Beta Interferon in Insect Cells Infected with a Vaculovirus Expression Vector,” Mol. Cell Biol. 3(12): 2156-2165 (1983).
[cited by applicant]
Smith et al., “Single-step purification of polypeptides expressed in
[cited by applicant]
Stemmer, “DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution,” Proc. Natl. Acad. Sci. USA. 91: 10747-10751 (1994).
[cited by applicant]
Studier et al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Methods in Enzymology, 185: 60-89 (1990).
[cited by applicant]
Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, pp. 60-89 (1990).
[cited by applicant]
Substantive Examination Adverse Report in My PI2014002773 dated Feb. 4, 2020 (2 pages).
[cited by applicant]
Wang et al., “The Gene Locus yijP Contributes to
[cited by applicant]
Wang et al., The gene locus yijP contributes to
[cited by applicant]
Zha et al., “Improving cellular malonyl-CoA level in
[cited by applicant]
Zhang et al.,j“Transcriptional Analysis of Essential Genes of the
[cited by applicant]
Zhu et al., “Functions of the Clostridium acetobutylicium FabF and FabZ proteins in unsaturated fatty acid biosynthesis”, BMC Microbiology 9:119 (2009).
[cited by applicant]
Non-Final Office Action issued in U.S. Appl. No. 15/650,285 dated Dec. 4, 2020.
[cited by applicant]
Final Office Action issued in U.S. Appl. No. 15/650,285 dated Feb. 13, 2020.
[cited by applicant]
Yu, Xingye et al. (2011). In vitro reconstitution and steady-state analysis of the fatty acid synthase from
[cited by applicant]
Jeon, Eunyoung et al. (2011). Development of
[cited by applicant]
Zhang, Xiujun et al. (2012). Improving fatty acid production in
[cited by applicant]