IP Library › Granted Patent US 12,203,102
Granted Patent B2
US 12,203,102 · App. 17/812,636 · Granted Jan 21, 2025

3-hydroxybutyryl-CoA dehydrogenase variants and methods of use

Inventors: Kevin Hoff (San Diego, CA); Cara Ann Tracewell (Solana Beach, CA); Kui Chan (Los Angeles, CA); Michael Kuchinskas (Franklin, MA); Harish Nagarajan (San Diego, CA)
Assignee: Genomatica, Inc.
C12N9/0006C12N9/1029C12P7/18C12P7/24C12P7/52C12P19/32C12Y101/01157C12Y203/01009
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Quick Facts
Patent No.
US 12,203,102
App. No.
17/812,636
Granted
Jan 21, 2025
Kind
B2
Abstract

The present disclosure provides thiolases and polypeptide variants of 3-hydroxybutyryl-CoA dehydrogenase, nucleic acids encoding the same, vectors comprising the nucleic acids, and cells comprising the polypeptide variants and/or thiolase, the nucleic acids, and/or the vectors. The present disclosure also provides methods of making and using the same, including methods for culturing cells, and for the production of various products, including 3-hydroxybutyryl-CoA (3-HB-CoA), 3-hydroxybutyraldehyde (3-HBal), 3-hydroxybutyrate (3-HB), 1,3-butanediol (1,3-BDO), and esters and amides thereof, and products made from any of these.

Claims (21)

1. An isolated 3-hydroxybutyryl-CoA dehydrogenase variant of a reference 3-hydroxybutyryl-CoA dehydrogenase, wherein:

(a) (i) the reference 3-hydroxybutyryl-CoA dehydrogenase has the amino acid sequence of SEQ ID NO: 2, and (ii) the 3-hydroxybutyryl-CoA dehydrogenase variant comprises one or more amino acid substitutions relative to SEQ ID NO: 2, and (iii) the one or more substitutions relative to SEQ ID NO: 2 comprise a substitution at position G35 selected from G35A, G35C, G35D, G35E, G35H, G35K, G35L, G35M, G35P, G35S, G35T, G35V, and G35Y, or

(b) (i) the 3-hydroxybutyryl-CoA dehydrogenase variant comprises one or more amino acid substitutions relative to SEQ ID NO: 2, the one or more amino acid substitutions comprise a substitution at position G35 selected from G35A, G35C, G35D, G35E, G35H, G35K, G35L, G35M, G35P, G35S, G35T, G35V, and G35Y, and (iii) has, other than the substitution at position G35, and

(ii) the 3-hydroxybutyryl-CoA dehydrogenase variant comprises one or more additional amino acid substitutions relative to SEQ ID NO: 2, wherein the one or more additional amino acid substitutions selected from C34, S38, and R40, wherein

(1) The amino acid corresponding to C34 is replaced with A, E, H, I, L, M, S, T, V, or Y;

(2) the amino acid corresponding to S38 is optionally replaced with A, C, D, F, G, H, I, K, L, M, Q, T, V, W, or Y;

(3) the amino acid corresponding to R40 is optionally replaced with D, F, G, H, I, K, L, M, N, P, Q, S, T, V, or Y; or

(4) optionally, two or more of (1)-(3) in any combination; or

(c) (i) the 3-hydroxybutyryl-CoA dehydrogenase variant comprises one or more amino acid substitutions relative to SEQ ID NO: 2, the one or more amino acid substitutions comprise a substitution at position G35 selected from G35A, G35C, G35D, G35E, G35H, G35K, G35L, G35M, G35P, G35S, G35T, G35V, and G35Y, and (iii) has, other than the substitution at position G35, a sequence having at least 85% identity to SEQ ID NO: 2; and

(ii) the 3-hydroxybutyryl-CoA dehydrogenase variant comprises 1, 2, 3, 4, 5, or more additional amino acid substitutions relative to SEO ID NO: 2 selected from: A103M, A107V, A156S, A158R, A193R, A204T, C20F, C34A, C34E, C34H, C34I, C34L, C34M, C34S, C34T, C34V, C34Y, D104A, D104E, D104Q, D106H, D129E, D236C, D236V, E215H, E215W, E42Q, E46C, E46S, E78K, F55I, G241G, G90V, K2090, L160A, L160M, L167Q, L213N, L243A, L243I, L243V, N112C, N112D, N112H, P207Q, Q1440, Q47L, O47L, Q480, R40D, R40F, R40G, R40H, R40I, R40K, R40L, R40M, R40N, R40P, R40Q, R40S, R40T, R40V, R40Y, R41H, R98C, S115R, S38A, S38C, S38D, S38E, S38F, S38G, S38H, S38I, S38K, S38L, S38M, S38Q, S38T, S38V, S38W, S38Y, T114C, T155C, T173S, T205N, T205Q, T92H, T92N, V203I, and V31I.

2. A method of use of the 3-hydroxybutyryl-CoA dehydrogenase variant of claim 1 as a biocatalyst.

3. A composition comprising the 3-hydroxybutyryl-CoA dehydrogenase variant of claim 1 and at least one substrate for said polypeptide variant.

4. A method for producing 3-hydroxybutyryl-CoA (3-HB-CoA), 3-hydroxybutyraldehyde (3-HBal), 3-hydroxybutyrate (3-HB), 1,3-butanediol (1,3-BDO), and/or an ester or amide thereof, the method comprising providing a substrate for the 3-hydroxybutyryl-CoA dehydrogenase variant of claim 1 , and converting the substrate to 3-HB-CoA, 3-HBal, 3-HB, or 1,3-BDO through one or more reactions

wherein the 3-hydroxybutyryl-CoA dehydrogenase variant is isolated and

wherein the 1,3-BDO is optionally at least about 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% R-1,3-butanediol (R-1,3-BDO).

5. A method for producing the 3-hydroxybutyryl-CoA dehydrogenase variant of claim 1 , the method comprising isolating the 3-hydroxybutyryl-CoA dehydrogenase variant.

6. The isolated 3-hydroxybutyryl-CoA dehydrogenase variant of claim 1 , wherein the 3-hydroxybutyryl-CoA dehydrogenase variant convert acetoacetyl-CoA to 3-hydroxybutyryl-CoA; and/or wherein the 3-hydroxybutyryl-CoA dehydrogenase variant of (a)-(d) is optionally a 3-hydroxybutyryl-CoA dehydrogenase having an increased ability to utilize NADH as a cofactor in a reaction converting acetoacetyl-CoA to 3-hydroxybutyryl-CoA, relative to a reference 3-hydroxybutyryl-CoA dehydrogenase encoded by SEQ ID NO: 2.

7. The composition of claim 3 , wherein said 3-hydroxybutyryl-CoA dehydrogenase variant can react with said substrate under in vitro conditions.

8. The composition of claim 7 , wherein the substrate is acetoacetyl-CoA.

9. The composition of claim 3 , wherein the composition further comprises a thiolase with at least 85% amino acid sequence identity to SEO ID NO: 4.

10. The composition of claim 9 , wherein the thiolase a reduced oxygen sensitivity in converting two acetyl-CoA molecules to acetoacetyl-CoA as compared to a thiolase comprising the amino acid sequence of SEQ ID NO: 6.

Assignments (3)
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 →
Continuity (3)
Division 16498336
Provisional Application 62480208 · Mar 31, 2017
Related Publication 20230139515A1 · May 4, 2023
References Cited (93)
US 7127379B2 · Palsson et al. · 2006 [cited by applicant]
US 9017983B2 · Burgard et al. · 2015 [cited by applicant]
US 20020012939A1 · Palsson · 2002 [cited by applicant]
US 20020168654A1 · Maranas et al. · 2002 [cited by applicant]
US 20030059792A1 · Palsson et al. · 2003 [cited by applicant]
US 20030224363A1 · Park et al. · 2003 [cited by applicant]
US 20030233218A1 · Schilling · 2003 [cited by applicant]
US 20040009466A1 · Maranas et al. · 2004 [cited by applicant]
US 20040029149A1 · Palsson et al. · 2004 [cited by applicant]
US 20040072723A1 · Palsson et al. · 2004 [cited by applicant]
US 20090047719A1 · Burgard et al. · 2009 [cited by applicant]
US 20130066035A1 · Burgard et al. · 2013 [cited by applicant]
US 20150164855A1 · Clarke et al. · 2015 [cited by applicant]
WO WO2002055995 · 2002 [cited by applicant]
WO WO2003106998 · 2003 [cited by applicant]
WO WO2006043555 · 2006 [cited by applicant]
WO WO2006043555AM3 · 2006 [cited by examiner]
WO WO2009094485 · 2009 [cited by applicant]
WO WO2010127319 · 2010 [cited by applicant]
WO WO2011156794 · 2011 [cited by applicant]
WO WO2013036764 · 2013 [cited by applicant]
WO WO2014190251 · 2014 [cited by applicant]
WO WO2018183628 · 2018 [cited by applicant]
WO WO2018183664 · 2018 [cited by applicant]
Whisstock et al. Quaterly Reviews of Biophysics, 2003, “Prediction of protein function from protein sequence and structure”, 36(3): 307-340. (Year: 2003). [cited by examiner]
Witkowski et al. Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine, Biochemistry. Sep. 7, 1999;38(36):11643-50. (Year: 1999). [cited by examiner]
Kisselev L., Polypeptide release factors in prokaryotes and eukaryotes: same function, different structure. Structure, 2002, vol. 10: 8-9. (Year: 2002). [cited by examiner]
Altschul et al., “Basic Local Alignment Search Tool,” [cited by applicant]
Bergquist et al., “Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDN): two complementary techniques for enzyme evolution,” [cited by applicant]
Bergquist et al., “Degenerate oligonucleotide gene shuffling,” [cited by applicant]
Burgard et al., “Minimal reaction sets for Escherichia coli metabolism under different growth requirements and uptake environments,” [cited by applicant]
Burgard et al., “OptKnock: A Bilevel Programming Framework for Identifying Gene Knockout Strategies for Microbial Strain Optimization,” [cited by applicant]
Coco et al., “DNA shuffling method for generating highly recombined genes and evolved enzymes,” [cited by applicant]
Colonna et al., “Synthesis and radiocarbon evidence of terephthalate polyesters completely prepared from renewable resources,” [cited by applicant]
Cosmetic Ingredient Review Board, “Final Report on the Safety Assessment of Butylene Glycol, Hexylene Glycol, Ethoxy diglycol, and Dipropylene Glycol,” [cited by applicant]
Currie et al., “Authentication and dating of biomass components of industrial materials; links to sustainable technology,” [cited by applicant]
Drake ed., [cited by applicant]
Fujii et al., “Error-prone rolling circle amplification: the simplest random mutagenesis protocol,” [cited by applicant]
Fujii et al., “One-step random mutagenesis by error-prone rolling circle amplification,” [cited by applicant]
GenBank Accession No. CCF95918, “Acetoacetyl-CoA reductase [Ralstonia solanacearum K60-1],” Feb. 20, 2012. [cited by applicant]
GenBank Accession No. SAL82526, “acetyacetyl-CoA reductase [Caballeronia choica],” Apr. 15, 2016. [cited by applicant]
GenBank Accession No. WP_009606749, “acetoacetyl-CoA reductase [Xanthomonas translucens],” Jun. 6, 2013. [cited by applicant]
GenBank Accession No. AGN95877, “chain dehydrogenase/reductase SDR [ [cited by applicant]
GenBank Accession No. AAB65780, “acetoacetyl-CoA reductase [ [cited by applicant]
GenBank Accession No. ETH84293, “acetoacetyl-CoA reductase [Bordetella pertussis STO1-CHOC-0017],” Dec. 13, 2013. [cited by applicant]
GenBank Accession No. SAK89949, “acetyacetyl-CoA reductase [Caballeronia arationis],” Nov. 22, 2016. [cited by applicant]
GenBank Accession No. AMP00485, “acetoacetyl-CoA reductase [Collimonas arenae],” Mar. 9, 2016. [cited by applicant]
Geneseq Accession No. AEH29045, “R. eutropha 3-keto acyl-CoA reductase mutant SEQ ID No. 2,” Jun. 29, 2006. [cited by applicant]
Geneseq Accession No. AEH29048, “R. eutropha 3-keto acyl-CoA reductase mutant SEQ ID No. 5,” Jun. 29, 2006. [cited by applicant]
Geneseq Accession No. AZQ30041, “R. eutrophus phaA gene encoded acetoacetyl-CoA thiolase/synthase, SEQ 16,” Feb. 2, 2012. [cited by applicant]
Geneseq Accession No. AZQ30043, “Rastonia eutrophus 3-hydroxybutyryl-CoA dehydrogenase, SEQ ID 18,” Feb. 2, 2012. [cited by applicant]
Gibbs et al., “Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling,” [cited by applicant]
Hayes et al., “Combining computational and experimental screening for rapid optimization of protein properties,” [cited by applicant]
Hibbert et al., “Directed evolution of biocatalytic processes,” [cited by applicant]
Hoffmeister et al., “Mitochondrial trans-2-Enoyl-CoA Reductase of Wax Ester Fermentation from Euglena gracilis Defines a New Family of Enzymes Involved in Lipid Synthesis,” [cited by applicant]
Houghten, “General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids,” [cited by applicant]
Huisman et al., “Enzyme Evolution for Chemical Process Applications,” [cited by applicant]
Jeon et al., “Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) from butyrate using engineered Ralstonia eutropha,” [cited by applicant]
Karlen et al., “Absolute determination of the activity of two C14 dating standards,” [cited by applicant]
Kretz et al., “Gene site saturation mutagenesis: a comprehensive mutagenesis approach,” [cited by applicant]
Lee et al., “A new approach to directed gene evolution by recombined extension on truncated templates (RETT),” [cited by applicant]
Lin et al., “Fed-Batch Culture of a Metabolically Engineered [cited by applicant]
Low et al., “Mimicking somatic hypermutation: affinity maturation of antibodies displayed on baceriophage using a bacterial mutator strain,” [cited by applicant]
Lutz et al., “Creating multiple-crossover DNA libraries independent of sequence identity,” [cited by applicant]
Lutz et al., “Rapid generation of incremental truncation libraries for protein engineering using α-phosphothioate nucleotides,” [cited by applicant]
Mann, “An International Reference Material for Radiocarbon Dating,” [cited by applicant]
Motsumoto et al., “Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics,” [cited by applicant]
Muller et al., “Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution,” [cited by applicant]
Ness et al., “Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently,” [cited by applicant]
Ostermeier et al., “A combinatorial approach to hybrid enzymes independent of DNA homology,” [cited by applicant]
Ostermeier et al., “Combinatorial protein engineering by incremental truncation,” [cited by applicant]
Otten et al., “Directed evolution: selecting today's biocatalysts,” [cited by applicant]
Pritchard et al., “A general model of error-prone PCR,” [cited by applicant]
Rajpal et al., “A general method for greatly improving the affinity of antibodies by using combinatorial libraries,” [cited by applicant]
Reetz et al., “Directed Evolution of an Enantioselective Enzyme through Combinatorial Multiple-Cassette Mutagenesis,” [cited by applicant]
Reetz et al., “Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes,” [cited by applicant]
Reetz et al., “Iterative Saturation Mutagenesis on the Basis of B Factors as a Strategy for Increasing Protein Thermostability,” [cited by applicant]
Reidhaar-Olson et al., “Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences,” [cited by applicant]
Reidhaar-Olson et al., “Random mutagenesis of protein sequences using oligonucleotide cassettes,” [cited by applicant]
Selifonova et al., “Rapid evolution of novel traits in microorganisms,” [cited by applicant]
Sen et al., “Developments in directed evolution for improving enzyme functions,” [cited by applicant]
Shao et al., “Random-priming in vitro recombination: an effective tool for directed evolution,” [cited by applicant]
Sieber et al., “Libraries of hybrid proteins from distantly related sequences,” [cited by applicant]
Stemmer, “DNA Shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution,” [cited by applicant]
Stemmer, “Rapid evolution of a protein in vitro by DNA shuffling,” [cited by applicant]
Volkov et al., “Random chimeragenesis by heteroduplex recombination,” [cited by applicant]
Volkov et al., “Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair,” [cited by applicant]
Wong et al., “Sequence satruation mutagenesis with tunable mutation frequencies,” [cited by applicant]
Whisstock et al., “Prediction of protein function from protein sequence and structure,” [cited by applicant]
Witkowski et al., “Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine,” [cited by applicant]
Wong et al., “Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution,” [cited by applicant]
Wong et al., “Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR,” [cited by applicant]
Zhao et al., “Molecular evolution by staggered extension process (StEP) in vitro recombination,” [cited by applicant]