Alcohol dehydrogenase variants

Patent metadata

Field	Value
Publication number	US-10563180-B2
Application number	US-201415027169-A
Country	US
Kind code	B2
Filing date	Oct 3, 2014
Priority date	Oct 4, 2013
Publication date	Feb 18, 2020
Grant date	Feb 18, 2020

Abstract

Described herein are non-natural NAD+-dependent alcohol dehydrogenases (ADHs) capable of at least two fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to its unmodified counterpart. Nucleic acids encoding the non-natural alcohol dehydrogenases, as well as expression constructs including the nucleic acids, and engineered cells comprising the nucleic acids or expression constructs are described. Also described are engineered cells expressing a non-natural NAD+-dependent alcohol dehydrogenase, optionally include one or more additional metabolic pathway transgene(s), methanol metabolic pathway genes, target product pathway genes, cell culture compositions including the cells, methods for promoting production of the target product or intermediate thereof from the cells, compositions including the target product or intermediate, and products made from the target product or intermediate.

First claim

What is claimed is:

1. An engineered microbial cell either (a) expressing a non-natural NAD + -dependent alcohol dehydrogenase comprising at least one amino acid substitution as compared to a corresponding alcohol dehydrogenase and capable of greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to an engineered microbial cell expressing the corresponding alcohol dehydrogenase without amino acid substitution or (b) expressing a first sequence that is a non-natural NAD + -dependent alcohol dehydrogenase comprising at least one amino acid substitution capable of greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, as compared to an engineered microbial cell expressing a second sequence that is an NAD + -dependent alcohol dehydrogenase, wherein the first and second sequences differ with regards to the at least one amino acid substitution,
wherein the non-natural alcohol dehydrogenase has a sequence identity of 75% or greater to an NAD + -dependent alcohol dehydrogenase template selected from the group consisting of SEQ ID NO: 1 (MDH MGA3_17392), SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, and SEQ ID NO: 77, or a fragment of said template having said dehydrogenase activity with an amino-terminal deletion, carboxy-terminal deletion, or both, the fragment having a sequence identity of 75% or greater, to the template, and
wherein the non-natural alcohol dehydrogenase comprises one or more amino acid substitutions based on formula: R 1 XR 2 , wherein R 1 is an original amino acid at position X of the template, and R 2 is the variant amino acid that replaces R 1 at a position on the template corresponding to X, wherein XR 2 is selected from the group consisting of (a) 11T, 38N, 42Q, 48D, 53I, 56K, 60E, 61A, 63F, 65Q, 70N, 71I, 71T, 71V, 74S, 81G, 84R, 86K, 87K, 94V, 99P, 99T, 103V, 106L, 107S, 108V, 108W, 109Y, 112K, 112R, 115H, 116F, 117D, 117Q, 117Y, 120H, 120R, 121A, 121D, 121E, 121L, 121M, 121R, 121S, 121T, 121V, 121W, 121Y, 122A, 122P, 123D, 123I, 123L, 123R, 123Y, 124I, 124L, 124R, 125C, 125G, 125W, 126G, 126V, 127C, 127R, 128A, 128R, 128S, 129A, 129M, 129P, 129S, 130F, 130I, 130Y, 134T, 143T, 145M, 146N, 147R, 148A, 148F, 148G, 148I, 148T, 148V, 148W, 149L, 149M, 149T, 149V, 150A, 150I, 152M, 155V, 157N, 158E, 158H, 158K, 158W, 161A, 161G, 161Q, 161S, 161V, 163F, 163N, 163Q, 163T, 164G, 164N, 165G, 181R, 184T, 186M, 190A, 190S, 199V, 217K, 226M, 256C, 267H, 269S, 270M, 270S, 270Y, 296S, 298H, 300T, 302V, 312V, 316V, 323M, 333L, 336L, 337C, 343D, 344A, 344G, 345E, 350K, 354M, 355D, 355I, 355K, 358G, 360A, 360G, 360K, 360R, 360S, 361N, 361R, 363K, and 379M.

2. The engineered microbial cell of claim 1 further comprising (c) one or more metabolic pathway transgene(s) encoding a protein of a metabolic pathway that promotes production of a target product or intermediate thereof, (d) a transgene encoding an enzyme to convert the formaldehyde to formate thereby generating reducing equivalents useful to product the target product and/or able to fix carbon of formate into the target product, or both (c) and (d).

3. The engineered microbial cell of claim 1 , wherein expression of the non-natural alcohol dehydrogenase provides an increased amount of reducing equivalents for an increase in a target product and/or for increased fixation of carbon from the formaldehyde into a target product.

4. The engineered microbial cell of claim 2 wherein the target product is selected from the group consisting of a diol, 1,4-butanediol, 1,3-butanediol, butadiene, succinate, adipate, HMDA, 6-aminocaproic acid (6ACA), methacrylic acid (2-methyl-2-propenoic acid), methacrylate, methyl methacrylate, 3-hydroxyisobutyrate, 2-hydroxyisobutyrate, or an intermediate compound thereof.

5. The engineered microbial cell of claim 1 further comprising (c) one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a), a formate dehydrogenase (EM8), a formaldehyde activating enzyme (EM10), a formaldehyde dehydrogenase (EM11), a S-(hydroxymethyl)glutathione synthase (EM12), a glutathione-dependent formaldehyde dehydrogenase (EM13), a S-formylglutathione hydrolase (EM14), a formate hydrogen lyase (EM15), and a hydrogenase (EM16); (d) one or more alcohol metabolic pathway gene(s) encoding a protein selected from the group consisting of a succinyl-CoA reductase (aldehyde forming) (EB3), a 4-hydroxybutyrate (4-HB) dehydrogenase (EB4), a 4-HB kinase (EB5), a phosphotrans-4-hydroxybutyrylase (EB6), a 4-hydroxybutyryl-CoA reductase (aldehyde forming) (EB7), a 1,4-butanediol dehydrogenase (EB8); a succinate reductase (EB9), a succinyl-CoA reductase (alcohol forming) (EB10), 4-hydroxybutyryl-CoA transferase (EB11), a 4-hydroxybutyryl-CoA synthetase (EB12), a 4-HB reductase (EB13), and a 4-hydroxybutyryl-CoA reductase (alcohol forming) (EB15), a succinyl-CoA transferase (EB1), and a succinyl-CoA synthetase (EB2A), or both (c) and (d), or (e) a formaldehyde assimilation pathway enzyme (FAPE) comprising a hexulose-6-phosphate (H6P) synthase (EF1), a 6-phospho-3-hexuloisomerase (EF2), a dihydroxyacetone (DHA) synthase (EF3) or a DHA kinase (EF4).

6. A composition comprising the engineered microbial cell of claim 1 , a cell culture composition, optionally comprising a target product or intermediate thereof, or a cell extract thereof.

7. A method for increasing the conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, comprising a step of (a) culturing the engineered microbial cell of claim 1 in a culture medium comprising methanol or ethanol, where in said culturing the cell provides greater conversion of the methanol or ethanol to formaldehyde or acetaldehyde respectively, as compared to an engineered microbial cell expressing the corresponding alcohol dehydrogenase without amino acid substitution.

8. The engineered microbial cell of claim 1 wherein the non-natural alcohol dehydrogenase which is capable of at least two fold greater, of at least three fold, of at least four fold, of at least five fold, of at least six fold, of at least seven fold, at least 8 fold, at least 9 fold, at least 10 fold, or at least 11 fold, or in the range of two fold to twelve fold greater, in the range of two fold to eleven fold greater, in the range of two fold to ten fold greater, in the range of two fold to nine fold greater, in the range of two fold to eight fold greater, in the range of two fold to seven fold greater, in the range of two fold to six fold greater, in the range of two fold to five fold greater, or in the range of two fold to four fold greater conversion of methanol or ethanol to formaldehyde or acetaldehyde, respectively, in vivo or in vitro, as compared to the corresponding alcohol dehydrogenase without amino acid substitution.

9. The engineered microbial cell of claim 1 wherein the NAD + -dependent non-natural alcohol dehydrogenase has a catalytic efficiency (k cat /K m ) for the conversion of methanol to formaldehyde of 8.6×10 −4 or greater.

10. A method of producing a target product or its intermediate comprising culturing the engineered microbial cell of claim 1 in a culture medium comprising methanol or ethanol to produce the target produ

Assignees

• Genomatica Inc

Inventors

• Andrae Stefan
• Kuchinskas Michael Patrick
• Li Jingyi
• Nagarajan Harish
• Pharkya Priti

Classifications

• C12Y101/01244

  Methanol dehydrogenase (1.1.1.244) · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12P7/18

  polyhydric · CPC title

• C12P7/24

  containing a carbonyl group · CPC title

• C12Y101/01001

  Alcohol dehydrogenase (1.1.1.1) · CPC title