ALDEHYDE DEHYDROGENASE VARIANTS AND METHODS OF USE
The invention provides polypeptides and encoding nucleic acids of aldehyde dehydrogenase variants. The invention also provides cells expressing aldehyde dehydrogenase variants. The invention further provides methods for producing 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof, comprising culturing cells expressing an aldehyde dehydrogenase variant or using lysates of such cells. The invention additional provides methods for producing 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof, comprising culturing cells expressing an aldehyde dehydrogenase variant or using lysates of such cells.
1 . An isolated nucleic acid molecule selected from:
(a) a nucleic acid molecule encoding an amino acid sequence referenced as SEQ ID NO: 1, 2 or 3 or 7-123, wherein said amino acid sequence comprises one or more amino acid substitutions selected from C174S, M204R, C220V, C267A, V315A, C356T, R396H, E437P, T440H, F442N, C464I and A467V as compared to wild type aldehyde dehydrogenase of SEQ ID NO:1, 2, 3 or 7-123;
(b) a nucleic acid molecule that hybridizes to the nucleic acid of (a) under highly stringent hybridization conditions and comprises a nucleic acid sequence that encodes the one or more amino acid substitutions;
(c) a nucleic acid molecule encoding an amino acid sequence comprising the consensus sequence of Loop A (SEQ ID NO:5) and/or Loop B (SEQ ID NO:6), wherein said amino acid sequence comprises one or more amino acid substitutions selected from C174S, M204R, C220V, C267A, V315A, C356T, R396H, E437P, T440H, F442N, C464I and A467V as compared to wild type aldehyde dehydrogenase of SEQ ID NO:1, 2, 3 or 7-123; and
(d) a nucleic acid molecule that is complementary to (a) or (b).
2 . The isolated nucleic acid molecule of claim 1 , wherein said amino acid sequence, other than the one or more amino acid substitutions, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acid sequence referenced in SEQ ID NO: 1, 2 or 3 or 7-123.
3 . The nucleic acid of claim 1 , wherein the amino acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the amino acid substitutions.
4 . A vector containing the nucleic acid molecule of claim 1 .
5 - 6 . (canceled)
7 . An isolated polypeptide comprising an amino acid sequence referenced as SEQ ID NO: 1, 2 or 3 or in Table 4, wherein said amino acid sequence comprises one or more amino acid substitutions selected from C174S, M204R, C220V, C267A, V315A, C356T, R396H, E437P, T440H, F442N, C464I and A467V as compared to wild type aldehyde dehydrogenase of SEQ ID NO:1, 2, 3 or 7-123.
8 . (canceled)
9 . An isolated polypeptide comprising an amino acid sequence referenced as SEQ ID NO: 1, 2 or 3 or in Table 4, wherein said amino acid sequence comprises one or more amino acid substitutions selected from C174S, M204R, C220V, C267A, V315A, C356T, R396H, E437P, T440H, F442N, C464I and A467V as compared to wild type aldehyde dehydrogenase of SEQ ID NO:1, 2, 3 or 7-123, wherein said amino acid sequence, other than the one or more amino acid substitutions, has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to an amino acids sequence referenced as SEQ ID NO: 1, 2 or 3 or 7-123.
10 . The isolated polypeptide of claim 9 , wherein said amino acid sequence further comprises a conservative amino acid substitution at from 1 to 100 amino acid positions, wherein said positions are other than the one or more amino acid substitutions.
11 . The isolated polypeptide of claim 7 , wherein said amino acid sequence comprises no modification from 2 to 300 amino acid positions compared to the parent sequence, other than the one or more amino acid substitutions.
12 . The isolated polypeptide of claim 7 , wherein the amino acid sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the amino acid substitutions set forth.
13 - 23 . (canceled)
24 . A cell comprising the nucleic acid of claim 1 .
25 - 40 . (canceled)
41 . A composition comprising the polypeptide of claim 7 and at least one substrate for said polypeptide.
42 - 25 . (canceled)
46 . Culture medium comprising the cell of claim 24 .
47 . (canceled)
48 . A method for producing 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof, or 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof, comprising culturing said cell of claim 24 to produce 3-HBal and/or 1,3-BDO, or an ester or amide thereof.
49 - 57 . (canceled)
58 . 3-Hydroxybutyraldeyde (3-HBal) and/or 1,3-butanediol (1,3-BDO) or the 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO) having a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source, wherein the 3-HBal and/or 1,3-BDO or the 4-HBal and/or 1,4-BDO is produced by the method of claim 48 , wherein the 3-HBal and/or 1,3-BDO or the 4-HBal and/or 1,4-BDO is enantiomerically enriched for the R form.
59 - 63 . (canceled)
64 . A composition comprising the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, of claim 58 , or a cell lysate or culture supernatant of a cell producing the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO.
65 . A product comprising the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, of claim 58 , wherein said product is a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-hydroxybutyrate (P4HB) or a co-polymer thereof, poly(tetramethylene ether) glycol (PTMEG), polybutylene terephthalate (PBT), polyurethane-polyurea copolymer, nylon, organic solvent, polyurethane resin, polyester resin, hypoglycaemic agent, butadiene or butadiene-based product.
66 - 69 . (canceled)
70 . A process for producing the product of claim 65 , comprising chemically reacting the 3-HBal and/or 1,3-BDO, or the 4-HBal and/or 1,4-BDO, with itself or another compound in a reaction that produces said product.
71 . (canceled)
72 . A method for producing 3-hydroxybutyraldehyde (3-HBal) and/or 1,3-butanediol (1,3-BDO), or an ester or amide thereof, comprising providing a substrate to the polypeptide of claim 7 and converting the substrate to 3-HBal and/or 1,3-BDO, wherein the substrate is a racemic mixture of 1,3-hydroxybutyryl-CoA.
73 . (canceled)
74 . A method for producing 4-hydroxybutyraldehyde (4-HBal) and/or 1,4-butanediol (1,4-BDO), or an ester or amide thereof, comprising providing a substrate to the polypeptide of claim 7 and converting the substrate to 4-HBal and/or 1,4-BDO, wherein the substrate is 1,4-hydroxybutyryl-CoA.
75 . (canceled)
76 . A method for producing 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO, comprising incubating a lysate of the cell of claim 24 to produce 3-HBal and/or 1,3-BDO, or 4-HBal and/or 1,4-BDO.
77 - 79 . (canceled)