3-hydroxybutyryl-coa dehydrogenase variants and methods of use

An isolated nucleic acid molecule comprising a sequence encoding an isolated polypeptide variant of a reference polypeptide, wherein:

• (a) (i) the reference polypeptide has an amino acid sequence of SEQ ID NO:
• 2, and (ii) the polypeptide variant is selected from Table 1 and has one or more amino acid substitutions relative to SEQ ID NO:
• 2,
• (b) (i) the polypeptide variant comprises a sequence having at least 50% identity to SEQ ID NO:
• 2 or to a variant selected from Table 1, optionally wherein the polypeptide variant comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:
• 2 or a variant selected from Table 1, and
• (ii) the polypeptide variant comprises an amino acid substitution corresponding to one or more positions of SEQ ID NO:
• 2 selected from C34, P36, N37, S38, and R40, wherein
• (1) the amino acid corresponding to C34 is replaced with A, D, E, H, I, L, M, S, T, V, or Y;
• (2) the amino acid corresponding to P36 is replaced with S;
• (3) the amino acid corresponding to N37 is replaced with K
• (4) the amino acid corresponding to S38 is optionally replaced with A, C, D, E, F, G, H, I, K, L, M, Q, T, V, W, or Y;
• (5) the amino acid corresponding to R40 is optionally replaced with A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, or Y;
• or
• (6) optionally, two or more of (1)-(5) in any combination;
• (c) (i) the polypeptide variant comprises a sequence having at least 50% identity to SEQ ID NO:
• 2 or a variant selected from Table 1,
• wherein the polypeptide variant optionally comprises a sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:
• 2 or to a variant selected from Table 1;
• and
• (ii) the polypeptide variant comprises 1, 2, 3, 4, 5, or more amino acid substitutions relative to SEO ID NO:
• 2 selected from:
• A103M, A107V, A156S, A158R, A193R, A204T, C20F, C34A, C34D, 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, L2431, L243V, N112C, N112D, N112H, N37K, P207Q, P36S, Q1440, Q47L, O47L, Q480, R40A, R40D, R40E, 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 V311;
• or
• (d) (i) the reference polypeptide is a first reference polypeptide, wherein the first reference polypeptide has an amino acid sequence of SEO ID NO:
• 2,
• (ii) the polypeptide variant comprises one or more amino acid substitutions relative to SEO ID NO:
• 2,
• (iii) the one or more amino acid substitutions are selected from Table 1, and
• (iv) the polypeptide variant has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a corresponding variant selected from Table 1,
• wherein the polypeptide variant of the first reference polypeptide optionally has at least 80%, 90%, or 95% sequence identity to the corresponding variant selected from Table 1;
• wherein the polypeptide variant of the first reference polypeptide further comprises a conservative amino acid substitution in from 1 to 100 modified amino acid positions relative to the corresponding first reference polypeptide, wherein said modified positions are other than the one or more amino acid substitutions of a second reference polypeptide;
• wherein the one or more amino acid substitutions of (a)-(d) optionally comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions selected from Table 1;
• wherein the polypeptide variant of (a)-(d) optionally comprises no modification at from 2 to 300 unmodified amino acid positions compared to the first reference polypeptide, wherein said unmodified positions are selected from those that are identical to between 2, 3, 4, or 5 amino acid sequences referenced in Table 2;
• wherein the polypeptide variant of (a)-(d) can optionally convert acetoacetyl-CoA to 3-hydroxybutyryl-CoA;
• and/or wherein the polypeptide 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 SEO ID NO:
• 2.

A method for producing 3-hydroxybutyryl-CoA (3-HB-CoA), 3-hydroxybutyraldehyde (3-HBal), 3-hydroxybutyrate (3-HB), and/or 1,3-butanediol (1,3-BDO), the method comprising:

• a. culturing a cell under conditions and for a sufficient period of time to produce 3-HB-CoA, 3-HBal, 3-HB, and/or 1,3-BDO;
• and
• b. isolating or purifying the 3-HB-CoA, 3-HBal, 3-HB, 1,3-BDO, and/or an ester or amide thereof, optionally comprising distillation;
• wherein the cell comprises a polynucleotide encoding a thiolase with at least 80%, 85%, 90%, 95%, 98% 99%, or 100% amino acid sequence identity to SEQ ID NO:
• 4
• wherein the thiolase optionally comprises the amino acid sequence of SEQ ID NO:
• 4 and/or exhibits 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;
• wherein the cell optionally further comprises a second polynucleotide encoding a polypeptide with at least 80%, 85%, 90%, 95%, 98% 99%, or 100% amino acid sequence identity to SEQ ID NO:
• 2;
• wherein the second polynucleotide is optionally (i) an exogenous polynucleotide, and/or (ii) integrated into a genome of the cell
• wherein the polynucleotide encoding the thiolase is optionally (i) an exogenous polynucleotide, and/or (ii) integrated into a genome of the cell;
• wherein the culturing optionally comprises a peak weight-based oxygen uptake rate of about 50 mmol O2/kg/hr, 40 mmol O2/kg/hr, 30 mmol O2/kg/hr, 25 mmol O2/kg/hr, or lower;
• wherein the cell optionally produces an increased amount of 1,3-BDO when cultured for at least 30 or 40 hours as compared to a cell in which the thiolase instead comprises the amino acid sequence of SEO ID NO:
• 6, optionally wherein the increased amount of 1,3-BDO is an increase of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50%;
• wherein the cell optionally produces a decreased amount of pyruvate when cultured for at least 30 or 40 hours as compared to a cell in which the thiolase instead comprises the amino acid sequence of SEO ID NO:
• 6, optionally wherein the decreased amount of pyruvate is a decrease of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80%;
• and/or
• wherein the cell optionally produces 1,3-BDO and pyruvate at an increased ratio of 1,3-BDO to pyruvate when cultured for at least 30 or 40 hours as compared to a cell in which the thiolase instead comprises the amino acid sequence of SEO ID NO:
• 6, optionally wherein the increased ratio is increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold.