3-hydroxybutyryl-CoA dehydrogenase variants and methods of use

An isolated nucleic acid molecule encoding a variant 3-hydroxybutyryl-CoA dehydrogenase selected from:

• (a) a first nucleic acid molecule encoding a first variant of a reference 3-hydroxybutyryl-CoA dehydrogenase, wherein (i) the reference 3-hydroxybutyryl-CoA dehydrogenase has the amino acid sequence of SEQ ID NO:
• 2, (ii) the first variant comprises one or more amino acid substitutions, but less than 20 amino acid substitutions, relative to SEQ ID NO:
• 2, and (iii) the one or more amino acid substitutions relative to SEQ ID NO:
• 2 comprises a substitution at position G35 selected from G35A, G35C, G35D, G35E, G35H, G35K, G35L, G35M, G35P, G35S, G35T, G35V, and G35Y;
• (b) a second nucleic acid molecule encoding a second variant of the reference 3-hydroxybutyryl-CoA dehydrogenase, wherein the second variant (i) comprises one or more amino acid substitutions relative to SEQ ID NO:
• 2, wherein the one or more amino acid substitutions relative to SEQ ID NO:
• 2 comprises a substitution at position G35 selected from G35A, G35C, G35D, G35E, G35H, G35K, G35L, G35M, G35P, G35S, G35T, G35V, and G35Y, and (ii) has, other than the substitution at position G35, at least 85% sequence identity to SEQ ID NO:
• 2, while retaining the substitution at position G35;
• (c) a third nucleic acid molecule that hybridizes to the first or second nucleic acid molecule of part (a) or (b), respectively, under highly stringent hybridization conditions and comprises a nucleic acid sequence that encodes the one or more amino acid substitutions relative to SEQ ID NO:
• 2;
• and
• (d) a fourth nucleic acid molecule that is fully complementary to the first nucleic acid molecule of part (a), the second nucleic acid molecule of part (b), or the third nucleic acid molecule of part (c);
• wherein the first nucleic acid molecule, the second nucleic acid molecule, the third nucleic acid molecule, and the fourth nucleic acid molecule are not naturally occurring, and wherein the first variant and the second variant comprise 3-hydroxybutyryl-CoA dehydrogenase enzyme activity.