	US 12,203,102 B2
	3-hydroxybutyryl-CoA dehydrogenase variants and methods of use
Kevin Hoff, San Diego, CA (US); Cara Ann Tracewell, Solana Beach, CA (US); Kui Chan, Los Angeles, CA (US); Michael Kuchinskas, Franklin, MA (US); and Harish Nagarajan, San Diego, CA (US)
Assigned to Genomatica, Inc., San Diego, CA (US)
Filed by Genomatica, Inc., San Diego, CA (US)
Filed on Jul. 14, 2022, as Appl. No. 17/812,636.
Application 17/812,636 is a division of application No. 16/498,336, abandoned, previously published as PCT/US2018/025086, filed on Mar. 29, 2018.
Claims priority of provisional application 62/480,208, filed on Mar. 31, 2017.
Prior Publication US 2023/0139515 A1, May 4, 2023
Int. Cl. C12N 9/04 (2006.01); C12N 9/10 (2006.01); C12P 7/18 (2006.01); C12P 7/24 (2006.01); C12P 7/52 (2006.01); C12P 19/32 (2006.01)

CPC C12N 9/0006 (2013.01) [C12N 9/1029 (2013.01); C12P 7/18 (2013.01); C12P 7/24 (2013.01); C12P 7/52 (2013.01); C12P 19/32 (2013.01); C12Y 101/01157 (2013.01); C12Y 203/01009 (2013.01)]

10 Claims

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.