US 12,460,239 B2
UDP-glycosyltransferase variants and uses thereof
Sean Lund, Emeryville, CA (US); and Gale Wichmann, Emeryville, CA (US)
Assigned to Corn Products Development, Inc., Westchester, IL (US)
Filed by CORN PRODUCTS DEVELOPMENT, INC., Westchester, IL (US)
Filed on Jul. 9, 2024, as Appl. No. 18/767,621.
Application 18/767,621 is a division of application No. 18/368,369, filed on Sep. 14, 2023, granted, now 12,065,685.
Application 18/368,369 is a continuation of application No. 16/871,324, filed on May 11, 2020, abandoned.
Claims priority of provisional application 62/846,909, filed on May 13, 2019.
Prior Publication US 2025/0313877 A1, Oct. 9, 2025
Int. Cl. C12P 19/56 (2006.01); C12N 15/81 (2006.01)
CPC C12P 19/56 (2013.01) [C12N 15/81 (2013.01); C12Y 106/02004 (2013.01); C12Y 114/14 (2013.01); C12Y 205/01029 (2013.01); C12Y 402/03019 (2013.01); C12Y 505/01012 (2013.01)] 20 Claims
 
1. A method for producing one or more steviol glycosides comprising
culturing a population of host cells in a medium with a carbon source under conditions suitable for making one or more steviol glycosides to yield a culture broth; and
recovering the one or more steviol glycosides from the culture broth,
wherein the host cells comprise:
(i) a variant UDP-glycosyltransferase polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with one or more amino acid substitutions, wherein the variant UDP-glycosyltransferase polypeptide produces a higher ratio of RebM/RebD compared to the UDP-glycosyltransferase polypeptide of SEQ ID NO: 1, and wherein the one or more amino acid substitutions are selected from the group consisting of (L85H), (L200G), (L379Q), (H155I), (H155V), (L200K), (V20G), (L200C), (A125F), (H155G), (H155T), (H155L), (L200E), (L200M), and (L379M);
(ii) a variant UDP-glycosyltransferase polypeptide comprising the amino acid sequence of SEQ ID NO: 1 with two or more amino acid substitutions, wherein the variant UDP-glycosyltransferase polypeptide produces a higher ratio of RebM/RebD compared to the UDP-glycosyltransferase polypeptide comprising SEQ ID NO: 1, and wherein the two or more amino acid substitutions are selected from the group consisting of (H155V, L200M), (H155G, L200E), (L85H, A125F, H155G, L200M), (A125F,H155G,L200K,L379M), (L200K,L379Q), (A125F,H155T,L200M), (L85H,L200M,L379Q), (L85H,H155G,L200G,L379M), (L85H,A125F,H155V,L200M), (L85H,A125F,H155I,L200C,L379Q), (A125F,H155L,L200E,L379Q), (H155T,L200G,L379M), (H155L,L200M), (H155V,L200C), (L85H,H155T,L200G), (L85H,H155I,L200C,L379Q), (H155I,L200M,L379Q), (L85H,A125F,H155T,L200E,L379M), (L85H,H155T,L200M), (H155V,L200C,L379M), (L85H,A125F,L200M,L379M), (H155V,L379Q), (L85H,L200K,L379M), (L85H,A125F,H155L,L200M,L379Q), (L85H,A125F,H155I,L200K,L379M), (H155I,L379M), (L200M,L379M), (L85H,H155V,L200G,L379Q), (H155T,L200E), (H155V,L379M), (A125F,H155I,L379M), (H155V,L200C,L379Q), (L85H,H155G,L200K,L379Q), (A125F,H155G,L200M,L379M), (H155T,L200E,L379Q), (L200K,L379G), (L85H,H155L,L200C,L379M), (A125F,H155I,L200K,L379M), (L85H,H155V,L200M), (L85H,H155V,L200E,L379V), (L85H,H155V,L200M,L379Q), (L85H,L200M,L379M), (H155V,L200E,L379M), (L85H,H155I,L200M,L379M), (L85H,H155I,L200M,L379Q), (L85H,A125F,H155T,L200C,L379M), (L85H,A125F,H155L,L200K,L379M), (V20L,L85M,A125P), (L85H,H155L,L200K,L379M), (L85H,H155V,L200C,L379Q), (H155I,L200K,L379M), (L85H,H155L,L200C), (L85H,A125F,H155L,L379M), (L85H,H155I,L200E,L379M), (L85H,A125F,H155G,L200M,L379M), (L85H,H155L,L200E,L379M), (H155L,L200C), (L85H,H155L,L200C,L379E), (L85H,H155V,L200K,L379Q), (H155T,L200K,L379M), (H155V,L200G,L379M), (L85H,A125F,H155V,L200M,L379M), (A125F,H155G,L200C,L379M), (A125F,H155L,L379M), (L85H,H155L,L200G,L379M), (L85H,H155L,L200E,L379Q), (H155G,L200E,L379M), (H155L,L200C,L379Q), (L85H,H155V,L379M), (L85H,A125F,H155I,L200C,L379M), (H155G,L200C,L379M), (A125F,H155V,L200M,L379M), and (H155L,L200E); or
(iii) a nucleic acid encoding the variant UDP-glycosyltransferase polypeptide of (i) or (ii);
wherein the host cells are capable of producing one or more steviol glycosides.