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US 12,460,239 B2Granted
UDP-glycosyltransferase variants and uses thereof
AssigneeCorn Products Development, Inc.
InventorsSean Lund, Gale Wichmann
Life of this patent
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Abstract
Provided herein are genetically modified host cells, compositions, and methods for improved production of steviol glycosides. The host cells are genetically modified to contain a heterologous nucleic acid that expresses novel and optimized variants of UGT76G1. The host cell further contains one or more heterologous nucleotide sequence encoding further enzymes of a pathway capable of producing one or more steviol glycosides in the host cell. The host cells, compositions, and methods described herein provide an efficient route for the heterologous production of rebaudioside M.
Claims20
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.
10 more elements
Dependencies are inferred from the claim text.