Uridine diphosphate-dependent glycosyltransferase enzyme
Inventors
DONALD, Jason Eric • KUMARAN, Ajikumar Parayil • Love, Aaron • Toomey, Christopher • Santos, Christine Nicole S.
Assignees
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Publication Number
US-11788070-B2
Publication Date
2023-10-17
Expiration Date
Abstract
In various aspects, the present invention provides uridine diphosphate-dependent glycosyltransferase (UGT) enzymes capable of catalyzing the transfer of a monosaccharide moiety from a NDP-sugar to the 3′ carbon of a sugar moiety of a substrate, such as a terpenoid glycan, thereby functioning as a “1-3 UGT.” In other aspects, the invention provides polynucleotides encoding the 1-3 UGT, and host cells comprising the same. In still other aspects, the invention provides methods for preparing glycosylated substrates, including steviol glycosides, using the enzyme and host cells of this disclosure.
Core Innovation
The invention describes uridine diphosphate-dependent 1-3 UGT enzymes that transfer a monosaccharide, for example glucose from UDP-sugar, to the 3′ carbon of a sugar moiety on terpenoid glycan substrates. This glycosylation activity enables biosynthesis of steviol glycosides from steviol glycoside intermediates and terpenoid glycan substrates, including conversion targets such as stevioside to RebA and RebD to RebM.
The invention provides engineered UGT sequence variants based on SrUGT76G1 and circular/permuted derivatives MbUGT1-3. Engineered embodiments include an E225–T232 loop deletion and specific substitutions such as S72Q, A305C, Y345F, and L428I, with sequence embodiments tied to particular constructs including MbUGT1-3 family members with indicated sequence IDs.
The invention additionally covers polynucleotides encoding the engineered 1-3 UGT enzymes and host cells, including microbial, yeast, or plant contexts, expressing the 1-3 UGT, optionally with other UGTs and steviol-biosynthesis enzymes. Documented concepts for producing glycosylated products are described at the level of in vitro reactions and whole-cell biotransformations, including use of fed steviol glycoside intermediates from stevia extracts.
Claims Coverage
The consolidated claim coverage identifies one independent feature set centered on a UDP-dependent 1-3 UGT enzyme defined by sequence identity to SEQ ID NO: 9 and related engineered variants. The coverage includes five inventive features.
Highly similar UDP-dependent glycosyltransferase sequence to SEQ ID NO: 9
A uridine diphosphate-dependent glycosyltransferase (UGT) enzyme comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9.
Engineered UGT sequence identity or exact match to SEQ ID NO: 9
An enzyme comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 9, or comprising the amino acid sequence of SEQ ID NO: 9.
Specific substitutions relative to a reference sequence
An enzyme engineered to include one or more substitutions relative to the amino acid sequence of SEQ ID NO: 6, including S72Q, A305C, Y345F, and L428I.
Bounded number of amino-acid modifications
An engineered enzyme including 1 to 10 amino acid modifications relative to the amino acid sequence of SEQ ID NO: 9, where the modifications include independently chosen substitutions, deletions, or insertions.
Recombinant microorganism expressing the encoding polynucleotide
An isolated recombinant microorganism that includes the polynucleotide recited in the dependent claim covering the enzyme.
Overall, the claims coverage centers on a UDP-dependent 1-3 UGT enzyme defined by high sequence identity, refined to at least 98% or exact SEQ ID NO: 9, with engineered variants defined by specific substitutions, bounded modification counts, and embodiments in isolated recombinant microorganisms via the corresponding encoding polynucleotides.
Stated Advantages
Improved RebA and RebM production
Reported conversion improvements, including RebD→RebM conversion and stevioside→RebA conversion
Documented Applications
Biosynthesis of steviol glycosides using the described 1-3 UGT activity on terpenoid glycan substrates, including conversion of RebD to RebM and stevioside to RebA.
Producing glycosylated products via in vitro reactions or whole-cell biotransformations, including use of fed steviol glycoside intermediates from stevia extracts.
Host cell expression of the 1-3 UGT, including microbial, yeast, or plant contexts, optionally together with other UGTs and steviol-biosynthesis enzymes.
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