Production of steviol glycosides through whole cell biotransformation of steviol glycoside intermediates
Inventors
KUMARAN, Ajikumar Parayil • Santos, Christine Nicole S. • DONALD, Jason Eric • FOWLER, Mary Elizabeth • PHILIPPE, Ryan N. • FREI, Christopher Scott • Love, Aaron
Assignees
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Publication Number
US-12012626-B2
Publication Date
2024-06-18
Expiration Date
Abstract
In various aspects and embodiments, the invention provides microbial cells and methods for producing advanced glycosylation products from lower glycosylated intermediates. The microbial cell expresses one or more UDP-dependent glycosyl transferase enzymes in the cytoplasm, for glycosylation of the intermediates. When incubating the microbial strain with a plant extract or fraction thereof comprising the intermediates, these glycosylated intermediates are available for further glycosylation by the cell, and the advanced glycosylation products can be recovered from the media and/or microbial cells.
Core Innovation
The invention relates to producing a glycosylated product from glycoside intermediates using a bacterial cell that expresses one or more UDP-dependent glycosyl transferase enzymes intracellularly. The UGT enzymes glycosylate steviol glycoside substrates, wherein the glycoside intermediates are steviol glycosides from stevia leaf extract. The method includes incubating the bacterial strain with the glycoside intermediates and recovering the glycosylated product.
The bacterial cell is engineered to support UDP-glucose availability and intracellular UGT activity by overexpression of phosphoglucomutase (pgm) and UTP-glucose-1-phosphate uridylyltransferase. In addition, the bacterial cell has a deletion, inactivation, or reduced expression of one or more genes encoding enzymes that consume UDP-glucose. The UDP-glucose-consuming enzymes include UDP-sugar hydrolase, an enzyme responsible for UDP-galactose biosynthesis from UDP-glucose, a trehalose-6-phosphate synthase, and a UDP-glucose-6-dehydrogenase.
The described approach emphasizes using live intracellular UGTs to act on externally provided plant glycosylated intermediates, while UDP-glucose cofactor recycling is supported by native metabolism. The document describes conversion toward advanced glycosylation products such as RebM, including monitoring of RebM and related components. The glycosylated product is recovered after incubation.
Claims Coverage
The document includes one independent claim (clm-00001), directed to a method for producing a glycosylated product from steviol glycoside intermediates using an engineered bacterial cell expressing intracellular UDP-dependent glycosyl transferase enzymes with an altered UDP-glucose supply/consumption profile. The inventive features are refined by dependent claims that specify fermentation mode, incubation time, bacterial genera, product components, and particular UGT enzyme sequence-identity/sequence options.
Intracellular UGT glycosylation of steviol glycoside intermediates
A method producing a glycosylated product from glycoside intermediates by providing a bacterial cell expressing one or more UDP-dependent glycosyl transferase enzymes (UGT enzymes) intracellularly, wherein the UGT enzymes glycosylate steviol glycoside substrates; incubating the bacterial strain with glycoside intermediates that are steviol glycosides from stevia leaf extract; and recovering the glycosylated product.
UDP-glucose supply via pgm and UTP-glucose-1-phosphate uridylyltransferase overexpression
The bacterial cell has an overexpression of phosphoglucomutase (pgm) and UTP-glucose-1-phosphate uridylyltransferase.
Reduced UDP-glucose consumption by deleting or inactivating selected UDP-glucose-consuming genes
The bacterial cell has a deletion, inactivation, or reduced expression of one or more genes encoding enzymes that consume UDP-glucose selected from a UDP-sugar hydrolase, an enzyme responsible for UDP-galactose biosynthesis from UDP-glucose, a trehalose-6-phosphate synthase, and a UDP-glucose-6-dehydrogenase.
Independent claim clm-00001 centers on intracellular UDP-dependent glycosyl transferase (UGT) glycosylation of steviol glycoside intermediates from stevia leaf extract using engineered bacterial cells that overexpress pgm and UTP-glucose-1-phosphate uridylyltransferase to support UDP-glucose availability and reduce UDP-glucose consumption by deleting, inactivating, or reducing expression of selected UDP-glucose-consuming genes, followed by incubation and recovery of the glycosylated product.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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