Pisum sativum kaurene oxidase for high efficiency production of rebaudiosides
Inventors
WICHMANN, Gale • KHANKHOJE, Aditi • MAHATDEJKUL-MEADOWS, Tina
Assignees
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Publication Number
US-12264351-B2
Publication Date
2025-04-01
Expiration Date
Abstract
Provided herein are compositions and methods for improved production of steviol glycosides in a host cell. In some embodiments, the host cell is genetically modified to comprise a heterologous nucleotide sequence encoding a Pisum sativum kaurene oxidase or its variant kaurene oxidase. In some embodiments, the host cell further comprises one or more heterologous nucleotide sequence encoding further enzymes of a pathway capable of producing steviol glycosides in the host cell. The compositions and methods described herein provide an efficient route for the heterologous production of steviol glycosides, including but not limited to, rebaudioside D and rebaudioside M.
Core Innovation
The disclosure describes genetically modified Saccharomyces cerevisiae host cells that produce one or more steviol glycosides by expressing a heterologous nucleic acid encoding a kaurene oxidase having at least 80% sequence identity to SEQ ID NO: 1. The host cell is capable of converting kaurene to kaurenoic acid at an efficiency greater than 30%, and the engineered yeast produces steviol glycosides including RebD and RebM.
The genetically modified Saccharomyces cerevisiae host cell further comprises heterologous nucleic acids encoding a geranylgeranylpyrophosphate synthase, a copalyl diphosphate synthase, an ent-kaurene synthase, a polypeptide having steviol synthase activity, and one or more polypeptides having UDP glycosyltransferase activity. The disclosure defines pathway components and steviol glycoside biosynthesis pathway activity requirements for producing steviol glycosides.
The document also describes fermentation and recovery of steviol glycosides produced by the genetically modified host cells, including fermentation composition comprising the genetically modified host cell and steviol glycosides produced from the host cell. In the described embodiments, RebM is reported as a major steviol glycoside component, with RebA, RebD, undetectable RebM2, and non-natural glycosides also noted.
Claims Coverage
The partial claim set includes two independent claims covering a genetically modified Saccharomyces cerevisiae host cell and a fermentation composition. Across these independent claims, the coverage includes six main inventive feature areas: kaurene oxidase sequence identity, kaurene-to-kaurenoic-acid conversion efficiency, additional diterpene-pathway enzymes, steviol synthase activity, UDP glycosyltransferase activity, and inclusion of the produced steviol glycosides in a fermentation composition.
Kaurene oxidase for kaurene-to-kaurenoic-acid conversion
A genetically modified Saccharomyces cerevisiae host cell comprising a heterologous nucleic acid encoding a kaurene oxidase having at least 80% sequence identity to SEQ ID NO: 1, wherein the genetically modified host cell is capable of converting kaurene to kaurenoic acid at an efficiency greater than 30%.
Diterpene pathway and steviol synthase activity genes
The genetically modified Saccharomyces cerevisiae host cell further comprises one or more heterologous nucleic acids encoding a geranylgeranylpyrophosphate synthase, a copalyl diphosphate synthase, an ent-kaurene synthase, and a polypeptide having steviol synthase activity.
UDP glycosyltransferase activity for steviol glycosides
The genetically modified Saccharomyces cerevisiae host cell further comprises one or more heterologous nucleic acids encoding one or more polypeptides having UDP glycosyltransferase activity, and the genetically modified host cell produces one or more steviol glycosides.
Fermentation composition with engineered host and produced steviol glycosides
A fermentation composition comprising a genetically modified host cell comprising a heterologous nucleic acid encoding a kaurene oxidase having at least 80% sequence identity to SEQ ID NO: 1 and capable of converting kaurene to kaurenoic acid at an efficiency greater than 30%, together with one or more heterologous nucleic acids encoding a geranylgeranylpyrophosphate synthase, a copalyl diphosphate synthase, an ent-kaurene synthase, a polypeptide having steviol synthase activity, and one or more polypeptides having UDP glycosyltransferase activity; and steviol glycosides produced from the genetically modified host cell.
Overall, the independent claims focus on an engineered Saccharomyces cerevisiae that expresses a kaurene oxidase meeting specified sequence-identity and conversion-efficiency thresholds, together with heterologous diterpene-pathway enzymes, steviol synthase activity, and UDP glycosyltransferase activity to produce steviol glycosides. The other independent claim turns this engineered cell into a fermentation composition defined by the included host cell and the steviol glycosides produced by the host cell.
Stated Advantages
Improved conversion of kaurene to kaurenoic acid when using the specified kaurene oxidase.
Reduced downstream purification burden due to little/no kaurenol/kaurenal residue from some KO enzymes.
Increased RebM titers versus Sr.KO in yeast engineering screens.
Documented Applications
Heterologous production of steviol glycosides, including rebaudioside D and rebaudioside M, in engineered Saccharomyces cerevisiae.
Fermentation and recovery of steviol glycosides produced by the genetically modified host cells.
Providing a fermentation composition comprising the genetically modified host cell and steviol glycosides produced from the host cell.
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