Stevia rebaudiana kaurenoic acid hydroxylase variants for high efficiency production of rebaudiosides

Inventors

WICHMANN, Gale A. • LI, Wenzong • LUND, Sean • JACKSON, Shaina J. • GARCIA DE GONZALO, Chantal V. • WARBINGTON, Hailley • Xiong, Yi • BORISOVA, Svetlana Alekseyevna

Assignees

Amyris Inc

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 Stevia rebaudiana kaurenoic acid hydroxylase. In some embodiments, the host cell further comprises 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 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 invention relates to genetically modified host-cell production of steviol glycosides, including rebaudioside D and rebaudioside M, by use of heterologous Stevia rebaudiana kaurenoic acid hydroxylase (KAH)/steviol synthase. It places the engineered KAH activity within a pathway context in which FPP is converted to steviol and then to glycosides, and identifies kaurenoic acid as a target substrate and steviol glycosides as the target products.

The disclosed subject matter relates to a kaurenoic acid hydroxylase polypeptide, according to SEQ ID NO: 1 residue numbering, comprising one, two, three, four, five, six, seven, eight, nine, or ten mutations selected from I166R, I153L, S158D, G306L, L232D, I333V, I350L, V316L, G447V, and M308L. The engineered polypeptide is capable of increased conversion of kaurenoic acid to steviol compared to the kaurenoic acid hydroxylase polypeptide of SEQ ID NO: 1.

The document further describes optional N-terminal domain swaps and chimeric KAH/heterologous amino terminal domain features, including an ER-targeting N-terminal transmembrane domain and examples such as ATR2 (Arabidopsis thaliana) and Aa.CPR (Artemisia annua). Across the host-cell framework, the document outlines the enzyme complement for steviol glycoside biosynthetic pathway steps, including KO/KAH, CPR, and UGTs, and references examples including UGT74G1, UGT76G1, UGT85C2, UGT91D, and UGTAD (UGT91C1/UGTSL2 examples).

Claims Coverage

The document’s independent claim specifies a kaurenoic acid hydroxylase polypeptide defined by SEQ ID NO:1 residue-numbered mutation content and characterized by increased conversion of kaurenoic acid to steviol. Across the consolidated content, one independent inventive feature is identified, with dependent refinements to mutation combinations and additional specified mutation positions.

Claim coverage centers on KAH polypeptides defined by SEQ ID NO:1 residue-numbered mutation combinations selected from a specified set, with the defining functional property of increased conversion of kaurenoic acid to steviol relative to the SEQ ID NO: 1 reference polypeptide.

Stated Advantages

Documented Applications