Microbial production of rotundone

Inventors

PHILIPPE, Ryan N. • PARAYIL KUMARAN, Ajikumar • Santos, Christine Nicole S. • Donald, Jason • Sarria, Stephen

Assignees

Manus Bio Inc

Abstract

The present disclosure provides methods and compositions for producing rotundone. In various aspects, the present disclosure provides enzymes, polynucleotides encoding said enzymes, and recombinant microbial host cells (or microbial host strains) for the production of rotundone. In some embodiments, the present disclosure provides microbial host cells for producing rotundone at high purity and/or yield, from either enzymatic transformation of α-guaiene, or from sugar or other carbon source. The present disclosure further provides methods of making products containing rotundone, including flavor or fragrance products, among others.

Core Innovation

The invention relates to microbial production of rotundone. A microbial host cell produces rotundone by expressing a heterologous α-guaiene synthase enzyme (αGTPS) and a heterologous α-guaiene oxidase enzyme (αGOX). The biosynthetic rationale connects α-guaiene formation and subsequent oxidation to rotundone, including an α-guaiene → rotundone transformation pathway and intermediate consideration such as rotundol.

The microbial system optionally incorporates additional enzyme activities to support generation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) from carbon sources, and then provides a pathway toward farnesyl diphosphate (FPP) and α-guaiene. The host cell may include engineered pathway elements for the methylerythritol phosphate (MEP) pathway and/or the mevalonic acid (MVA) pathway, and the invention discusses coupling to oxidations that can reduce formation of rotundol while increasing rotundone.

The microbial host cell and its enzymes are further defined by sequence-identity constraints. In particular, the αGOX enzyme is defined by an amino acid sequence that is at least 80% identical to SEQ ID NO: 51 or SEQ ID NO: 52. The disclosure also includes experimental screening of αGTPS variants and co-expression concepts for αGOX systems, with rotundone confirmation by GC/MS and mention of alternative oxidation systems.

Claims Coverage

The independent claim coverage focuses on a microbial host cell for producing rotundone, with inventive features centered on heterologous expression of αGTPS and αGOX and a defined αGOX sequence-identity requirement. The identified independent claim includes two core inventive features, with dependent claims refining host type, sequence-identity thresholds, and expression or recovery arrangements.

Across the independent claim, the inventive concept is a rotundone-producing microbial host cell that expresses heterologous αGTPS and αGOX, together with an αGOX amino-acid sequence-identity requirement (at least 80% identical to SEQ ID NO: 51 or SEQ ID NO: 52). Dependent claims further constrain sequence identity, select host organisms, and refine co-expression and recovery features.

Stated Advantages

Documented Applications