Genetically Modified Yeast for Gamma-Decalactone Biosynthesis

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Solution Overview

Problem

Current methods for producing γ-decalactone in fermented beverages, such as beer and wine, are limited by the need for fatty acid supplementation, which poses cost and regulatory challenges, and result in low concentrations below the human detection threshold, failing to effectively enhance stone fruit flavors.

Innovation Solution

Genetically modified yeast cells expressing a heterologous gene encoding oleate 12-hydroxylase activity, along with additional genes for deregulated transcription factors and enzymes like acyl-CoA desaturase 1 and alcohol-O-acyltransferase, capable of producing increased levels of γ-decalactone without supplementation, thereby enhancing stone fruit flavors.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If fatty acid supplementation is used to produce γ-decalactone in fermented beverages, then γ-decalactone production is enabled, but production costs increase and regulatory challenges arise

Engineering Contradiction:

Improveγ-decalactone concentrationVSAvoidproduction cost and regulatory compliance

Core Design Contradiction:

Quantity of substanceVSEase of manufacture

Solution Approach 1:

The yeast cells are genetically modified to express oleate 12-hydroxylase and other enzymes, enabling them to autonomously synthesize γ-decalactone from endogenous fatty acids without requiring external supplementation. The system uses its own metabolic resources to produce the desired flavor compound, eliminating the need for costly exogenous fatty acid additions and associated regulatory compliance issues.

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The invention changes the biochemical parameters of the yeast by introducing heterologous genes (oleate 12-hydroxylase, acyl-CoA desaturase 1, alcohol-O-acyltransferase) and deregulating transcription factors (ADR1, PIP2, OAF1, OAF3). This genetic modification alters the yeast's metabolic capabilities, enabling it to produce γ-decalactone at detectable concentrations using its own fatty acid metabolism rather than requiring external supplementation.

Inventive Principle:

Principle #35Parameter changes

2Quantity of substance

If conventional fermentation methods are used, then fermented beverages are produced, but γ-decalactone concentrations remain below human detection threshold

Engineering Contradiction:

Improveγ-decalactone concentrationVSAvoidflavor enhancement effectiveness

Core Design Contradiction:

Quantity of substanceVSDifficulty of detecting and measuring

Solution Approach 1:

The yeast cells are pre-engineered with the complete enzymatic pathway for γ-decalactone biosynthesis before fermentation begins. The heterologous genes and deregulated transcription factors are introduced in advance, allowing the yeast to immediately produce γ-decalactone at enhanced concentrations during fermentation, rather than relying on trace amounts that naturally occur or would require post-fermentation addition.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The genetically modified yeast continuously produces γ-decalactone throughout the fermentation process through the expressed oleate 12-hydroxylase and other enzymes. The deregulated transcription factors ensure sustained high-level expression of the biosynthetic pathway, maintaining continuous production of the flavor compound at concentrations above the human detection threshold throughout the fermentation period.

Inventive Principle:

Principle #20Continuity of useful action

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The modified yeast cells produce γ-decalactone at concentrations above the odor threshold, effectively enhancing the stone fruit flavor profiles in fermented beverages without the need for fatty acid supplementation, addressing the limitations of existing methods.

Implementation Method 1

a heterologous gene encoding an enzyme having oleate 12-hydroxylase activity

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 2

enzyme having oleate 12-hydroxylase activity

Methodology Applied

Scientific EffectHydroxylation: Oxidation

Implementation Method 3

enzymes like acyl-CoA desaturase 1

Methodology Applied

Scientific EffectDesaturation:

Implementation Method 4

alcohol-O-acyltransferase

Methodology Applied

Scientific EffectTransferase reaction:

Implementation Method 5

capable of producing a fermented product

Methodology Applied

Scientific EffectFermentation: Fermentation

Data Source

PatentUS20240400953A1Methods and compositions for gamma-decalactone biosynthesis in fermented beverages

Publication Date: 2024.12.05 BERKELEY BREWING SCI INC

AI summary

Provided herein are genetically modified yeast cells that recombinantly express a gene encoding a fatty acid hydroxylase (FAH) enzyme, such as an oleate 12-hydroylase, and produce y-decalactone levels above an odor-threshold. Also provided herein are genetically modified yeast cells that recombinantly express genes encoding a fatty acid hydroxylase (FAH) enzyme, and one or more additional genes, such as an acyl-CoA desaturase 1 (OLE1) enzyme, a deregulated transcription factor, and/or an alcohol-O-acyltransferase (AAT) enzyme. Also provided are methods of producing fermented beverages and compositions comprising ethanol using the genetically modified yeast cells described herein.