Recombinant Yeast NADH Reduction for Ethanol Yield

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Solution Overview

Problem

Xylose-assimilating yeast strains introduced with the xylose isomerase gene have insufficient ethanol fermentation ability, resulting in low ethanol production efficiency.

Innovation Solution

A recombinant yeast is developed by introducing the xylose isomerase gene and modifying the acetohydroxy acid reductoisomerase enzyme to reduce NADH production, either by lowering its activity, expression level, or introducing a mutant form with enhanced NADP+ dependence, thereby improving xylose assimilation and ethanol yield.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If the xylose isomerase gene is introduced to enable xylose utilization, then xylose assimilation ability is improved, but ethanol fermentation ability deteriorates

Engineering Contradiction:

Improvexylose assimilation abilityVSAvoidethanol fermentation ability

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The invention changes the cofactor preference parameter of acetohydroxy acid reductoisomerase from NAD+-dependent to NADP+-dependent through site-directed mutagenesis. This parameter change resolves the contradiction by redirecting NADH consumption away from the mutated enzyme toward acetate formation, thereby improving both xylose assimilation and ethanol fermentation simultaneously

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If acetaldehyde dehydrogenase is introduced to consume excessive NADH, then xylose assimilation is improved, but ethanol production efficiency deteriorates

Engineering Contradiction:

Improvexylose assimilationVSAvoidethanol production efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The invention extracts the NADH consumption function from the acetate metabolism pathway by mutating acetohydroxy acid reductoisomerase to use NADP+ instead of NAD+. This separates the NADH consumption needed for xylose assimilation from the acetate formation pathway, allowing ethanol production to proceed efficiently without excessive NADH consumption interfering with the process

Inventive Principle:

Principle #2Taking out (Extraction)

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 recombinant yeast exhibits enhanced xylose assimilation and ethanol production efficiency, achieving a remarkable improvement in ethanol yield in xylose-containing media.

Implementation Method 1

xylose isomerase (XI), which is an isomerase that converts xylose into xylulose

Methodology Applied

Scientific EffectIsomerization:

Implementation Method 2

a production of NADH as a result of the enzymatic reaction of acetohydroxy acid reductoisomerase is lowered

Methodology Applied

Scientific EffectEnzymatic reaction: Enzyme

Implementation Method 3

method for producing ethanol comprising a step of culturing the recombinant yeast in a xylose-containing medium to perform ethanol fermentation

Methodology Applied

Scientific EffectFermentation: Fermentation

Data Source

PatentUS10017788B2Recombinant yeast and method for producing ethanol using the same

Publication Date: 2018.07.10 TOYOTA JIDOSHA KK

AI summary

The invention is intended to improve xylose assimilation ability and ethanol fermentation ability in a xylose-assimilating yeast into which a xylose isomerase gene has been introduced. The amount of NADH produced by the recombinant yeast into which the xylose isomerase gene had been introduced as a result of the enzymatic reaction of acetohydroxy acid reductoisomerase is lowered.