Recombinant Yeast Acetate Tolerance via Redox Balancing

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Solution Overview

Problem

Current methods for producing ethanol from cellulosic feedstocks face challenges due to microbial inhibitors like acetate, particularly in maintaining robustness and efficient anaerobic acetate consumption, which is limited in wild-type Saccharomyces cerevisiae strains.

Innovation Solution

A recombinant microbial host cell with genetic modifications to enhance the activity of proteins in multiple metabolic pathways: converting acetate into ethanol, importing glycerol, converting C5 carbohydrates into ethanol, and natively producing glycerol, using heterologous proteins like acetylating acetaldehyde dehydrogenase, glycerol facilitators, and xylose isomerase to improve ethanol production and robustness.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If AADH is introduced to enable anaerobic acetate conversion, then acetate consumption capability is improved, but cellular robustness deteriorates

Engineering Contradiction:

Improveacetate consumption capabilityVSAvoidcellular robustness

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent changes the redox balance parameters by introducing NADH-producing pathways (xylose isomerase, xylulokinase, transketolase, transaldolase) to compensate for the redox imbalance caused by AADH expression, thereby maintaining cellular robustness while enabling acetate consumption

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The engineered yeast strain performs multiple functions simultaneously: it ferments glucose, consumes acetate, ferments xylose, and produces ethanol, making the system universally applicable to cellulosic hydrolysates with multiple substrates and inhibitors

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Productivity

If glycerol production is abolished to increase ethanol titer, then ethanol production is improved, but cellular robustness deteriorates

Engineering Contradiction:

Improveethanol titerVSAvoidcellular robustness

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

Instead of completely abolishing glycerol production, the patent uses partial suppression combined with alternative NADH-producing pathways, achieving sufficient ethanol production while maintaining enough glycerol for cellular protection and robustness

Inventive Principle:

Principle #16Partial or excessive action

3Productivity

If multiple genetic modifications are introduced to enhance metabolic pathways, then ethanol production and acetate consumption are improved, but system complexity increases

Engineering Contradiction:

Improveethanol production efficiencyVSAvoidgenetic modification complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent segments the metabolic engineering into distinct functional modules: acetate activation (ACS), redox balancing (XYL1, XKS, TKL, TAL), and ethanol production (ADH), allowing independent optimization and characterization of each pathway component

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent uses heterologous enzymes from S. stipitis (XYL1, XKS, TKL, TAL) as intermediary components to bridge the metabolic gap between glucose/xylose fermentation and acetate consumption, providing a modular solution that can be transferred between strain backgrounds

Inventive Principle:

Principle #24Intermediary (Mediator)

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 approach enables efficient anaerobic acetate consumption and increased ethanol production while maintaining cellular robustness, independent of specific co-substrates, enhancing the conversion of acetate and C5 carbohydrates into ethanol.

Implementation Method 1

introducing a heterologous NADH-specific acetylating acetaldehyde dehydrogenase (AADH) makes it thermodynamically possible for yeasts to anaerobically convert acetate into ethanol during glucose fermentation

Methodology Applied

Scientific EffectFermentation: Fermentation

Implementation Method 2

increasing the activity of one or more proteins that function in a second metabolic pathway to import glycerol in the recombinant microbial host cell

Methodology Applied

Scientific EffectFacilitated diffusion: Diffusion

Implementation Method 3

converting a C5 carbohydrate into the alcohol in the microbial host cell

Methodology Applied

Scientific EffectIsomerization:

Data Source

PatentUS11739338B2Acetate toxicity tolerance in recombinant microbial host cells

Publication Date: 2023.08.29 DANSTAR FERMENT AG

AI summary

Acetate is a potent microbial inhibitor which can affect the performance of yeast in ethanolic fermentation. The present disclosure provides a recombinant microbial host cell having (i) a first genetic modification for increasing the activity of one or more proteins that function in a first metabolic pathway to convert acetate into an alcohol in the microbial host cell; (ii) a second genetic modification for increasing the activity of one or more proteins that function in a second metabolic pathway to import glycerol in the recombinant microbial host cell (iii) a third genetic modification for increasing the activity of one or more proteins that function in a third metabolic pathway to convert a C5 carbohydrate into ethanol in the microbial host cell. The recombinant microbial host cell comprises and natively expresses native proteins that function in a fourth native metabolic pathway to produce glycerol in the microbial host cell.