Eukaryotic Cell Acetate Detoxification via NAD+ Enzymes

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Solution Overview

Problem

Current methods for producing second-generation bioethanol from lignocellulosic biomass face challenges due to inhibitory effects of acetic acid and other toxic by-products, which limit the fermentation efficiency of eukaryotic cells, and existing detoxification processes are costly and inefficient.

Innovation Solution

Genetically modified eukaryotic cells expressing NAD+ dependent enzymes such as D-glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose dehydrogenase, gluconolactonase, and gluconate kinase, which enhance acetate conversion and ethanol production while reducing glycerol production.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Object-affected harmful factors

If chemical or biological detoxification procedures are used to remove acetic acid from hydrolysates, then the inhibitory effect of acetic acid is reduced, but the process becomes costly and fermentable substrate is lost

Engineering Contradiction:

Improveinhibitory effect of acetic acidVSAvoidcost and substrate loss

Core Design Contradiction:

Object-affected harmful factorsVSEase of manufacture

Solution Approach 1:

The yeast strain is genetically engineered to possess endogenous acetate-consuming pathways, enabling it to detoxify acetic acid in situ during fermentation without requiring external detoxification treatments. The cell uses its own metabolic machinery to convert acetic acid into useful fermentation products, making the system self-sufficient and eliminating the need for costly pretreatment steps

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The harmful acetic acid inhibitor is converted into a beneficial substrate that the engineered yeast can metabolize to produce ethanol and other fermentation products. By introducing pathways that allow acetate to serve as a carbon source, the system transforms the toxic byproduct into a valuable resource, simultaneously achieving detoxification and increased product yield

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

2Productivity

If Saccharomyces cerevisiae is used under anaerobic conditions, then ethanol fermentation occurs, but the cell cannot naturally consume acetic acid

Engineering Contradiction:

Improveethanol fermentationVSAvoidacetic acid accumulation

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The metabolic parameters of S. cerevisiae are fundamentally altered through genetic engineering to enable acetate consumption under anaerobic conditions. By introducing heterologous genes encoding enzymes for the Wood-Ljungdahl pathway and modifying existing metabolic pathways, the cell's physiological capabilities are changed to allow simultaneous ethanol production and acetate utilization in the absence of oxygen

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

Heterologous enzyme pathways serve as intermediaries to bridge the gap between S. cerevisiae's natural metabolism and the desired acetate-consuming capability. The introduced enzymes from other organisms act as catalysts that enable the yeast to process acetic acid through pathways not naturally present in Saccharomyces, facilitating the conversion of acetate to ethanol and other products

Inventive Principle:

Principle #24Intermediary (Mediator)

3Productivity

If genes involved in glycerol production are deleted to solve redox balance, then ethanol yield increases, but acetic acid tolerance does not improve

Engineering Contradiction:

Improveethanol yieldVSAvoidacetic acid tolerance

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The solution separates the two functions of redox balancing and acetate consumption into distinct metabolic pathways. While glycerol production genes are deleted to maintain ethanol yield, separate heterologous pathways are introduced that specifically handle acetate metabolism, allowing the cell to address acetate tolerance through a dedicated mechanism rather than relying on glycerol production

Inventive Principle:

Principle #1Segmentation

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 cells demonstrate improved acetate consumption and increased ethanol yield, reducing fermentation time and costs, making the process more efficient and cost-effective for biofuel production.

Implementation Method 1

Second generation bioethanol is produced from e.g. lignocellulosic fractions of plant biomass that is hydrolyzed into free monomeric sugars, such as hexoses and pentoses, for fermentation into ethanol

Methodology Applied

Scientific EffectFermentation: Fermentation

Implementation Method 2

expression of mhpF from E. coli, encoding for a NAD+-dependent acetylating acetaldehyde dehydrogenase, enabled anaerobic growth of a gpd1Δ gpd2Δ strain by coupling the reduction of acetate to acetaldehyde with NAD+ regeneration

Methodology Applied

Scientific EffectOxidation: Oxidation

Data Source

PatentUS11136600B2Eukaryotic cell with increased production of fermentation product

Publication Date: 2021.10.05 DANISCO US INC

AI summary

The present invention relates to a eukaryotic cell that is genetically modified comprising one or more heterologous gene encoding:a) D-glucose-6-phosphate dehydrogenase and/orb) 6-phosphogluconate dehydrogenase; and/orc) glucose dehydrogenase, gluconolactonase and gluconate kinase,wherein a), b) and glucose dehydrogenase in c) are NAD+ dependent.