Recombinant Yeast With Anaerobic TKL Expression for Lower Glycerol
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Solution Overview
Problem
Existing recombinant yeast cells used for ethanol production face challenges in reducing glycerol production under anaerobic conditions, leading to reduced overall sugar conversion to ethanol and increased waste treatment costs, especially under high dry solids and high temperature conditions.
Innovation Solution
Promoting transketolase activity in recombinant yeast cells using a specific promoter (TKL promoter) with an anaerobic/aerobic expression ratio of 2 or more, combined with heterologous nucleic acid sequences encoding ribulose-1,5-phosphate carboxylase/oxygenase (Rubisco) and phosphoribulokinase (PRK), to enhance redox balancing and reduce glucose accumulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional yeast cells are used for ethanol production under anaerobic conditions, then ethanol is produced, but substantial amounts of glycerol are formed as a by-product reducing overall sugar conversion efficiency
Solution Approach 1:
The invention changes the metabolic parameters of the yeast cell by introducing heterologous nucleic acid sequences encoding for transketolase, Rubisco, and PRK. This genetic modification alters the redox metabolism pathway, enabling the yeast to convert more sugar to ethanol while reducing glycerol formation by changing the NADH/NAD+ balance through enhanced pentose phosphate pathway activity.
Solution Approach 2:
The invention introduces intermediary enzymes (transketolase, Rubisco, and PRK) that mediate metabolic transformations. These heterologous enzymes act as intermediaries to redirect metabolic flux from glycerol production toward ethanol production by providing alternative pathways for NADH reoxidation and carbon flow.
2Reliability
If transketolase is promoted under aerobic conditions, then redox balancing is improved, but transketolase expression is insufficient under anaerobic conditions where it is most needed for ethanol production
Solution Approach 1:
The invention applies dynamic gene expression control by using the anaerobically inducible PGK1 promoter to drive transketolase expression. This promoter dynamically responds to oxygen availability, strongly inducing transketolase expression under anaerobic conditions when it is most needed for ethanol production, while maintaining lower expression under aerobic conditions.
Solution Approach 2:
The invention changes the expression parameter of transketolase by coupling it to an anaerobic promoter (PGK1). This creates condition-dependent expression levels that match the metabolic needs: high expression under anaerobic conditions to maximize ethanol production and redox balancing, and reduced expression under aerobic conditions to avoid unnecessary metabolic burden.
3Productivity
If high dry solids and high temperature conditions are applied to increase fermentation efficiency, then productivity improves, but yeast cell robustness and performance deteriorate
Solution Approach 1:
The invention changes the metabolic parameters of the yeast cell through genetic modification, introducing heterologous pathways that improve redox balancing and reduce by-product formation. This metabolic reprogramming enhances the yeast's ability to maintain performance under stressful fermentation conditions including high dry solids and elevated temperatures.
Solution Approach 2:
The heterologous enzymes (transketolase, Rubisco, PRK) act as intermediary components that mediate metabolic adjustments in response to environmental stress. These intermediaries provide alternative metabolic routes that help maintain redox balance and cellular function under the challenging conditions of high dry solids and high temperature fermentation.
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 cells maintain robustness and continued ethanol production performance even under high dry matter content and elevated temperatures, achieving low residual glucose concentrations at the end of fermentation.
Implementation Method 1
Promoting transketolase activity in recombinant yeast cells using a specific promoter (TKL promoter) with an anaerobic/aerobic expression ratio of 2 or more
Implementation Method 2
During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via alcoholic fermentation
Implementation Method 3
the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction
Implementation Method 4
the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate to ethanol via NAD+-dependent alcohol dehydrogenase
Implementation Method 5
Glycerol formation is initiated by reduction of the glycolytic intermediate dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (glycerol-3P), a reaction catalyzed by NAD+-dependent glycerol 3-phosphate dehydrogenase
Implementation Method 6
Subsequently, the glycerol 3-phosphate formed in this reaction is hydrolysed by glycerol-3-phosphatase to yield glycerol and inorganic phosphate
Data Source
AI summary
A recombinant yeast cell functionally expressing:a) a heterologous nucleic acid sequence encoding a protein having ribulose-1,5-phosphate carboxylase/oxygenase activity (EC4.1.1.39; Rubisco), a heterologous nucleic acid sequence encoding a protein having phosphoribulokinase activity (EC2.7.1.19; PRK) and optionally one or more nucleic acid sequences encoding for molecular chaperones for the protein having ribulose-1,5-phosphate carboxylase/oxygenase activity; andb) a nucleic acid sequence encoding a protein having transketolase activity (EC 2.2.1.1), wherein the expression of the nucleic acid sequence encoding the protein having transketolase activity is under control of a promoter (the “TKL promoter”), which TKL promoter has an anaerobic/aerobic expression ratio for the transketolase of 2 or more.