Genetically Modified Yeast for Anaerobic Succinic Acid Fermentation
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Solution Overview
Problem
Current methods are unable to fermentatively convert glycerol into C4 dicarboxylic acids, such as succinic acid, using yeast, as they require aerobic conditions and supplemental additives, limiting their efficiency and applicability.
Innovation Solution
A genetically modified Saccharomyces yeast with blocked glycerol-3-phosphate pathway, expressing a heterologous glycerol uptake facilitator, glycerol dehydrogenase, dihydroxyacetone kinase, and a C4-dicarboxylic acid transporter, optimized for anaerobic conditions to produce succinic acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If yeast is used for glycerol fermentation, then production of C4-dicarboxylic acids is enabled, but aerobic conditions are required which limits fermentative capability
Solution Approach 1:
The patent changes the metabolic parameters of yeast by blocking the glycerol-3-phosphate pathway and introducing alternative pathways (DHA pathway and glyceraldehyde pathway) that enable fermentative metabolism under anaerobic conditions, transforming the yeast from aerobic to fermentative capability for glycerol utilization
Solution Approach 2:
The patent extracts and removes the limitation of aerobic requirement by eliminating the dependency on the glycerol-3-phosphate pathway and mitochondrial respiration, allowing the yeast to function under anaerobic conditions through alternative cytosolic pathways
2Productivity
If glycerol-3-phosphate pathway is used for glycerol degradation, then glycerol metabolism is enabled, but supplemental additives are required which reduces efficiency
Solution Approach 1:
The patent extracts and eliminates the requirement for supplemental additives by using alternative glycerol degradation pathways that do not depend on the glycerol-3-phosphate pathway, allowing growth on minimal media containing only glycerol as carbon source
Solution Approach 2:
The patent introduces heterologous pathway enzymes (glycerol dehydrogenase and dihydroxyacetone kinase from other organisms) to copy functional capability that bypasses the need for complex media supplements required by the native glycerol-3-phosphate pathway
3Productivity
If glycerol is fermented anaerobically, then fermentative production is achieved, but redox balance must be optimized which complicates metabolic engineering
Solution Approach 1:
The patent changes the redox parameters by introducing pathways that generate NADH in the cytosol (DHA pathway) and balancing it with NADH-consuming reactions (succinate production, lactate production, or ethanol production), achieving redox neutrality without complex regulatory mechanisms
Solution Approach 2:
The patent segments the metabolism into distinct functional modules: glycerol uptake, glycerol degradation via DHA pathway, NADH generation, and NADH consumption through succinate/lactate/ethanol production, allowing independent optimization of each module for redox balance
4Productivity
If C4-dicarboxylic acid production is enhanced, then product yield is improved, but export capability must be increased which requires additional genetic modifications
Solution Approach 1:
The patent introduces heterologous C4-dicarboxylic acid transporters as intermediary proteins that facilitate the export of succinic acid from the yeast cell, acting as a bridge between intracellular production and extracellular accumulation
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
Enables efficient, anaerobic production of succinic acid from glycerol, improving redox balance and export of C4-dicarboxylic acids, reducing the need for neutralizing compounds and enhancing metabolic pathways for redox neutrality.
Implementation Method 1
The first type relies on facilitated diffusion, requires no energy, and depends on glycerol facilitators
Implementation Method 2
a heterologous glycerol dehydrogenase catalyzing the oxidation of glycerol to dihydroxyacetone is expressed
Implementation Method 3
A key benefit of using glycerol as the sole or additional carbon source in industrial biotechnology is that glycerol catabolism yields more reduction equivalents per C-mole compared to glucose
Implementation Method 4
a heterologous dihydroxyacetone kinase is expressed or a native dihydroxyacetone kinase is overexpressed
Implementation Method 5
Enables efficient, anaerobic production of succinic acid from glycerol
Data Source
Figure 1
Figure 2A~2C
AI summary
The invention relates to a genetically modified yeast for fermenting glycerol. The aim of the invention is to improve the capability of yeast to ferment glycerol into C4 dicarboxylic acids. This is achieved by a genetically modified yeast cell of the genus Saccharomyces. The yeast cell is genetically modified in that i) the decomposition of glycerol into dihydroxyacetone phosphate via the glycerol-3-phosphate pathway is blocked, ii) a heterologous glycerol uptake facilitator protein is expressed, iii) a heterologous glycerol dehydrogenase that catalyzes the oxidation of glycerol into dihydroxyacetone is expressed or a native glycerol dehydrogenase that catalyzes the oxidation of glycerol into dihdroxyacetone is over-expressed, iv) a heterologous dihydroxyacetone kinase is expressed or a native dihydroxyacetone kinase is over-expressed, v) a heterologous C4 dicarboxylic acid transporter is expressed, vi) a heterologous glycerol dehydratase is expressed, and vii) a heterologous 1,3-PDO oxidoreductase is expressed.