Engineered GPD Enzyme for Butanol Yield
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Solution Overview
Problem
Current methods for producing butanol, such as chemical synthesis from petrochemicals, are expensive and environmentally unfriendly, and biological production faces yield losses due to glycerol production, which diverts carbon and reducing equivalents from glycolysis.
Innovation Solution
Engineering glycerol-3-phosphate dehydrogenase (GPD) enzymes with specific substitutions and recombinant microorganisms to optimize NADH and NADPH affinity, reducing glycerol production while maintaining growth requirements, and using these organisms in butanol biosynthetic pathways to enhance butanol yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If glycerol production pathway is inactivated by deleting GPD genes, then butanol yield is improved, but microorganism growth and osmoprotection are compromised
Solution Approach 1:
The patent extracts the harmful glycerol production function from the GPD enzyme by deleting the native GPD1 and GPD2 genes, while separately providing the essential growth function through a heterologous GPD gene from E. coli that does not compete with the butanol pathway for substrates
Solution Approach 2:
The patent segments the glycerol production function from the butanol production pathway by using a heterologous GPD gene that is spatially and functionally separated from the native yeast GPD genes, allowing independent optimization of each pathway
2Reliability
If native GPD enzymes are used, then growth and osmoprotection are maintained, but carbon and reducing equivalents are diverted to glycerol production
Solution Approach 1:
The patent introduces a heterologous GPD gene from E. coli as an intermediary enzyme that performs the essential glycerol production function without competing with the butanol pathway, since it uses different substrate availability patterns and is not regulated by the same mechanisms as native yeast GPD enzymes
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
Increased butanol production with reduced glycerol levels, improving the butanol to glycerol molar ratio and overall yield in microbial fermentation processes.
Implementation Method 1
engineered glycerol-3-phosphate dehydrogenase (GPD) enzyme... catalyzes the conversion of dihydroxyacetone phosphate to glycerol-3-phosphate
Implementation Method 2
fermentative production of butanol... microbial fermentation processes
Data Source
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AI summary
Provided herein are glycerol-3 -phosphate dehydrogenase (GPD) enzymes with increased KM for NADH and GPD enzymes with substantially the same affinity for NADH and NADPH and/or are feedback inhibited by glycerol-3-phosphate. Also provided herein are recombinant microorganisms comprising a heterologous gene encoding GPD and a deletion or disruption in an endogenous gene encoding GPD. Also provided are recombinant microorganisms comprising a heterologous gene encoding GPD and a butanol biosynthetic pathway. Further provided are methods of producing butanol comprising providing the recombinant microorganisms described herein and contacting the recombinant microorganism with at least one fermentable carbon substrate under conditions wherein butanol is produced.