Engineered Zymomonas Mobilis for 2,3-Butanediol Production
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Solution Overview
Problem
Native Zymomonas mobilis strains are limited in their ability to produce 2,3-butanediol and produce significant amounts of ethanol, which is toxic and difficult to purify, while attempts to knockout the pdc gene have failed to redirect carbon flux effectively for 2,3-butanediol production.
Innovation Solution
Engineered Zymomonas mobilis strains with exogenous genes for 2,3-butanediol production, including acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase, integrated into the chromosome or on a plasmid, knocking out the pyruvate decarboxylase gene to eliminate ethanol production and enhance 2,3-butanediol titers, using glucose and xylose as carbon sources.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If exogenous genes for 2,3-butanediol production are introduced into Zymomonas mobilis, then 2,3-butanediol titer is improved, but ethanol production increases which is toxic and difficult to purify
Solution Approach 1:
The pyruvate decarboxylase gene (pdc), which is responsible for ethanol production in Zymomonas mobilis, is knocked out or inactivated. This extraction of the harmful ethanol-producing pathway allows the engineered strain to produce 2,3-butanediol without generating toxic ethanol byproducts, directly resolving the contradiction between improving 2,3-butanediol titer and avoiding ethanol toxicity
Solution Approach 2:
The metabolic pathway parameters are fundamentally changed by introducing exogenous genes (acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase) and simultaneously knocking out the pdc gene. This parameter change redirects carbon flux from ethanol production to 2,3-butanediol production, achieving high 2,3-butanediol titers while eliminating ethanol generation
2Object-generated harmful factors
If pyruvate decarboxylase gene is knocked out to eliminate ethanol production, then ethanol toxicity is reduced, but carbon flux redirection for 2,3-butanediol production is insufficient
Solution Approach 1:
Before attempting to produce 2,3-butanediol, the pyruvate decarboxylase gene is preemptively knocked out to eliminate the competing ethanol production pathway. This preliminary action ensures that all carbon flux from glucose metabolism is directed toward 2,3-butanediol production through the introduced exogenous genes, maximizing productivity without ethanol interference
Solution Approach 2:
The engineered Zymomonas mobilis strain achieves multi-functionality by combining the knockout of pdc gene with the introduction of complete 2,3-butanediol biosynthesis genes (als, aldc, bdh). This universal engineering approach simultaneously eliminates ethanol production and establishes efficient 2,3-butanediol production, resolving the contradiction between reducing harmful factors and maintaining productivity
3Productivity
If native Zymomonas mobilis is used for fermentation, then rapid ethanol production is achieved, but 2,3-butanediol production capability is limited
Solution Approach 1:
The metabolic parameters of Zymomonas mobilis are fundamentally changed by knocking out the pdc gene and introducing exogenous 2,3-butanediol biosynthesis genes. This parameter change transforms the organism from an ethanol-producing strain to a 2,3-butanediol-producing strain, achieving both rapid production (maintaining Z. mobilis's metabolic speed) and new product capability (2,3-butanediol production)
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
Achieves high titers of 2,3-butanediol production, up to 120 g/L, with reduced ethanol production, simplifying downstream processes and enabling efficient use of biomass-derived sugar streams, and allowing for further catalytic upgrading to hydrocarbon fuels.
Implementation Method 1
Zymomonas mobilis is a gram-negative, facultative anaerobic microorganism. The optimal temperature for growth of most strains of Z. mobilis is between 25 and 30° C., and optimal pH is about 5.0. Z. mobilis has the ability to rapidly and efficiently produce ethanol.
Implementation Method 2
the non-naturally occurring Zymomonas species has exogenous genes that encode for acetolactate synthase (ALS), acetolactate decarboxylase (ALDC), and butanediol dehydrogenase (BDH)
Data Source
AI summary
Non-naturally occurring Zymomonas strains useful for the production of 2,3-butanediol are provided.