Engineering and evolving glyoxylate utilization in Ethylmalonyl-CoA Pathway-deficient Methylobacterium extorquens PA1 for the production of crotonate from methanol
Abstract
The development of methylotrophic microbial platforms for the production of value-added compounds is a crucial step towards a methanol-based bioeconomy. Methanol is a promising substrate for the synthesis of such compounds as methods for its sustainable production continue to advance. One of the most straight-forward routes to green methanol includes the use of renewable energy to power electrolysis of water, generating green hydrogen that can subsequently be used for the hydrogenation of CO2. Moreover, methanol does not compete with the food industry and reduces the risk of contamination during fermentation. One promising organism capable of efficiently utilizing C1 substrates is the natural methylotroph Methylobacterium extorquens, one of the most well-studied organisms for methylotrophy. The central carbon metabolism of M. extorquens contains intermediates, most interestingly CoA esters in the Ethylmalonyl-CoA Pathway (EMCP), which could be harnessed for the production of value-added compounds using methanol as a substrate. However, rewiring of central carbon metabolism is tedious and comes with multiple drawbacks in strain performance. I therefore chose the M. extorquens PA1 strain, which grows faster on minimal medium and has a smaller genome compared to the more commonly used AM1 strain, benefiting genetic engineering work. To develop an EMCP-deficient strain of M. extorquens PA1, I introduced the heterologous glyoxylate shunt, which would allow to exploit the EMCP for the accumulation of CoA esters and the subsequent conversion to the carboxylic acid, here crotonate. To test this, the plasmid containing the glyoxylate shunt also harbored the thioesterase encoding gene for the YciA from E. coli, which catalyzes the reaction of crotonylCoA to crotonate. I further aimed to employ CRISPR interference (CRISPRi) constructs on plasmids with varying expression levels for the targeted knockdown of the competing Polyhydroxybutyrate (PHB) pathway in order to facilitate crotonate production. In summary, this work advanced the development of a potential production strain for EMCP-derived carboxylic acids and introduced a new approach for the downregulation of the competing PHB pathway – an alternative to the highly unstable genetic knockouts.
Review
Metadata
Contributors
Supervisor:
Erb, Tobias
Becker, Anke
Dates
Issued: 2026-02-18
Relations
Has part: New central carbon metabolic pathways in Alphaproteobacteria: from natural to synthetic metabolism; Francesca Severi; https://doi.org/10.17192/z2022.0099
Faculty
FB17:Biologie
Language
en
Keywords
Methylobacterium extorquensPolyhydroxybutyrateMetabolic EngineeringMethanolAdaptive Laboratory EvolutionCRISPR interference
DFG-subjects
2.21-02 - Mikrobielle Ökologie und Angewandte Mikrobiologie
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Schrodt, Alina: Engineering and evolving glyoxylate utilization in Ethylmalonyl-CoA Pathway-deficient Methylobacterium extorquens PA1 for the production of crotonate from methanol. : 2026-02-18.
License
Except where otherwised noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International