Genetic optimization of the human gut bacterium Phocaeicola vulgatus for enhanced succinate production

Abstract The demand for sustainably produced bulk chemicals is constantly rising. Succinate serves as a fundamental component in various food, chemical, and pharmaceutical products. Succinate can be produced from sustainable raw materials using microbial fermentation and enzyme-based technologies. Bacteroides and Phocaeicola species, widely distributed and prevalent gut commensals, possess enzyme sets for the metabolization of complex plant polysaccharides and synthesize succinate as a fermentative end product. This study employed novel molecular techniques to enhance succinate yields in the natural succinate producer Phocaeicola vulgatus by directing the metabolic carbon flow toward succinate formation. The deletion of the gene encoding the methylmalonyl-CoA mutase (Δ mcm, bvu₀309-0310) resulted in a 95% increase in succinate production, as metabolization to propionate was effectively blocked. Furthermore, deletion of genes encoding the lactate dehydrogenase (Δ ldh, bvu₂499) and the pyruvate: formate lyase (Δ pfl, bvu₂880) eliminated the formation of fermentative end products lactate and formate. By overproducing the transketolase (TKT, BVU₂318) in the triple deletion mutant, succinate production increased from 3. 9 mmol/g dry weight in the wild type to 10. 9 mmol/g dry weight. Overall, succinate yield increased by 180% in the new mutant strain P. vulgatus Δ mcm Δ ldh Δ pfl pG106_ tkt relative to the parent strain. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus, and forms the basis for targeted genetic optimization. The increase of efficiency highlights the huge potential of P. vulgatus as a succinate producer with applications in sustainable bioproduction processes. Key points • Deleting methylmalonyl-CoA mutase gene in P. vulgatus doubled succinate production • Triple deletion mutant with transketolase overexpression increased succinate yield by 180% • P. vulgatus shows high potential for sustainable bulk chemical production via genetic optimization