Genetic optimization of metabolic pathways in Phocaeicola vulgatus for enhanced succinate production

Abstract

Due to the growing challenges posed by climate change, the significance for alternative production methods to the conventional petrochemical production of bulk chemicals and the utilization of renewable materials is rapidly increasing. This includes the use of microbial fermentation and enzyme-based technologies to convert natural resources to key platform chemicals like succinate. As a salt of succinic acid, succinate serves as a fundamental component in various compounds across the food, chemical, and pharmaceutical sectors. Bacteroides and Phocaeicola species are widely distributed and prevalent members of the human gut microbiota and possess enzyme systems for the metabolization of complex plant polysaccharides. As a natural succinate producer, P. vulgatus (formerly Bacteroides vulgatus), seems to be a promising candidate for succinate production. This study aimed to enhance succinate yields in P. vulgatus by channeling the metabolic carbon flow in favor of succinate formation. The methodology involved employing a markerless gene deletion method, as described in earlier studies, in combination with the shuttle vector pG106, previously identified as a suitable overexpression system for P. vulgatus. Specifically, the deletion of the methyl-malonyl Co-A-mutase (Bvu_0309-0310) resulted in a 100% increase of succinate production in P. vulgatus, as metabolization to propionate was effectively blocked. Furthermore, deletion of the lactate-dehydrogenase (Bvu_2499) and the pyruvate-formate-lyase (Bvu_2880) has successfully eliminated the formation of fermentative end products lactate and formate. Moreover, the overexpression of the transketolase (Bvu_2318) from the pentose-phosphate shunt contributed to an additional increase in succinate production. In summary, a total increase of the succinate yield by 150% compared to the wildtype strain has been achieved through a combination of genetic deletions and overexpression. Additionally, the formation of undesired fermentation byproducts has been successfully prevented, highlighting the potential of P. vulgatus as an efficient succinate producer with applications in sustainable bioproduction processes.