Metabolic Engineering of Mannheimia succiniciproducens for Enhanced Succinic Acid Production

Abstract

Succinic acid is a four-carbon dicarboxylic acid synthesized as an intermediate of the tricarboxylic acid cycle and also as one of the mixed-acid fermentation products, which can be used many applications. Most of Succinic acid production is perfomed by petroleum-based process. But it has some problems such as limited source and causing environmental pollution. To solve these problems, recently many groups studied bio-based process for succinic acid production by microbial fermentation. It is an impact techinique to resolve the problems caused by petroleum-based process. However, because of low productivity and expensive purification cost, It is unreasonable to construct bio-based process factory. Therefore, we have to develop a high productivity and homo succinic acid producer to commercialize in the world. Mannheimia succiniciproducens which is a capnophilic Gram-negative bacterium isolated from bovine rumen was able to produce a large amount of succinic acid as a major fermentation product under anaerobic conditions in the presence of $CO_2$. Based on complete genome sequence of M. succiniciproducens gene work studies were advanced to develop producing homo succinic acid strain. In the previous study, Our group constructed metabolically engineered LPK7(LdhA::Kmr PflB::Cmr Pta-ackA::Spr) and PALK(LdhA::Kmr Pta-ackA::Spr)strains. Two strains didn’t produce three major by-products such as lactic acid, formic acid and acetic acid when are formed during anaerobic fermentation of wild type strains under $CO_2$ -rich condition any more. But, large amounts of pyruvic acid is accumulated and cell growth is much slower than wild type strain owing to imbalanced metabolic fluxes. Here, we report the strategies for the metabolic engineering of M. succiniciproducens based on the genome sequence and construction of a homo succinic acid producer. To overcome these problems, ldhA and ackA knockout strain ALK(AckA::Cmr) was constructed by genome-based metabolic engineering. ...