Introduction of glycerol utilization system and improvement of succinate transporter system through metabolic engineering of Mannheimia succiniciproducens

Abstract

Glycerol has been a by-product from biodiesel industry. Its economical viability and natural potential as a carbon source have motivated engineering of Mannheimia succiniciproducens for the succinic acid production through glycerol fermentation; succinic acid is a dicarboxylic precursor for industrially important chemicals. We describe in silico analysis with genome-scale M. succiniciproducens metabolic model to understand the metabolic flux capacity of fermentative products including succinic acid and undesired biochemical for several carbon sources, and to confirm the effects of glycerol as a suitable carbon source for succinic acid production rationally and systematically. The rate of glycerol utilization in M.succiniciproducens is too slow to directly employ it as an industrial strain. Prior to metabolic engineering of M.succiniciproducens, we performed its metabolic adaptive evolution on glycerol for 50 days to obtain mutant strains in which both cell growth and glycerol utilization were increased. We also proposed several gene overexpression strategies to enhance the utilization rate of glycerol based on the metabolic pathway of the wild type M.succiniciproducens MBEL55E, and conducted wet experiments required to construct a new metabolic pathway of glycerol. Fed-batch fermentations of an enhanced recombinant strain of M.succiniciproducens using glycerol as a carbon source revealed remarkable improved succinic acid productivity. In addition, we achieved homo-production of succinic acid without by-products through the entire fermentation, thus facilitating the purification of succinic acid. Usually, at the final step of fed-batch cultivation, severe inhibition of cell growth has been observed by the accumulation of intracellular succinic acid and redox-imbalance. In this regard, we performed gene knockout studies of three dicarboxylate transporters selected from genome annotation data, so as to obtain an acid-tolerant strain and elucidate the function of ...