Metabolic engineering of Escherichia coli for malate production with a temperature sensitive malate dehydrogenase

Malate has been widely used as a fine chemical and its production by microbial fermentation has attracted significant attention recently. Succinate producer E. coli SBS550MG-cms243/pHL413 K was engineered to produce malate through a three stage fermentation strategy in this study. Firstly, we examined the effects of employing a temperature sensitive malate dehydrogenase (Mdh) to limit the consumption of the product, two were tested for malate accumulation from succinate aerobically, and the RvMdh from Rhodomicrobium vannielii ATCC 17100 was found to perform better than RcMdh from Rhodobacter capsulatus SB 1003. Malate production was further improved by overexpression of native Fumarase C and deactivation of malic enzymes SfcA and MaeB. Up to 196.9 mM (26.4 g/L) malate can be produced within 48 h by using 30 g/L glucose as a carbon source, the malate yield is up to 1.25 mol/mol fed glucose (0.93 g/g) when 40 g/L glucose was used. In this study, a temperature sensitive Mdh was employed for malate production, which enables more efficient malate production through a three stage fermentation process and this approach can be combined with other developed strategies.