Construction and Application of a Multienzyme System for Synthesis of L-malate

This study aimed to develop a multienzymatic system for synthesis of L-malate. First, recombinant Escherichia coli strains were constructed expressing maleic acid cis-trans isomerase (MaiA) or fumarase C (FumC) from different sources. Serratia marcescens MaiA (SMaiA) and E. coli FumC (ECFumC) showed good catalytic performance. Next, six co-expression systems for SMaiA and ECFumC were constructed. E. coli BL21 (DE3)-pRSF_Duet-1-ecfumC-smaiA (named strain pFM2) had the highest L-malate catalytic activity. In 7-L fed-batch fermentation, the SMaiA and ECFumC activities of strain pFM2 wet cells were 43.4 and 154.5 U/g, respectively, 2.4- and 10.7-fold the values that were obtained in shaken flasks. Finally, a whole-cell catalytic process was established for the production of L-malate by strain pFM2 with maleate as the substrate. When the dose of pFM2 wet cells was 0.5 g/100 mL and 1 mol/L maleate was the substrate, the catalytic process was completed within 4 h. Notably, the intermediate fumarate was almost absent during the conversion process. The concentration of L-malate reached 143.8 g/L with a yield of 0.60 g/(L·min). The molar conversion rate of the substrate was 98.4%. These findings lay a foundation for the industrial application of multienzymatic synthesis of L-malate.