Biosynthesis Of L-Alanine From Cis-Butenedioic Anhydride Catalyzed By A Triple-Enzyme Cascade Via A Genetically Modified Strain

In industry, l-alanine is biosynthesized using fermentation methods or catalyzed from l-aspartic acid by aspartate beta-decarboxylase (ASD). In this study, a triple-enzyme system was developed to biosynthesize l-alanine from cis-butenedioic anhydride, which was cost-efficient and could overcome the shortcomings of fermentation. Maleic acid formed by cis-butenedioic anhydride dissolving in water was transformed to l-alanine via fumaric acid and l-asparagic acid catalyzed by maleate isomerase (MaiA), aspartase (AspA) and ASD, respectively. The enzymatic properties of ASD from different origins were investigated and compared, as ASD was the key enzyme of the triple-enzyme cascade. Based on cofactor dependence and cooperation with the other two enzymes, a suitable ASD was chosen. Two of the three enzymes, MaiA and ASD, were recombinant enzymes cloned into a dual-promoter plasmid for overexpression; another enzyme, AspA, was the genomic enzyme of the host cell, in which AspA was enhanced by a T-7 promoter. Two fumarases in the host cell genome were deleted to improve the utilization of the intermediate fumaric acid. The conversion of whole-cell catalysis achieved 94.9% in 6 h, and the productivity given in our system was 28.2 g (L h)(-1), which was higher than the productivity that had been reported. A catalysis-extraction circulation process for the synthesis of l-alanine was established based on high-density fermentation, and the wastewater generated by this process was less than 34% of that by the fermentation process. Our results not only established a new green manufacturing process for l-alanine production from cis-butenedioic anhydride but also provided a promising strategy that could consider both catalytic ability and cell growth burden for multi-enzyme cascade catalysis.