Abstract:[Background] L-malic acid is widely used in biomedicine, food, and chemical industries. However, the wild-type Escherichia coli hardly accumulates malic acid. The T7 expression system has the ability to strictly control and induce expression at a high level, while its application is influenced by the presence of T7 RNA polymerase. [Objective] To construct a high-yield L-malic acid-producing chassis strain based on the T7 expression system. [Methods] Firstly, multi-gene knockouts in the malate consumption pathway of E. coli MG1655 were performed. Then, the gene expression cassette of T7 RNA polymerase was introduced into the chromosome of E. coli MG1655, and thus a high-yield L-malic acid-producing chassis strain MG1655W(DE3) was successfully constructed. [Results] The expression of the reporter gene in the chassis strain increased the fluorescence intensity to 114.0% compared with that of E. coli BL21(DE3). The L-malic acid accumulation of the recombinant strain was 12.9 times that of the original strain, and further overexpression of malate dehydrogenase via the T7 expression system increased the L-malic acid production to 26.7 times that of the original strain. [Conclusion] We successfully constructed a high-yield L-malic acid-producing chassis strain MG1655W(DE3) that could utilize the T7 expression system. This strain shows high application values in the production of metabolites such as malic acid and provides a new strategy for constructing excellent chassis strains for malic acid production.
