Abstract:[Objective] Heterofermentative lactic acid bacteria (LAB) can convert fructose to mannitol efficiently using the intracellular mannitol dehydrogenase enzyme, but fructose as a substrate is relatively expensive and not suitable for industrial production. In order to reduce cost, inexpensive substrates must be selected. Sucrose, abundantly in nature, is relatively cheap and can be used by recombinant Escherichia coli to produce mannitol. Sucrose hydrolase and mannitol dehydrogenase are the key enzymes to convert sucrose into mannitol. This paper was focused on the construction of sucrose hydrolase and mannitol dehydrogenase co-expression strain. [Methods] The target genes sacA and mdh, 1 502 bp and 1 032 bp respectively, were derived from the Lactobacillus plantarum and Lactobacillus buchneri. The genes were cloned into the expression vector pET-28a(+), resulting in the generation of the recombinant expression vector pET-28a-sacA-mdh, which was transformed into the E. coli BL21. The expression of the target protein was analysed by SDS-PAGE and the enzyme activity measurement. [Results] SDS-PAGE showed that the molecular weight of the expressed protein was 55.1 kD and 37.8 kD, respectively, which was consistent with the expected molecular weight, demonstrating the expression of genes sacA and mdh. The enzyme activity of sucrose hydrolase and mannitol dehydrogenase was 25.78 U/mL and 14.56 U/mL, respectively. The concentration of mannitol reached 45.19 g/L and the conversion was 37.66% after optimizing the fermentation conditions. [Conclusion] Compared with the mannitol production by LAB fermentation using sucrose, the yield was increased six-fold, with the advantages of short fermentation period and high stability. The successful construction of recombinant strain laid the foundation for the industrial production of mannitol.
