1. College of Enology, Northwest Agriculture and Forestray University, Yangling 712100, China; 2. Institute of Microbiology，Chinese Academy of Sciences，Beijing 100080, China
Abstract：The key enzymes for malolactic fermentation (MLF) are malolactic enzyme and malate permease，coded by mleA gene and mleP gene respectively. The mle locus about 2.6 kb containing mleA and mleP genes, coming from an excellent Oenococcus oeni strain of SD-2a screened by College of Enology, NAFU, in China，was used together with PGK1 promoter and ADH1 terminator were ligated and inserted into YEp352 (yeast-Escherichia coli shuttle plasmid), which was named pYELmleAP. Yeast transformants were screened on SD/-Ura. Malolactic enzyme gene, one of the target genes, was detected in the yeast transformants by Dot blotting hybridization. The target protein of malolactic enzyme was expressed by SDS-PAGE detecting. After transformants were cultured in media containing L-malate for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. The results indicated that the functional expression of mleA gene was achieved in recombinants S. cerevisiae, turning L-malic acid into L-lactic acid. L-malate contents of the transformants showed extra significant difference with the control ones in t test, while L-lactic contents of the transformants showed significant difference with the control ones. In the culture supernatants adding L-malate, 1249~1368 mg/L L-lactic acids were detected and the comparative drop rates of L-malate were 19.33%-19.42%, while no L-lactic was detected from control transformants.
