关键词: 酒酒球菌, 苹果酸－乳酸酶基因, 整合重组表达, 酿酒酵母

Abstract:

【Objective】 In this paper, researches concerning the malolactic enzyme gene mleA cloning from a patent strain O. oeni SD-2a screened from Chinese wine and integrated expressing in S. cerevisiae were made in order to perform alcoholic fermentation (AF) and malolactic fermentation (MLF) simultaneously during winemaking. 【Method】 Cloned malolactic enzyme gene mleA from Oenococcus oeni SD-2a, PGK1 promoter and ADH1 terminator were ligated and inserted into integrating vector YIp5 to construct the expression plasmid named pYILmleA. When transformed into S. cerevisiae YS59, the resulted yeast transformants YS59/pYILmleA were screened on SD/-Ura and identified by auxotrophic test, mating type test and colony PCR. Target protein was detected by SDS-PAGE and the targeted gene integrated to the chromosome was detected by dot blotting hybridization. The transformants were cultured in medium containing L-malate and the culture supernatants were collected and L-malate and L-lactic acid content were detected by HPLC to confirm if functional expression were achieved. 【Result】 Auxotrophic test, mating type test and colony PCR showed positive transformants were obtained. Target protein was detected by SDS-PAGE and the targeted gene was integrated into the chromosome detected by dot blotting hybridization. When the transformants of YS59/pYILmleA were cultured in SD/-Ura added with 10% glucose and 5648 mg?L-1 L-malate for 4d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC, 1278-1312 mg?L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18%-20.85%. L-malate contents and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t, test respectively. 【Conclusion】 The result indicated that the integrated expressive plasmid containing mleA gene from patent strain O.oeni SD-2a screened from Chinese wine was constructed and the functional expression was achieved in recombinants S. cerevisiae.

Key words: Oenococcus oeni, malolactic enzyme gene, integrated recombinant expression, Saccharomyces cerevisiae