Integrated Expression of the Oenococcus oeni mleA Gene in Saccharomyces cerevisiae  

作　　者：LIU Yan-lin LI Hua 

机构地区：[1]College of Enology, Northwest A ＆F University/Shaanxi Engineering Research Center for Viti-Viniculture, Yangling 712100, P.R. China

出　　处：《Agricultural Sciences in China》2009年第7期821-827,共7页中国农业科学（英文版）

基　　金：supported by the National High-Tech Research and Development Program of China (863 Program of China, 2007AA10Z314);andthe Earmarked Fund for Modern Agro-Industry Technology Research System, China (Z225020801)

摘　　要：Malolactic enzyme is the function enzyme which catalyses the reaction for L-malate converting to L-lactic during malolactic fermentation （MLF）. In this paper, researches concerning the malolactic enzyme gene mleA cloned from a patent strain Oenococcus oeni SD-2a screened in Chinese wine and integrated expressing in Saccharomyces cerevisiae were performed in order to carry out both alcoholic fermentation （AF） and malolactic fermentation （MLF） during winemaking, with a view to achieving a better control of MLF in enology. To construct the expression plasmid named pYILmleA, cloned mleA gene, PGK1 promoter, and ADH1 terminator were ligated and inserted into integrating vector YIp5. Yeast transformants 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 bloting hybridization. After the transformant was cultured in SD/-Ura adding glucose （10%） and L-malate （5 648 mg L-1） for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. 1 278-1 312 mg L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18-20.85%. L-malate and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t-test respectively. The result indicated that the functional expression was achieved in recombinants S. cerevisiae.Malolactic enzyme is the function enzyme which catalyses the reaction for L-malate converting to L-lactic during malolactic fermentation （MLF）. In this paper, researches concerning the malolactic enzyme gene mleA cloned from a patent strain Oenococcus oeni SD-2a screened in Chinese wine and integrated expressing in Saccharomyces cerevisiae were performed in order to carry out both alcoholic fermentation （AF） and malolactic fermentation （MLF） during winemaking, with a view to achieving a better control of MLF in enology. To construct the expression plasmid named pYILmleA, cloned mleA gene, PGK1 promoter, and ADH1 terminator were ligated and inserted into integrating vector YIp5. Yeast transformants 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 bloting hybridization. After the transformant was cultured in SD/-Ura adding glucose （10%） and L-malate （5 648 mg L-1） for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. 1 278-1 312 mg L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18-20.85%. L-malate and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t-test respectively. The result indicated that the functional expression was achieved in recombinants S. cerevisiae.

关 键 词：Oenococcus oeni malolactic enzyme gene integrated recombinant expression Saccharomyces cerevisiae 

分 类 号：S661.1[农业科学—果树学]