【作者】 刘延琳；

【导师】 李华； 张博润；

【作者基本信息】 西北农林科技大学 ， 果树学， 2004， 博士

【摘要】 酒精发酵和苹果酸-乳酸发酵是葡萄酒酿造的两个重要生化过程,通常由酿酒酵母(Saccharomyces cerevisiae)和酒酒球菌(Oenococcus oeni)完成。苹果酸-乳酸酶基因(mleA)和苹果酸通透酶基因(mleP)是O. oeni 进行苹果酸-乳酸发酵(MLF)的两个关键基因。为了避免乳酸菌在发酵过程中产生不必要或不安全次生代谢物质,减少细菌引发葡萄酒微生物破败的危害,经济有效地控制葡萄酒发酵,缩短葡萄酒酿造周期,本项目利用前期研究筛选出的酒酒球菌优良菌系,克隆其mleA 基因和mleP 基因并转化S.cerevisiae,进行它们在S.cerevisiae 中的转化、表达、相互作用及共表达研究,得出的主要结论如下: 1. 酒酒球菌mleA 基因的克隆及其在酿酒酵母中的转化与表达苹果酸-乳酸酶是进行MLF 的功能酶。本研究利用西北农林科技大学葡萄酒学院首次在国内筛选出的酒酒球菌优良菌系Oenococcus oeni SD-2a,进行其苹果酸-乳酸酶基因mleA 的克隆及其在S.cerevisiae 中的转化与表达研究: (1)构建了O.oeni SD-2a苹果酸-乳酸酶基因的重组质粒pLmleA并测序(GenBank登录的序列号为AY786176);测序分析表明克隆到的基因序列与GenBank 已报导序列(序列号X82326)同源性为99%,有两个碱基发生变化,其中一个为错义突变,编码的氨基酸由Asp 变为Glu,并缺少了BamHI 位点; (2)以PGK1 强启动子和ADH1 终止子为调控元件,以大肠杆菌-酵母菌附加体型穿梭质粒YEp352 及整合型质粒YIp5 为载体,构建了包含mleA 基因的重组表达质粒pYELmleA、pYELmleAP 和pYILmleA; (3)用重组表达质粒pYELmleA、pYELmleAP 及pYILmleA转化酿酒酵母YS58,用SD/-Ura 培养基平板筛选鉴定,得到了相应的酵母转化子YS58/pYELmleA、YS58/ pYELmleAP 和YS58/pYILmleA;更多还原

【Abstract】 Alcoholic fermentation (AF) and malolactic fermentation (MLF) are the two main biochemical processes in winemaking, and are mostly induced by Saccharomyces cerevisiae and Oenococcus oeni respectively. MLF of wine, which is the bacterial conversion of L-malic acid to L-lactic acid and carbon dioxide, results in a natural decrease in acidity, leads to biological stability increasing and wine flavor changing. Usually MLF develops naturally, but several factors, such as ethanol, sulfur dioxide concentration and pH, may affect this reaction. At the present time, this reaction is not entirely controlled, and processing problems, microbial alteration of wine, unpredictable production of sub-metabolites and time consumption occur frequently in winemaking. Malolactic enzyme gene(mleA)and malate permease gene(mleP)are the two main important genes of O.oeni involving in MLF. Malolactic enzyme is the function enzyme to turn L-malic acid into L-lactic acid, while malate permease performs malic acid and lactic acid transport. In this paper, research concerning the mleA and mleP genes cloning from O.oeni SD-2a and expressing in S.cerevisiae is performed. 1. Cloning of mleA gene from Oenococcus oeni and Expression in Saccharomyces cerevisiae In this study, mleA gene is amplified by PCR from O. oeni SD-2a, an excellent O. oeni strain screened in China. The PCR primers used were PmleaL(5’-CGGAATTCATGACAGATCCAGTAAGTAT-3’) and PmleaR(5’-TAGGTACCACACTCTCAACACTCGTAAT-3’), designed according to the GenBank reported sequences (Accession number is X82326). The amplified DNA fragment of mleA, about 1.62kb, was reclaimed and digested with EcoRI and KpnI. Using the unique restriction sites introduced by the PCR, the DNA fragment of mleA gene was inserted into yeast-Escherichia coli shuttle vector YEp352, resulted in recombinant plasmid pLmleA. mleA gene from O.oeni was sequenced and the Accession number is AY786176 registered in GenBank. Sequence analysis showed 99% identity of the two sequences, AY786176 and X82326. Two bases in the sequence of O.oeni SD-2a mleA show difference from X82326. The amino acid coded in the position of 1614 is Glu instead of Asp, and the site of BamHI does not exist for the base changing. In order to be expressed properly in S.cerevisiae, the mleA gene from pLmleA, PGK1 promoter from plasmid pVC727 and ADH1 terminator from plasmid pEA-1 were ligated and inserted into YEp352 and integrating vector YIp5. The resulted plasmids were named pYELmleA and pYILmleA. Another plasmid named pYELmleAP containing mle locus (mleA+mleP) was also constructed using YEp352 by the same way. When transformed into S.cerevisiae YS58, transformants were screened on SD/-Ura (YNB media containing leucine, histidine and tryptophan). Yeasts transformants of YS58/ pYELmleA, YS58/ pYILmleA and YS58/pYELmleAP were obtained. These three kinds of transformants were cultured in medium containing L-malate for 4d, the culture supernatants were collected and L-malate and L-lactic acid content were determined by HPLC. The result indicated that the functional expression of mleA gene was achieved in recombinants S.cerevisiae which can turn L-malic acid into L-lactic acid. L-malate contents of the transformants show extra significant difference with the control ones. In the SD/-Ura culture supernatant of YS58/pYELmleA and YS58/pYELmleAP, 1002-1106mg/L and 1249-1368mg/L L-lactic acids were detected respectively, while the comparative drop rates of L-malate were 19.18%-22.34% and 19.33%-19.42% respectively. In the SD/-Ura culture supernatant of pYILmleA, 345mg/L L-lactic acids were detected and the comparative drop rates of L-malate was 6.29%, while no L-lactic was detected from control transformants YS58/YIp5. 2. Cloning of Malate Permease Gene mleP from Oenococcus oeni and Expression in Saccharomyces cerevisiae The malate permease, coded by mleP gene, is important for assisting malolactic fermentation (MLF). In this paper, mleP gene was amplified by PCR from O. oeni SD-2a. The amplified DNA fragment, about 0.95kb, was inserted into pBluscript M13 to construct recombinant plasmid pBMmleP. In order to be expressed properly in S.cerevisiae, the mleP gene was inserted into YEp352 together with PGK1 promoter from pVC727 and ADH1 terminator from pEA-1, yielding expression plasmid pYEmleP. When transformed into S. cerevisiae strain YS58, transformants were screened on SD/-Ura. Transformants were cultured 4d in medium YEPD and SD/-Ura, both of which adding 5g/L L-malate. Thetransformants supernatant was collected and L-malate and L-lactic contents were detected by HPLC. The result showed that L-malate contents in supernatant fluid of the transformants showed significant difference with the control ones. L-malate transporting ability of YS58/pYEmleP (transformed YS58 with pYEmleP) was improved by 10.74%-12.99% in YEPD and 11.06%-12.30% in SD/-Ura, comparing with the control transformants. 3. Cloning of malolactic enzyme and malate permease genes and their co-expression in S. cerevisiae It is the first time to co-express mleA gene and mleP gene in this study. Two plasmids pGBKT7 and pGADT7 in yeast two-hybrid system were used to clone mleA and mleP gene. The mleA and mleP genes were inserted into expression vector pGBKT7 and pGADT7 respectively, resulting recombinant expression plasmid pGBLmleA and pGALmleP. When they were transformed into S.cerevisiae strain YS58, transformants were screened on SD/-Trp or SD/-Leu selective medium respectively. Recombinant yeasts YSGA and YSGP were obtained. Meanwhile, two plasmids pGBLmleA and pGALmleP containing mleA and mleP gene were co-transformed into S. cerevisiae YS58 to yield co-recombinant yeasts YSGA-GP. Both mleA and mleP gene can be expressed in S.cerevisiae without autonomous activation of the trported genes. While they were co-expressed in S.cerevisiae, the reported genes, His3 and LacZ, were activated, indicating that protein interaction exists between malolactic enzyme and malate permease. After transformants containing either recombinant expression plasmids or vectors were cultured in medium containing certain amount of L-malate, L-malate and L-lactate contents were detected by HPLC after collecting culture supernatant fluid. The results indicated that L-malate residual contents in culture supernatant fluid of recombinants YSGA, YSGP, YSGA-GP showing extra significant differences with the control ones. The ability of exogenous L-malate transportation of YSGP was improved comparing with the control YSDT7. To YSGA, cultured 4d in SD/-Trp medium (10% glucose) with about 13g/L L-malate, the L-lactate contents were 0.82-1.63g/L, corresponding L-malate reductions were 2.38-3.54g/L and the drop rates of L-malate were 17.95%-26.70%, while the comparative drop rates were 15.20%-24.24% comparing with the control YSKT7. To YSGP, also expressing mleP gene alone, the comparative L-malate drop rates were 6.78%-10.74% in SD/-Trp medium. YSGA-GP was yielded by co-transforming of mleA and mleP gene. The plasmid stability tests indicate that plamids are relatively stable in selective medium than that of innon-selective medium. The co-expression of mleA and mleP gene in YSGA-GP results in a much higher MLF activity. YSGA-GP transformants were cultured 8d in selective medium (10% glucose) with about 13g/L L-malate adding. The L-lactate content in the culture supernatant fluid increased gradually while the L-malate content decreased. The drop rate of L-malate reached 53.32%, and the comparative drop rate of L-malate was 51.49% comparing with the control YSKT7-DT7. However, there is further work needed for realization of complete degradation of L-malate. Unicellular clones of YSGA-GP were used for the fermentation study after unicellular separation and incubation. The result showed that MLF was induced with AF simultaneously by YSGA-GP. Besides the time advantage of the simultaneous AF/MLF-procedure, it achieved more microbiologically stable wines with low volatile acid and tolerance to SO2 and low pH, which provides the great potential of industrial application.更多还原