The Surface Display Of Malolactic Enzyme On Saccharomyces Cerevisiae

Malolactic fermentation induced by lactic acid bacteria is always slow due to the unsuitable environment, because of which, lactic acid bacteria could produce biogenic amines, urethane and other harmful substances during fermentation. Usually, biological hazard was caused concomitantly by incomplete fermentation. To solve these problems, it seems that malolactic enzyme (MLE) can be added to the wine to transform malic acid but with low conversion efficiency. It can also construct reducing-acid Saccharomyces cerevisiae utilizing cell fusion and genetic engineering. But it is difficult to obtain reducing-acid S. cerevisiae by cell fusion technology. Gene engineering S. cerevisiae was succeed to construct but the conversion of malic acid was low for the barriers of the malic acid transform.In the research, the yeast surface displaying plasmid would be constructed. As a reporter, enhanced green fluorescent protein (EGFP) was displayed on the surface of S. cerevisiae to verify the display plasmid. Then, mle gene was inserted into the display plasmid, expressed and displayed on the surface of S. cerevisiae. If genetically engineered yeast was successfully constructed, it can be used for alcoholic fermentation and MLF during fermetation. The main results were as following:1 Construction of surface displaying plasmid pADH1-AGGThe a-factor secretion signal gene (267 bp) was amplifyed from plasmid pPIC9K, and the gene (agg) encoding 3’-half of a-agglutinin (1623 bp) was amplified from S. cerivisiae genome. Then, the signal peptide gene was inserted into plasmid pADH1 for constructing a new plasmid named pADHl-signal. On this basis, the agg gene was inserted into plasmid pADHl-signal for constructing a new plasmid named pADH1-AGG. Subsequently, plasmid pADH1-AGG was successfully constructed by PCR verification and enzyme digestion.2 Functional verification of pADHl-AGGThe enhanced green fluorescent protein gene (717 bp) was amplified from plasmid pEGFP-C1 and inserted into plasmid pADHl-AGG. The new plasmid was named pADH1-GFP. In order to examine whether EGFP was displayed on the surface of S. cerivisiae, the yeast was detected by fluorescent microscope, protease K and fluorescence spectrophotometer experiment. The results were as follows:The positive transformants carrying pADH1-GFP and pADHl-AGG (negative) were observed under fluorescent microscope at blue light respectively. The results indicated that brighter green-fluorescence was discovered on yeast cells carrying pADHl-GFP, but no fluorescence on the negative yeast. It could be confirmed that the EGFP was expressed successfully in yeast.The positive transformants carrying pADH1-GFP and pADHl-AGG (negative) were treated with protease K. The results showed that green fluorescence was disappeared, which was similar to the negative control. It indicated that EGFP was successfully displayed on S. cerevisiae.Yeast cells were ruptured, located by fluorescence spectrophotometer. It was obviously that the fluorescence value of cell wall extract was 408.936, and that of cell-free extract was 73.936. Thus, it could be confirmed that EGFP was anchored on the cell wall of S. cerevisiae successfully. Hence, we can confirmed that pADH1-AGG can be used to express and display foreign proteins on the surface of S. cerevisiae successfully.3 Surface display of MLE on S. cerivisiaeThe mle gene (1600 bp) was amplified from Oenococcus oeni genome and inserted into pADHl-AGG for constructing a new plasmid named pADH1-MLE. After that, pADH1-MLE was transformed into S. cerivisiae. It was validated that there were three transformants named Fm1, Fm4 and Fm5. The MLE was successfully displayed on S. cerivisiae utilizing immunofluorescence. Subsequently, the culture supernatants was detected by high-pressure liquid chromatography. The result indicated that the functional expression of MLE was achieved. And 488.6 mg/L L-lactic acids were detected while the drop rates of L-malate were 21.11%.