Screening And Application Of Acid-resistant Yeast

Acid and yeast had got much attantion because of its caracter that it can be grown and fermented under acidic conditions. The objective of this work was to screen acid-resistant yeast and reseach its application in the fermentation, and we used the mtabolic engineering for producing L-lactic acid by wild acid-resistant yeast, and discussed primary factor of L-lactic acid fermentation of recombinant. It was basic work as the L-lactic acid industrial production in the future. The mainly researching contents are blow.Two wild acid-resistant yeast strains isolated from 96 natural samples, was found to be able to grow well at YEPD medium(containing 68 g/L the lactic acid). Based on further molecular biological tests, one strain was primarily identified as Candida.sp; the other one was primarily identified as Issatchenkia orientalis.The acid-resistant yeast strains were taken as the further research strains. In the result of aerobic fermentation research, it was found that, I. orientalis could be used for producing the single-cell protein in the cassava-alcohol wastewater. The cell dry weight of it can be up to 7.40 g/L, and the pH of the wastewater pH can rose to 6.5 from 3.9. On the study of facultative anaerobic fermentation, the malic acid-degradation could better be operated in the wild grape wine by I. orientalis. At the end of fermentation, the malic acid content can be decreased to 1 g/L, and the malic acid had been degradated about 80%, and pH values had slightly increased. In this study, acid-resistant yeast which was screened out as the research object, by metabolic engineering to expression of exogenous L-lactate dehydrogenase gene(ldhA), increased the lactic acid metabolic pathway, and the metabolic flux lead to the direction of L-lactic acid formation,and in order to achieve the purpose of producing lactic acid at low pH. After that, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae As3.819, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into acid-resistant yeast strains by electroporation transformation. Subsequently, a recombinant yeast C4-2 was obtained which could produce L-lactic acid.It was shown by the fermentation results that the initial sugar concentration,pH value of medium and the time of ventilation fermentation could be the factors of effect the concentration of lactic acid. The highest yield was up to 42.4 g/L. The tandem expression of lactate dehydrogenase gene was studied in this work. The resultant tandem restructuring expression plasmid pYX212-TPI-ldhA-TTA-TPI-ldhA- TTB-kanMX was successfully constructed and introduced into acid-resistant yeast strains by electroporation transformation, and subsequently a recombinant yeast C4-E was obtained. It was shown by the fermentation results lactic acid yield by C4-E was slightly lower than that of C4-2. It showed that the copies of ldhA gene were not the main reason for the yield of L-lactic acid being low.In summary, this study verified the prospect of acid-resistant yeast by experiments in ventilation and anaerobic conditions, and it will be important inspiration to study the organic acid production of yeasts.