Construction of lactose-fermenting and high-ethanol-producing hybrid yeast by protoplast fusion of Saccharomyces cerevisiae and Kluyveromyces fragilis and the analysis of beta-galactosidase gene in the hybrid yeast

The availability of a yeast strain which is capable of fermenting lactose and at the same time is tolerant to high concentrations of ethanol would be useful for the production of ethanol from lactose. Kluyveromyces fragilis is capable of fermenting lactose, but it is not as tolerant as Saccharomyces cerevisiae to high concentrations of ethanol. In this study, we have used the protoplast fusion technique to construct hybrids between auxotrophic strains of Saccharomyces cerevisiae having high ethanol tolerance and an auxotrophic strain of lactose fermenting Kluyveromyces fragilis isolated by ethyl methanesulfonate mutagenesis. The fusants obtained were prototrophic and capable of assimilating lactose and producing ethanol in excess of 13% (v/v). The complementation frequency of fusion was about 0.7%. Formation of fusants was confirmed by the increased amount of chromosomal DNA per cell. Fusants contained 8-16 {dollar}times{dollar} 10{dollar}sp8{dollar} {dollar}mu{dollar}g DNA/cell as compared to about 4 {dollar}times{dollar} 10{dollar}sp8{dollar} {dollar}mu{dollar}g DNA/cell for the parental strains suggesting that multiple fusions had taken place.; The fusant obtained by protoplast fusion of Saccharomyces cerevisiae and Kluyveromyces fragilis was further studied. The gene coding for the enzyme {dollar}beta{dollar}-alactosidase was isolated by cloning in E. coli YMC9 using recombinant DNA techniques. The K. fragilis chromosomal DNA was partially digested with the restriction endonuclease Sau3A and the fragments were ligated into BamHI-digested pBR322 plasmid. The plasmid library pFR-1 was transformed into {dollar}lacsp{lcub}-{rcub}{dollar} E. coli strain YMC9 and {dollar}lacsp{lcub}+{rcub}{dollar} transformants were selected. Restriction mapping of the inserted DNA fragment revealed that the size of the cloned fragment was 7.6 Kbp. The recombinants were able to synthesize active {dollar}beta{dollar}-galactosidase enzyme, which showed that the cloned gene was expressed in the prokaryotic host, albeit not very efficiently. Furthermore, the DNA-DNA hybridization study revealed that the probe (the cloned gene from the fusant yeast) hybridized to five fragments in the yeast genomic DNA. This further confirmed that the {dollar}beta{dollar}-galactosidase gene which was cloned came from the parent strain of yeast and not from E. coli.