Construction of stable laboratory and industrial yeast strains expressing a foreign gene by integrative transformation using a dominant selection system.
An expression cassette of mouse dihydrofolate reductase (Mdhfr) cDNA under control of the yeast cytochrome c promoter was inserted in a yeast plasmid containing the ARS1 sequence. The ARS replicating function was destroyed by BglII treatment prior to yeast transformation. Using this linearized plasmid, genomic transformants could be obtained from either laboratory or industrial strains of bakers' yeast based on direct methotrexate (MTX)-resistance selection. The entire sequence of the linearized plasmid was integrated by homologous recombination at the ARS region of the host chromosome. The results indicate that repetitive and homologous recombination occurs readily in such transformations. The stability of the constructed integrants was more than 99.95% per generation in non-selective medium, and tandem repeats of up to six copies (i.e., about 44 kb) were not changed even after 30 generations in rich medium. Expression in rich medium of cointegrated, human interleukin 2 cDNA under control of the triose phosphate isomerase promoter was shown by Western blot experiments in both laboratory and industrial yeast strains. Furthermore, a comparison of the transcription efficiency of the Mdhfr gene in the chromosome with that in the plasmid revealed that the efficiency was almost proportional to the number of gene copies, irrespective of the location of the transcription unit. These results show that by using the MTX/Mdhfr dominant selection-amplification system one can construct stable recombinant yeast strains suitable for heterologous gene expression in laboratory as well as in industrial fermentation conditions.
D004271:DNA, Fungal / Q000235:genetics; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D007376:Interleukin-2 / Q000235:genetics; D008727:Methotrexate / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012641:Selection, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D004274:DNA, Recombinant; D007376:Interleukin-2; D012331:RNA, Fungal; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate