A variant of Saccharomyces cerevisiae pep4 strain with improved oligotrophic proliferation, cell survival and heterologous secretion of α-amylase
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A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse α-amylase, is described. Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain. To use the NI cells as a host for heterologous expression, we tried to amend the introduced pYAS/12S vector and obtain a host strain, NI-C, with stable NI phenotype and trp1 marker restored. Southern analysis of genomic DNA of NI-C suggested that the original pYAS/12S was abnormally rearranged and not completely corrected. Further assay showed that the viability and mitotic ability of the NI-C strain were increased. While using the NI-C strain as host for plasmid transformation and heterologous expression of mouse α-amylase, we observed that transformed colonies grew more quickly and secreted more α-amylase than general yeast strains. A further test showed that the NI-C strain was able to use mouse α-amylase as a positive selection marker to form transformed colonies on nitrogen-starved plates that contain starch as the sole carbon source. The results imply that the NI-C variant is an improved pep4 strain that can be used for heterologous expression and for the development of new selective markers in the yeast transformation system.
KeywordsSaccharomyces Cerevisiae Sole Carbon Source Heterologous Expression Selective Marker General Yeast
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