A variant of Saccharomyces cerevisiae pep4 strain with improved oligotrophic proliferation, cell survival and heterologous secretion of alpha-amylase.
A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse alpha-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 alpha-amylase, we observed that transformed colonies grew more quickly and secreted more alpha-amylase than general yeast strains. A further test showed that the NI-C strain was able to use mouse alpha-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.
Chen
D C
DC
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC. chendzch@ccvax.sinica.edu.tw
Chen
S Y
SY
Gee
M F
MF
Pan
J T
JT
Kuo
T T
TT
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D015139:Blotting, Southern; D002455:Cell Division; D015169:Colony Count, Microbial; D005819:Genetic Markers; D014644:Genetic Variation; D051379:Mice; D010957:Plasmids / Q000235:genetics; D006656:Protamine Kinase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000378:metabolism*
D005819:Genetic Markers; D029701:Saccharomyces cerevisiae Proteins; D006656:Protamine Kinase; D000516:alpha-Amylases; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases