High-level production of recombinant Geotrichum candidum lipases in yeast Pichia pastoris.
We describe the heterologous high-level expression of the two Geotrichum candidum lipase (GCL) isoenzymes from strain ATCC 34614 in the methylotrophic yeast Pichia pastoris. The lipase cDNAs were placed under the control of the methanol-inducible alcohol oxidase promoter. The lipases expressed in P. pastoris were fused to the alpha-factor secretion signal peptide of Saccharomyces cerevisiae and were secreted into the culture medium. Cultures of P. pastoris expressing lipase accumulated active recombinant enzyme in the supernatant to levels of approximately 60 mg/L virtually free from contaminating proteins. This yield exceeds that previously reported with S. cerevisiae by a factor of more than 60. Recombinant GCL I and GCL II had molecular masses of approximately 63 and approximately 66 kDa, respectively, as determined by SDS-PAGE. The result of endoglucosidase H digestion followed by Western blot analysis of the lipases suggested that the enzymes expressed in P. pastoris received N-linked high-mannose-type glycosylation to an extent, 6-8% (w/w), similar to that in G. candidum. The specific activities and substrate specificities of both recombinant lipases were determined and were found to agree with what has been reported for the enzymes isolated from the native source.
Holmquist
M
M
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada.
Tessier
D C
DC
Cygler
M
M
D003001:Cloning, Molecular; D015870:Gene Expression; D005848:Geotrichum / Q000201:enzymology* / Q000235:genetics; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics*; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008049:Lipase; C065602:lipase II, Geotrichum candidum; C085712:lipase III, Geotrichum candidum