Heterologous protein expression in Pichia thermomethanolica BCC16875, a thermotolerant methylotrophic yeast and characterization of N-linked glycosylation in secreted protein.
This study describes Pichia thermomethanolica BCC16875, a new methylotrophic yeast host for heterologous expression. Both methanol-inducible alcohol oxidase (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters from Pichia pastoris were shown to drive efficient gene expression in this host. Recombinant phytase and xylanase were expressed from both promoters as secreted proteins, with the former showing different patterns of N-glycosylation dependent on the promoter used and culture medium. In addition, growth temperature also had an effect on N-glycan modification of cell wall mannoproteins. The major glycoprotein oligosaccharide species produced from P. thermomethanolica BCC16875 is Man(8-12) GlcNAc(2) , which is similar to that from other methylotrophs. Moreover, mannosylphosphate and α-1,6- and α-1,2-linked mannose modifications of heterologous secreted protein were also detected. The attainably high level of protein production in complement to distinctive thermotolerance rarely found in other industrial yeasts makes this microorganism an attractive host for large-scale fermentation.
Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand. sutipa@biotec.or.th
Sutipa
S
Tanapongpipat
Peerada
P
Promdonkoy
Toru
T
Watanabe
Witoon
W
Tirasophon
Niran
N
Roongsawang
Yasunori
Y
Chiba
Lily
L
Eurwilaichitr
D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006031:Glycosylation; D006358:Hot Temperature; D000432:Methanol / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D011134:Polysaccharides / Q000032:analysis; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011134:Polysaccharides; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010832:6-Phytase; D014995:Xylosidases; D000432:Methanol