Construction of protease-deficient Candida boidinii strains useful for recombinant protein production: cloning and disruption of proteinase A gene (PEP4) and proteinase B gene (PRBI).
The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed. The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRBI gene (1683 bp encoding a protein of 560 amino acids) were found. The deduced amino acid sequences of PrA and PrB are very similar to Saccharomyces cerevisiae PrA and PrB (64% and 61% identities, respectively). Both PEP4 and PRBI genes were disrupted in the C. boidinii genome by one-step gene disruption. The resultant pep4delta and the pep4delta prb1delta strains lost protease activity when compared with the wild-type original strain. The constructed C. boidinii strains are expected to be useful hosts for heterologous protein production.
Komeda
Toshihiro
T
Central Laboratories for Key Technology, Kirin Brewery Co, Yokohama-shi, Kanagawa, Japan. t-komeda@kirin.co.jp
Sakai
Yasuyoshi
Y
Kato
Nobuo
N
Kondo
Keiji
K
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics*; D002175:Candida / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D010450:Endopeptidases / Q000172:deficiency*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D016282:Aspartic Acid Endopeptidases; D004271:DNA, Fungal; D010450:Endopeptidases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C023302:yeast proteinase B; D029701:Saccharomyces cerevisiae Proteins; C022442:aspergillopepsin II