Construction of protease-deficient Candida boidinii strains useful for recombinant protein production:: Cloning and disruption of proteinase A gene (PEP4) and proteinase B gene (PRB1)

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 PRB1 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 cereviside PrA and PrB (64% and 61% identities, respectively). Both PEP4 and PRB1 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.