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)

Abstract

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 cerevisiae 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 pep4Δ and the pep4Δ prb1Δ 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. © 2002 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.