High-level secretion of fungal glucoamylase using the Candida boidinii gene expression system

The methylotrophic yeast, Canadida boidinii, was investigated as an expression host for secretory enzyme production. The cDNA of Rhizopus oryzae glucoamylase was placed under the C. boidinii alcohol oxidase (AODl) promoter. A transformant integrated with a single-copy expression cassette to the chromosome produced glucoamylase into the medium to a high amount when the cells were grown on methanol or methanol plus glycerol as (a) carbon source(s). The transformant C. boidinii cells were grown up to ca. 95 g dry cell weight/liter medium, and the concentration of glucoamylase in the medium reached 3.4 g/liter. This showed that the signal sequence from Rhizopus glucoamylase functioned very efficiently in C. boidinii. Next, secreted glucoamylase from C. boidinii was purified and compared with the enzyme produced in S. cerevisiae. The enzyme produced in C. boidinii was found to have higher molecular weight than that produced in S. cerevisiae, which was due to the difference of the N-linked glycosylated sugar structure of the produced proteins.