Breeding of a new type of baker's yeast by δ-integration for overproduction of glucoamylase using a homothallic yeast

Abstract

With the aim of developing a new type of bread that can be produced from raw materials such as rice, new recombinant yeast strains producing glucoamylase were constructed. The yeast OC-2, which is used for wine making, was selected as a host strain in a screening test because of its superiority in flavor formation in bread. OC-2 is a homothallic and diploid strain, and has no auxotrophic marker. First, tryptophan auxotrophic mutants (Δtrp1/Δtrp1) were constructed from OC-2 by the gene disruption method. Conventional YEp- and YIp-type yeast expression plasmids containing glucoamylase cDNA from Aspergillus oryzae, and the TRP1 gene as a selective marker were introduced into the recipient strain. Both transformants with the YEp- or YIp-type plasmid showed low glucoamylase productivity in the culture broth, probably due to very low plasmid stability, and a low copy number, respectively. A new type of integrative plasmid was thus constructed by the δ-integration system using a δ sequence of the yeast retrotransposon Ty1 to obtain transformants with higher glucoamylase production ability. These transformants produced higher amounts of glucoamylase and could grow in synthetic medium containing starch as a carbon source. Furthermore, using the integrated transformants, duplication of copies of the integrated glucoamylase cDNA by spore formation and autodiploidization was carried out by utilizing the homothallic characteristics of the strain. By Southern blot analysis, the copy number was estimated to be about twice that of the original transformant, and the glucoamylase productivity was proportionally increased. The fact that the constructed strains could grow in synthetic medium containing soluble starch as a sole carbon source suggested that they will be useful for making new types bread.