Construction of brewing-wine Aspergillus oryzaepyrG- mutant bypyrG gene deletion and its application in homology transformation

pyrG- host cells are indispensable for pyrG- based transformation system. Isolations ofpyrG- host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG- mutant by sitedirected mutation of pyrG gene deletion which would be used as a host for further transformation, pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to con- struct pyrG- mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating thatpyrG was completely excised. The ApyrG mutants were applied as pyrG- host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production ofA. Oryzae. The xdh disrup- tion mutants were efficiently constructed by transforming a pMD-pyrC-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Axdh mutant was three times as much as that of the ApyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG- host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding ofA. Oryzae.