title: Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation.
abstract: pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) 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 site-directed 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 construct 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 that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG 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 of A. oryzae.
authors.affiliation: Department of Bioengineering & Biotechnology, College of Chemical Engineering, Huaqiao University, Xiamen 361021, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Du
authors.affiliation: Department of Bioengineering & Biotechnology, College of Chemical Engineering, Huaqiao University, Xiamen 361021, China.
authors.forename: Guizhen
authors.initials: G
authors.lastname: Xie
authors.affiliation: Department of Bioengineering & Biotechnology, College of Chemical Engineering, Huaqiao University, Xiamen 361021, China.
authors.forename: Chunfa
authors.initials: C
authors.lastname: Yang
authors.affiliation: College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China chenhw@hqu.edu.cn fbs@xmu.edu.cn.
authors.forename: Baishan
authors.initials: B
authors.lastname: Fang
authors.affiliation: Department of Bioengineering & Biotechnology, College of Chemical Engineering, Huaqiao University, Xiamen 361021, China chenhw@hqu.edu.cn fbs@xmu.edu.cn.
authors.forename: Hongwen
authors.initials: H
authors.lastname: Chen
mesh_terms: D001236:Aspergillus oryzae / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D005285:Fermentation; D017353:Gene Deletion; D005800:Genes, Fungal; D009154:Mutation; D010957:Plasmids; D014920:Wine
chemical_list: D017931:DNA Primers
keywords: Aspergillus oryzae; gene deletion; pyrG gene; uridine auxotrophs