[Construction and functional analysis of the pepB gene disruptant in Aspergillus niger].
An integration plasmid pMW1-pepB for the pepB gene disruption in Aspergillus was constructed. The plasmid contained the pepB gene upstream (P) 1.4kb and downstream (T) 1.3kb homologous fragments with insertion of the expression unit of the hygromycin resistance gene (hph) between them. P and T DNA fragments were synthesized by PCR from Aspergillus niger chromosomal DNA. The integration plasmid was digested with the Hpa I restriction enzyme, the resultant 4.2kb linear fragment was introduced into the Aspergillus niger strain GICC2773 which expressing the glucoamylase/laccase fusion protein by PEG-mediated transformation. 62 Hygromycin resistance transformants were screened, and from them one strain named pepB29 was identified to be the pepB disruptant by PCR analysis. Data of functional assay of the pepB29 strain indicated that the disruption of the pepB gene secreted reduced acid proteolytic activity, and improved the heterologous protein laccase production.
Sun
Jing
J
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Li
Jing-Peng
JP
Wang
Ao-Quan
AQ
Tang
Guo-Min
GM
Wang
Hua-Ming
HM
D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000502:physiology; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D002934:Cinnamates / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D042845:Laccase / Q000096:biosynthesis; D010957:Plasmids; D016133:Polymerase Chain Reaction
D002934:Cinnamates; D006921:Hygromycin B; C026273:hygromycin A; D042845:Laccase; D016282:Aspartic Acid Endopeptidases