Exploitation of a KU70-deficient mutant for improving gene deletion frequency in aspergillus niger

Abstract

Filamentous fungus Aspergillus niger CGMCC 10142 was successfully transformed with Agrobacterium tumefaciens AGL-1 for nonhomologous end-joining (NHEJ) or homologous recombination (HR) of transforming DNA (T-DNA). To improve targeting frequency, the KU70-deficient mutant (Δku70) in A. niger CGMCC 10142 was deleted, obtaining the new strain A. niger Δku70. Co-cultivation of A. niger conidia with A. tumefaciens in the proper concentration of acetosyringone resulted in the formation of Δku70 with the hygromycin B-resistance. It showed a dramatically improved targeted gene deletion frequency, with other transformants such as tpsA, cs, pd, and aox1 gene being true knockout when bleomycin resistant was used based on Δku70. All the selected resistant transformants were proved to be stable through subculture and the knocking-out integration of the G418 gene into the genome was determined by PCR. The results showed that the efficiency of HR was 40–80% higher comparing with original strain CGMCC 10142, which suggests that the deficiency of ku70 can improve gene deletion efficiency in A. niger.