INFLUENCE OF KU70 GENE KNOCKOUT ON TRANSFORMATION FREQUENCY OF PENICILLIUM VERRUCULOSUM MYCELIAL FUNGI

Abstract

To increase the frequency of homologous recombination (HR) during the transformation of the industrial strain Penicillium verruculosum 221-151 (VKM F-3972D), the ku70 gene encoding the Ku70, which binds at the sites of double-stranded DNA breaks and is involved in the repair process by the non-homologous end joint (NHEJ), was knocked out by the CRISPR/CAS9 method. Presumably, the new host strain, P. verruculosum ΔniaDΔku70, should have had an increased frequency of homologous recombination during transformation in comparison with the host strain P. verruculosum ΔniaD due to the integrative insertion of the expression cassette only by the HR mechanism. The pep1 gene encoding homologous aspartate protease was chosen as a marker. However, it was shown that the knockout of the ku70 gene led to a dramatic decrease in the frequency of co-transformation in the P. verruculosum ΔniaDΔku70 strain compared to the P. verruculosum ΔniaD strain at the same load of exogenous DNA (3 μg). The number of copies of the pep1 gene in recombinant strains of the P. verruculosum Pep1 (with a native Ku70) series ranged from 3 to 28 copies, which indicated the predominance of the non-homologous recombination mechanism.