Influence of ku70 Gene Knockout on the Transformation Frequency of Penicillium verruculosum Mycelial Fungi

Abstract

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