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).

中文翻译：

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ku70基因敲除对疣状青霉菌丝体转化频率的影响

摘要

为了增加工业疣状青霉221-151 菌株 (VKM F-3972D)转化过程中同源重组 (HR) 的频率，编码Ku70蛋白的 ku70 基因结合在双链 DNA 断裂位点并参与其中在通过非同源末端连接 (NHEJ) 的修复过程中，被 CRISPR/CAS9 方法敲除。据推测，与宿主菌株P. verruculosum ΔniaD 相比，新宿主菌株P. verruculosum ΔniaDΔku70 在转化过程中同源重组的频率应该更高，因为表达盒仅通过 HR 机制进行整合插入。选择编码其同源天冬氨酸蛋白酶的pep1基因作为标记。然而，结果表明，在相同的外源 DNA 负载量 (3 μg) 下，与P. verruculosum ΔniaD菌株相比，ku70基因的敲除导致P. verruculosum ΔniaDΔku70 菌株的共转化频率显着降低。同时，P. verruculosum Pep1系列重组菌株（带有天然Ku70）中pep1基因的拷贝数范围为3至28个拷贝，这表明非同源重组机制（NHEJ）占主导地位。