Characterization of the Ku70 homologue HdfA deletion in Penicillium chrysogenum

Targeting an engineered DNA fragment to a specific site in chromosomes in order to disrupt, overexpress or modify the nucleotide sequence of a gene requires homologous recombination repair mechanism. This DNA repair mechanism is not predominant in fungi, resulting in extremely low targeting efficiency. To increase this efficiency, it is becoming common practice to disable the non homologous end joining (NHEJ) pathway that causes random integration, by deleting homologous gene to human KU70 and KU80 which encode proteins functioning in the NHEJ pathway. These genes have been successfully deleted in several organisms, including the yeast Kluyveromyces lactis and the fungi Neurospora Crassa and several Aspergilli species. In this study we investigated the behavior of high penicillinG-producing Penicillium chrysogenum strains, in which the KU70 or KU80 homologues, HdfA or HdfB, had been deleted. Targeting efficiency in these mutant strains was significantly increased relative to the reference strain. Both physiological and transcriptome data of chemostat cultivations of the hdfA deletion strain and the reference strain showed minimal differences. However, in a direct competition experiment to assess global strain fitness, the reference strain had a clear advantage over the deletion strain. The full characterization of these recombinant host strains is an essential step to guide the future construction of a whole genome knock-out mutant collection. Penicillium chrysogenum is a filamentous fungus that is used to industrially produce the beta-lactam antibiotics. After decades of classical strain improvement, the genome sequence has been deciphered and annotated. The combination of the P. chrysogenum genome sequence and a strain exhibiting increased targeting efficiency would paved the way to the construction of deletion or expression libraries that would be a tremendous help in understanding the nature of the productive capabilities of this organism. This study reports the effect of the deletions of the Ku70 and Ku80 homologues, HdfA and HdfB respectively, in P. chrysogenum. A detailed analysis of the physiology, transcriptome and phenotype was performed to assess the use of these strains as platform for functional genomics study.

将工程化DNA片段靶向整合至染色体特定位点，以实现基因的敲除、过表达或核苷酸序列修饰，需依赖同源重组修复（homologous recombination repair, HRR）机制。该DNA修复通路在真菌中并不占主导地位，因此靶向整合效率极低。为提升靶向效率，当前通行的研究策略是通过敲除与人类KU70、KU80同源的基因——这两个基因编码非同源末端连接（non-homologous end joining, NHEJ）通路中的功能蛋白——来阻断该介导随机整合的NHEJ通路。上述同源基因已在多种生物体中成功实现敲除，包括乳酸克鲁维酵母（Kluyveromyces lactis）、粗糙脉孢霉（Neurospora crassa）以及多种曲霉（Aspergilli）物种。本研究针对高产青霉素G的产黄青霉（Penicillium chrysogenum）菌株展开探究，该菌株中KU70、KU80的同源基因HdfA或HdfB已被敲除。相较于参考菌株，上述突变菌株的靶向整合效率显著提升。对HdfA敲除菌株与参考菌株的恒化培养物的生理特性数据与转录组数据进行分析，结果显示二者差异微乎其微。然而在评估菌株全局适应性的直接竞争实验中，参考菌株相较于敲除菌株展现出明显的生长优势。对这类重组宿主菌株进行全面表征，是指导未来构建全基因组敲除突变体库的必要步骤。产黄青霉是一种丝状真菌，被工业应用于β-内酰胺类抗生素的生产。经过数十年的经典菌株选育改良，其基因组序列已完成解析与注释。将产黄青霉的基因组序列与具备高靶向整合效率的菌株相结合，将为构建基因敲除或表达文库铺平道路，而这类文库将极大助力解析该菌株生产能力的本质机制。本研究报道了在产黄青霉中分别敲除Ku70、Ku80的同源基因HdfA与HdfB所产生的影响，并通过生理特性、转录组与表型的详细分析，评估了这些菌株作为功能基因组学研究平台的应用潜力。