Marker genes are essential for gene modification and genome editing of microorganisms. In Aspergillus oryzae, a widely used host for enzyme production, only a few marker genes can be used for positive selection. One of these genes, the pyrithiamine (PT) resistance marker gene thiA, is not useful for CRISPR/Cas9 genome editing because of its unique resistance-conferring mechanism. In this study, a novel PT resistance marker was investigated considering its potential applications in genome editing. A mutant resistant to PT was selected from UV-mutagenized A. oryzae RIB40. Whole genome analysis was conducted on the mutants, and a novel candidate gene for PT resistance was identified. This candidate gene exhibited similarity to the thiamine transporter gene thi9 of Schizosaccharomyces pombe and was designated as thiI. A thiI loss-of-function mutant was generated using the CRISPR/Cas9 genome editing system to investigate its effect on PT resistance. This mutant showed PT resistance and exhibited no growth defect or auxotrophy. The thiI gene was further investigated for its use as a selection marker in genome co-editing. Ribonucleoprotein complex comprising recombinant Cas9 nuclease and sgRNA targeting thiI or another target gene (wA or sreA) was prepared and simultaneously introduced into A. oryzae RIB40. thiI and target gene double loss-of-function mutants were efficiently selected on PT-containing medium. thiI was shown to be a useful marker gene in A. oryzae for use in genome editing. This study is expected to provide insights, which will promote basic research and industrial applications of A. oryzae.

标记基因对于微生物的基因修饰和基因组编辑至关重要。在米曲霉（Aspergillus oryzae）中，广泛用于产生酶的宿主中，只有少数标记基因可用于阳性选择。这些基因之一，吡硫胺（PT）抗性标记基因thiA，由于其独特的抗性赋予机制，因此无法用于CRISPR / Cas9基因组编辑。在这项研究中，考虑到其在基因组编辑中的潜在应用，研究了一种新型的PT抗性标记。从紫外线诱变的A中选择对PT具有抗性的突变体。水稻RIB40。对突变体进行了全基因组分析，并鉴定了新的抗PT候选基因。该候选基因与粟酒裂殖酵母的硫胺素转运蛋白基因thi9相似，被命名为thiI。甲THII使用CRISPR / Cas9基因组编辑系统以研究其对PT电阻效应产生丧失功能的突变体。该突变体显示出PT抗性，并且没有生长缺陷或营养缺陷。的THII基因进一步研究其作为在基因组中共同编辑的选择标记使用。核糖核蛋白复合物，包含靶向thiI或另一个靶基因的重组Cas9核酸酶和sgRNA （wA制备或将sreA）同时引入米曲霉RIB40中。在含PT的培养基上有效地选择了thiI和靶基因双重功能丧失突变体。THII被证明是一个有用的标记基因的米曲霉用于基因组编辑使用。预期该研究将提供见解，这将促进米曲霉的基础研究和工业应用。