摘要

产黄青霉不仅是生产青霉素的工业上重要的丝状真菌，而且还是生产天然产物的有前途的细胞工厂。开发具有合适选择标记的高效转化系统对于黄褐青霉的遗传操作至关重要。在这项研究中，我们建立了一个新的高效的根癌农杆菌介导的转化（ATMT）系统，该系统具有两个不同的选择标记，赋予了对产黄青霉对神经丝菌素和新霉素的抗性。在乙酰丁酮浓度为200μM的22°C共培养60 h的最佳条件下，ATMT系统的转化效率可达到每106个孢子5009±96个转化子。获得的转化子可以用作T-DNA插入突变体，用于筛选参与形态发生和次级代谢的基因。特别地，所构建的ATMT系统成功地应用于在产黄青霉的野生株中产生laeA调节基因的敲除突变体和相关的互补株。我们的结果表明，LaeA调节剂控制着产黄青霉的生长，孢子形成，渗透胁迫响应和抗生素产生，但其功能依赖于氮源。此外，我们表明，来自柑橘采后病原体P. digitatum和工业真菌黑曲霉的laeA直系同源基因可以恢复产黄青霉LaeA缺失突变体的表型缺陷。最终，这项工作提供了一种新的ATMT系统，可用于T-DNA插入诱变，异源基因表达或与产黄青霉二级代谢相关的潜在基因的分子检查。

Penicillium chrysogenum is not only an industrially important filamentous fungus for penicillin production, but it also represents as a promising cell factory for production of natural products. Development of efficient transformation systems with suitable selection markers is essential for genetic manipulations in P. chrysogenum. In this study, we have constructed a new and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system with two different selection markers conferring the resistance to nourseothricin and phleomycin for P. chrysogenum. Under the optimized conditions for co-cultivation at 22 °C for 60 h with acetosyringone concentration of 200 μM, the transformation efficiency of the ATMT system could reach 5009 ± 96 transformants per 106 spores. The obtained transformants could be exploited as the T-DNA insertion mutants for screening genes involved in morphogenesis and secondary metabolism. Especially, the constructed ATMT system was applied successfully to generate a knockout mutant of the laeA regulatory gene and relevant complementation strains in a wild strain of P. chrysogenum. Our results indicated that the LaeA regulator controls growth, sporulation, osmotic stress response and antibiotic production in P. chrysogenum, but its function is reliant on nitrogen sources. Furthermore, we showed that the laeA orthologous genes from the citrus postharvest pathogen P. digitatum and from the industrial fungus Aspergillus niger could recover the phenotypic defects in the P. chrysogenum laeA deletion mutant. Conclusively, this work provides a new ATMT system, which can be employed for T-DNA insertional mutagenesis, heterologous gene expression or for molecular inspections of potential genes related to secondary metabolism in P. chrysogenum.

https://www.ncbi.nlm.nih.gov/pubmed/31627859