目的:这项工作旨在构建一个通用的,有效,和食品级根癌农杆菌介导的转化(ATMT)系统,用于在丝状真菌青霉(也称为黄青霉)中重组表达。
结果:在这项研究中,通过多位点测序分析,将野生型产黄假单胞菌VTCC31172菌株重新分类为P.rubens.Further,通过同源重组,在VTCC31172菌株中成功删除了尿苷/尿嘧啶生物合成所需的pyrG基因,以产生稳定的尿苷/尿嘧啶营养缺陷型突变体(ΔpyrG)。通过补充尿苷/尿嘧啶可以恢复P.rubensΔpyrG菌株的生长,并针对该菌株建立了基于尿苷/尿嘧啶营养缺陷机制的新型ATMT系统。对于106个孢子,最佳的ATMT效率可以达到1750个转化体(相当于0.18%)。此外,在共培养过程中,以0.005-0.02%的浓度补充尿苷/尿嘧啶可显着提高转化效率。尤其是,我们证明了曲霉菌米曲霉的pyrG标记和amyB启动子在P.rubensΔpyrG中具有完全功能。DsRed报告基因在米曲霉amyB启动子的调控下的表达在荧光显微镜下以强烈的红色信号照亮了P.rubens的菌丝体。此外,在amyB启动子的控制下,烟曲霉phyA基因的多个拷贝的基因组整合显着增强了P.rubens的植酸酶活性。
结论:我们的工作中开发的ATMT系统为在不使用耐药性标记的情况下在P.rubens中生产重组产品提供了安全的遗传平台。
OBJECTIVE: This work aimed to construct a versatile, effective, and food-grade Agrobacterium tumefaciens-mediated transformation (ATMT) system for recombinant expression in the filamentous fungus Penicillium rubens (also known as Pencillium chrysogenum).
RESULTS: In this study, the wild-type P. chrysogenum VTCC 31172 strain was re-classified as P. rubens by a multilocus sequencing analysis. Further, the pyrG gene required for uridine/uracil biosynthesis was successfully deleted in the VTCC 31172 strain by homologous recombination to generate a stable uridine/uracil auxotrophic mutant (ΔpyrG). The growth of the P. rubens ΔpyrG strain could be restored by uridine/uracil supplementation, and a new ATMT system based on the uridine/uracil auxotrophic mechanism was established for this strain. The optimal ATMT efficiency could reach 1750 transformants for 106 spores (equivalent to 0.18%). In addition, supplementation of uridine/uracil at the concentrations of 0.005-0.02% during the co-cultivation process significantly promoted transformation efficiency. Especially, we demonstrated that the pyrG marker and the amyB promoter from the koji mold Aspergillus oryzae were fully functional in P. rubens ΔpyrG. Expression of the DsRed reporter gene under the regulation of the A. oryzae amyB promoter lighted up the mycelium of P. rubens with a robust red signal under fluorescence microscopy. Furthermore, genomic integration of multiple copies of the Aspergillus fumigatus phyA gene under the control of the amyB promoter significantly enhanced phytase activity in P. rubens.
CONCLUSIONS: The ATMT system developed in our work provides a safe genetic platform for producing recombinant products in P. rubens without using drug resistance markers.