An efficient Agrobacterium-mediated system based on the pyrG auxotrophic marker for recombinant expression in the filamentous fungus Penicillium rubens

• Published in: Biotechnology letters Vol. 45; no. 5-6; pp. 689 - 702
• Main Authors: Tran, Van-Tuan, Thai, Hanh-Dung, Vu, Tao Xuan, Vu, Ha Hong, Nguyen, Giang Thu, Trinh, Minh Thi, Tran, Huyen Thi Thanh, Pham, Huong Thi Thu, Le, Nhung Thi Hong
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.06.2023; Springer Nature B.V
• Subjects: Agrobacterium tumefaciens - genetics; Antibiotics; Applied Microbiology; Biochemistry; Biomedical and Life Sciences; Biosynthesis; Biotechnology; Drug resistance; Enzymes; Fluorescence; Fluorescence microscopy; Fungi; Genes; Genetic engineering; Genetic transformation; Genomes; Homologous recombination; Life Sciences; Metabolites; Microbiology; Original Research Paper; Penicillin; Penicillium - genetics; Penicillium - metabolism; Penicillium rubens; PHYA gene; Phytase; PyrG gene; Reporter gene; RNA polymerase; Sequence analysis; Spores; Transformation, Genetic; Uracil; Uracil - metabolism; Uridine; Uridine/uracil auxotrophic; Recombinant expression; Phytase; promoter; mediated transformation; DsRed fluorescent protein; AmyB promoter; Agrobacterium tumefaciens-mediated transformation; Uridine/uracil auxotrophic Penicillium rubens
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-023-03374-y

Objectives 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 (Δ pyrG ). The growth of the P. rubens Δ pyrG 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 10 6 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 Δ pyrG . 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.