Development of an Efficient and Stable Transformation System for Aspergillus Oryzae Based on the pyrG Gene

Abstract Background Aspergillus oryzae is an ideal host for expressing heterologous and homologous genes. An efficient and stable transformation system is the key to the successful expression of the gene of interest in A. oryzae.Results To improve the expression efficiency of the gene of interest in A. oryzae, we constructed the uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG by Ultraviolet (UV) mutagenesis of pyrG gene deletion which would be used as a host for further transformation. In addition, a novel and efficient expression vector pBC-hygro.4 was constructed, including the pyrG cassette gene, His-Tag, amyB promoter and terminator,and green fluorescent protein GFP marker. pBC-hygro.4 transformed A. oryzae RIB40ΔpyrG efficiently via the PEG-CaCl2-mediated transformation method, and the stability of pBC-hygro.4 was tested by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verification, we successfully obtained a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG. At the same time, the developed vectors are fully functional for heterologous expression of the GFP fluorescent proteins in the A. oryzae RIB40ΔpyrG.Conclusion Our work provides a new method that can be applied to other filamentous fungi to develop similar fungal transformation systems based on auxotrophic/nutritional markers for food-grade recombination applications.