Construction of Aspergillus Oryzae food-grade expression system based on auxotrophic markers

Abstract

ABSTRACT Aspergillus oryzae is an ideal host for expressing heterologous and homologous genes. Due to the risk of antibiotics to food safety, it is banned for use in resistance screening with food-grade expression system. Therefore there merit and value in exploring the use of Aspergillus oryzae food-grade expression system based on auxotrophic markers. In this study uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG were generated by ultraviolet mutagenesis of pyrG gene deletion which would then be used as a host for further transformation analysis and applications. Meanwhile, a novel and efficient expression vector pBC-hygro.4 was constructed, which included the pyrG cassette gene, His-Tag, amyB promoter and terminator, and green fluorescent protein GFP marker. pBC-hygro.4 was successfully transferred to A. oryzae RIB40ΔpyrG via the PEG-CaCl2-mediated transformation method, and the stability of pBC-hygro.4 was evaluated by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verification, a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG were successfully generated. In addition, the developed vectors were fully functional for heterologous expression of the GFP fluorescent proteins in A. oryzae RIB40ΔpyrG and the recombinant A. oryzae RIB40ΔpyrG cultures indicated pronounced green fluorescence in the mycelia. Based on auxotrophic/nutritional markers, this study provides an effective method that can be applied to develop similar fungal transformation systems in other filamentous fungi, which will be beneficial for food-grade applications.