Efficient generation of uridine/uracil auxotrophic mutants in homokaryotic Cordyceps militaris strains for constructing a food-grade expression platform.

Cordyceps militaris is a well-known medicinal mushroom widely exploited in traditional medicine and nutraceuticals. In this study, we aim to establish a new platform for improving the production of beneficial ingredients in this fungus. We successfully generated uridine/uracil auxotrophic mutants (ΔpyrG) in five homokaryotic C. militaris strains. The efficiency of the pyrG deletion by homologous recombination reached 100% in all the C. militaris strains. Genetic transformation of the C. militaris ΔpyrG strains mediated by Agrobacterium tumefaciens using the native pyrG auxotrophic marker resulted in high transformation yields of 109-810 transformants per 10⁵ conidia. Additionally, the pyrG marker from Aspergillus oryzae was also functional for the genetic transformation of C. militaris ΔpyrG. We further showed that the gpd1 and tef1 genes were strongly expressed during the mycelial growth of C. militaris, and their promoter sequences were integrated into binary vectors for enhancing recombinant expression. With the constructed platform, the strong heterologous expression of the DsRed protein was proven, and the genomic integration of the endogenous CmFE gene encoding a serine protease under the regulation of the tef1 promoter significantly increased the activity of this enzyme in C. militaris. Our work provides a promising platform for food-grade recombinant expression in C. militaris.