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

<i>Cordyceps militaris</i> 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 (Δ<i>pyrG</i>) in five homokaryotic <i>C. militaris</i> strains. The efficiency of the <i>pyrG</i> deletion by homologous recombination reached 100% in all the <i>C. militaris</i> strains. Genetic transformation of the <i>C. militaris</i> Δ<i>pyrG</i> strains mediated by <i>Agrobacterium tumefaciens</i> using the native <i>pyrG</i> auxotrophic marker resulted in high transformation yields of 109-810 transformants per 10⁵ conidia. Additionally, the <i>pyrG</i> marker from <i>Aspergillus oryzae</i> was also functional for the genetic transformation of <i>C. militaris</i> Δ<i>pyrG.</i> We further showed that the <i>gpd1</i> and <i>tef1</i> genes were strongly expressed during the mycelial growth of <i>C. militaris</i>, 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 <i>CmFE</i> gene encoding a serine protease under the regulation of the <i>tef1</i> promoter significantly increased the activity of this enzyme in <i>C. militaris</i>. Our work provides a promising platform for food-grade recombinant expression in <i>C. militaris</i>.