Generating a construct harboring the rpb1 promoter for enhancing recombinant protein expression in the medicinal mushroom Cordyceps militaris

Abstract

Cordyceps militaris is a valuable medicinal mushroom that contains numerous bioactive ingredients such as cordycepin, adenosine, pentostatin, polysaccharides, and carotenoids. However, the contents of these substances in wild-type strains of C. militaris are relatively low. Recently, the genome of C. militaris has been fully sequenced, and genes involved in the biosynthesis of beneficial constituents have been identified. Therefore, research on enhancing the biosynthesis of the bioactive compounds in this fungus by recombinant DNA technology has advantages. In the present study, we have successfully generated a binary vector with a T-DNA region carrying a construct regulated by the native rpb1 promoter that can enhance gene expression in C. militaris. The T-DNA structure from the binary vector was then transferred into the C. militaris genome by Agrobacterium tumefaciens-mediated transformation. Analysis of some fungal transformants by PCR using the specific primer pairs indicated that the T-DNA structure was successfully integrated into the fungal genome. Observation of the transformants under fluorescence microscopy confirmed the overexpression of the DsRed fluorescent protein under the regulation of the rpb1 promoter. The binary vector carrying the rpb1 promoter constructed in this study can be employed to promote the expression of target genes in the medicinal mushroom C. militaris.