Overlapping promoter library designed for rational heterogenous expression in Cordyceps militaris

Cordyceps militaris, a kind of edible and medicinal fungus widely accepted in East Asia, has attracted much attention as a potential cell factory for producing adenosine analogs. Despite the rapid development in gene editing techniques and genome modeling, the diversity of DNA elements in C. militaris was too short to achieve rational heterogeneous expression for metabolic engineering studies.

In this study, P_trpC, a kind of promoter with a relatively appropriate expression level and small size, was selected as a monomer for promoter library construction. Through in vitro BioBricks assembly, 9 overlapping P_trpC promoters with different copy numbers as well as reporter gene gfp were connected and subsequently integrated into the genome of C. militaris. Both the mRNA transcription level and the expression level of gene gfp gradually increased along with the copy number of the overlapping promoter NP_trpC and peaked at 7. In the meantime, no significant difference was found in either the biomass or morphological characteristic of engineered and wild-type strains.

This study firstly expanded the overlapping promoter strategy used in model microorganism in C. militaris. It was a proof-of-concept in fungi synthetic biology and provide a general method to pushed the boundary of promoter engineering in edible mushroom.