Gentic overexpression increases production of hypocrellin A in Shiraia bambusicola S4201.

Hypocrellin A (HA) is a perylenequinone (PQ) isolated from Shiraia bambusicola that shows antiviral and antitumor activities, but its application is limited by the low production from wild fruiting body. A gene overexpressing method was expected to augment the production rate of HA in S. bambusicola. However, the application of this molecular biology technology in S. bambusicola was impeded by a low genetic transformation efficiency and little genomic information. To enhance the plasmid transformant ratio, the Polyethylene Glycol-mediated transformation system was established and optimized. The following green fluorescent protein (GFP) analysis showed that the gene fusion expression system we constructed with a GAPDH promoter Pgpd1 and a rapid 2A peptide was successfully expressed in the S. bambusicola S4201 strain. We successfully obtained the HA high-producing strains by overexpressing O-methyltransferase/FAD-dependent monooxygenase gene (mono) and the hydroxylase gene (hyd), which were the essential genes involved in our putative HA biosynthetic pathway. The overexpression of these two genes increased the production of HA by about 200% and 100%, respectively. In general, this study will provide a basis to identify the genes involved in the hypocrellin A biosynthesis. This improved transformation method can also be used in genetic transformation studies of other fungi.