Establishment of Transformation System in Cordyceps Militaris by using Integration Vector with Benomyl Resistance Gene

To enhance the production of bioactive compounds efficiently from filamentous fungi, the secondary metabolism was activated by genetic engineering. laeA gene which is regarded as a global regulator of secondary metabolism in filamentous fungi was introduced into five strains of entomopathogenic fungi Cordyceps militaris. Since there was no reported transformation systemin in C. militaris, this research aimed at establishing the transformation system in this species. Benomyl and its resistance gene were adopted as a marker system to facilitate the introduction of laeA fragments into C.militaris under the control of Bauveria bassiana pGPD promoter. The minimum inhibitory concentration of benomyl against C.militaris was 1.5 μg/ml and a transformation efficiency of 7 colonies/μg DNA was obtained by the protoplast-PEG method with the vector pBT6, containing the benomyl resistance gene. Southern analysis of the transformants confirmed that pBT6 exists as an integration vector, and laeA integrated in the genome at random. The observation of the changes in metabolism of laeAtransformants was conducted using HPLC equipped with photodiode array method. The transformants harboring heterogeneous laeA showed enhanced productivity of secondary metabolites, compared to their wild type strains.