NRPS (non-ribosomal pepetide synthetase) plays an important role in the biosynthesis of secondary metabolites, which includes economically important compounds such as antibiotics, antitumor agents, immune-suppressants, siderophores, toxins, etc. In most cases, however, their functions and biosynthesizing pathways have been still remained riddles because of their cryptic expression and recalcitrant genetics. In our previous genome analyses of Cordyceps bassiana C101, we predicted the sequences of secondary metabolite biosynthesizing gene clusters, which contain non-ribosomal pepetide synthetases whose functions are already known such as beauvericin, bassianolide, bassiatin, tenellin, beauverolides, and oosporein. The authors focused on nrps6 that has not been functionally studied. The single-gene deletion and overexpression mutants were established via Agrobacterium-mediated transformations. PCR, quantitative real-time PCR, and southern blot analyses were carried out to confirm the correct single-gene integrated homologous recombination. In further study, comparative metabolic profiling between wild-type and mutant strains will be followed by their detailed chemical structure analysis through NMR spectroscopy.

Cordyceps bassiana (asexual stage Beauveriabassiana) is a widely distributed entomopathogenic fungus that infects hundreds of various insects. It produces a number of secondary metabolites, including insect toxins beauvericin and bassianolide and pigment tenellin. In the present study, members of NRPS and PKS-NRPS hybrid genes were predicted from whole genome sequences of eighteen different fungal species belonging to five families of Hypocreales (Cordycipitaceae, Clavicipitaceae, Ophiocordycipitaceae, Hypocreaceae and Nectriaceae) using antiSMASH ver. 1.2.2. Phylogeny using all predicted adenylation (A) domains was drawn in order to identify potential orthologous genes of beauvericin synthetase (BbBEAS) and bassianolide synthetase (BbBSLS) in Hypocreales. The orthologs were examined to understand the evolution of beauvericin and bassianolide synthetase genes. Phylogenetic tree shows grouping of A1 and A2 domains of BbBEAS, BbBSLS and enniatin synthetases in two separate clades indicating the origin of genes from fusion of two distantly related modules. Species phylogeny of C. bassiana and its allies is congruent with gene trees of BbBEAS and BbBSLS, suggesting that the gene fusion event predates the species divergence. In the inferred phylogeny, A1 domain of BbBEAS showed highest similarity with that of BbBSLS, followed by those of Fusarium equiseti enniatin synthetase and Xylariasp. bassianolide synthetase. Syntenic analysis of beauvericin gene clusters of C. bassiana and closely related species represents that C. militaris lost the BbBEAS gene in evolution. Synteny of bassianolide gene clusters shows the loss of NRPS genes encoding BbBSLS in B. pseudobassiana, Isaria tenuipes, I. farinosa and C. pruinosa. BbBEAS and 2-ketoisovalerate reductase (kivr) genes are found conserved in beauvericin and enniatin synthesizing gene clusters.