Studies On The Biosynthesis Mechanism Of The Fungal Alkaloid Brevianamides

Brevianamides is a class of prenylated indole alkaloids generated by Penicillium. or Aspergillus. Brevianamide A and Brevianamide D have significant insecticidal activity to the larvae of Spodoptera frugiperda, Heliothis virescens and other lepidopteran insects.In this study, the whole genome of Penicillium brevicompactum NRRL 864 was analyzed. Four genes related to the biosynthesis of Brevianamide A were dug out from Penicillium brevicompactum NRRL 864 using the gene cluster of notoamide A produced by Aspergillus versicolor as a probe through bioinformatics methods, and their functions were also investigated. Three genes except BvnA among the 4 genes related to the biosynthesis of Brevianamide A were cloned by polymerase chain reaction?PCR? using the cDNAs of Penicillium brevicompactum NRRL 864 as template. These 3 genes were cloned into pET28 b expression vector and transformed into E.coli BL21?DE3? to construct expressing bacteria, and the gene products were overexpressed after the expressing bacteria were induced by Isopropyl ?-D-thiogalactopyranoside?IPTG?, respectively. The recombinant soluble proteins of BvnB and BvnC were purified by Ni²⁺-NTA Resin, while recombinant BvnD protein appeared in inclusion bodies. No soluble BvnD protein was got even through co-expressing it with soluble tags or through refolding treatment.Finally, the substrate specificity, product structure and enzymatic kinetic of the reaction catalyzed by recombinant BvnC protein were analyzed. The results showed that only Brevianamide F was the appropriate substrate of recombinant BvnC in the tested 10 compounds, and BvnC protein was able to specifically catalyze the prenylation of Brevianamide F at the C-? position when dimethylallyl diphosphate?DMAPP? was used as the prenyl group donor. The product was structurally determined to be Deoxybrevianamide E using MS and NMR analysis. The apparent kinetic constants and kcat for Brevianamide F transformation reaction were 33.1 ?mol/L and 1.5/ min, respectively. Due to insolubility of recombinant Bvn D proteins, other related studying is still undegoing. This study laid foundations for studying the biosynthesis of Brevianamides and exploration of novel pesticides based on the structure of Brevianamides.