Biosynthesis Of Dimerized Cyclodipeptide Alkaloids

The structural diversity of natural products stems from the fact that they can be built into complex and highly functional skeletons using simple modules.Natural products,especially microbial natural products,have always been an important source of discovery and development of new drugs.The use of natural products as lead compounds is an important way for drug research and development.The alkaloid natural products containing the diketopiperazine structure represent a large class of secondary metabolites mainly produced by microorganisms,and its stable six-membered ring skeleton structure makes diketopiperazine compounds an important drug effect group in medicinal chemistry.Diketopiperazines are classified into non-ribosomal peptide synthetases and cycliodipeptide synthetases by the mechanism of biosynthesis.The cycliodipeptide synthetases directly use the aminoacyl-t RNA as a substrate to produce a cycliodipeptide which is typically further modified by at least one custom enzyme,like cytochrome P450,found in the corresponding cycliodipeptide synthase gene cluster.Nasc B is one of the cytochrome P450 which is used to catalyze the synthesis of pyrroloindole structures.In the biosynthesis of Naseseazine C,this C3-aromatic pyrroloindole alkaloid can be synthesized by only one cycliodipeptide synthase and one cytochrome P450 enzyme-Nasc B.In this study,the P450 genes containing a tryptophan cycliodipeptide synthetase are screened from the gene bank by genomic mining method,and their phylogenetic relationships are simulated on the phylogenetic tree.The P450 gene and its corresponding cycliodipeptide synthetases are constructed into mycobacteria for co-expression.The functions of a series of cycliodipeptide synthetases and P450 enzymes were identified,and a variety of novel natural products were discovered through substrate screening.Subsequently,the protein of CYP450 was engineered by means of homologous modeling and residue substitution to find some important sites for substrate binding.This study provides a new reference for functionalized natural product skeletons and mining more biologically active natural products.In this study,we established a mycobacterial catalytic system,verified the functions of a series of genes,analyzed the structure of some products,and obtained a variety of non-natural dimerized cycliodipeptide alkaloids,which extended the pyrroindole family.This strategy of biochemical combination has greatly improved the discovery process of natural products,provides ideas for mining new natural products,and it’s of great theoretical significance and important application value for the discovery of new microbial drugs.