Molecular cloning, sequence analysis and functional characterization of the gene cluster for biosynthesis of K-252a and its analogs

Among the indolocarbazole alkaloids of antitumor antibiotics, K-252a represents a structurally unique indolocarbazole glycoside and exhibits potent neuroprotective and broad anticancer activities. K-252a consists of K-252c and the unusual dihydrostreptose moiety, linked together with oxidative and glycosidic C-N bonds. Herein, we reported a complete sequence of an approximately 45 kb genomic fragment harboring the gene cluster for the biosynthesis of indolocarbazole alkaloids in Nocardiopsis sp. K-252 (NRRL15532). The sequence of 35 open reading frames discovered several new, critical genes, hence shedding new light on biosynthesis, resistance and regulation of K-252a and its analogs. To functionally characterize the gene cluster in vitro and in enzyme level, a multigene expression cassette containing the K-252c biosynthetic genes was constructed and successfully overexpressed in Escherichia coli to yield soluble proteins for cell-free tandem enzymatic assays. Consequently, the heterologous expression with soluble NokA and NokB led to in vitro production of chromopyrrolic acid (CPA), thereby providing functional evidence for K-252c biosynthesis. Moreover, a facile production of CPA in culture broth was successfully accomplished by using an in vivo biotransformation of L-tryptophan with E. coli harboring the gene cassette. Importantly, by sequence analysis and the functional characterization here and in the companion paper, biosynthetic pathways leading to formation of K-252a and its analogs were hence proposed. Together, the results provide critical information and materials useful for combinatorial biosynthesis of K-252a and its analogs for therapeutic applications.