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Issue 10, 2009
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Molecular cloning, sequence analysis and functional characterization of the gene cluster for biosynthesis of K-252a and its analogs

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Abstract

Among the indolocarbazole alkaloids of antitumor antibiotics, K-252a represents a structurally unique indolocarbazoleglycoside 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 enzymaticassays. 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 vivobiotransformation 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.

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

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Article information


Submitted
19 Mar 2009
Accepted
24 Jun 2009
First published
04 Aug 2009

Mol. BioSyst., 2009,5, 1180-1191
Article type
Paper

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

H. Chiu, Y. Chen, C. Chen, C. Jin, M. Lee and Y. Lin, Mol. BioSyst., 2009, 5, 1180
DOI: 10.1039/B905293C

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