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

Hsien-Tai Chiu, Yi Lin Chen, Chien Yu Chen, Chyn Jin, Meng Na Lee, Yu Chin Lin

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1180-1191
Number of pages12
JournalMolecular BioSystems
Volume5
Issue number10
DOIs
Publication statusPublished - 2009 Sep 28

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Molecular Cloning
Multigene Family
Sequence Analysis
Alkaloids
Escherichia coli
Genes
staurosporine aglycone
Biosynthetic Pathways
Enzyme Assays
Glycosides
Biotransformation
Tryptophan
Open Reading Frames
Anti-Bacterial Agents
Enzymes

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Molecular Biology

Cite this

Chiu, Hsien-Tai ; Chen, Yi Lin ; Chen, Chien Yu ; Jin, Chyn ; Lee, Meng Na ; Lin, Yu Chin. / Molecular cloning, sequence analysis and functional characterization of the gene cluster for biosynthesis of K-252a and its analogs. In: Molecular BioSystems. 2009 ; Vol. 5, No. 10. pp. 1180-1191.
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Molecular cloning, sequence analysis and functional characterization of the gene cluster for biosynthesis of K-252a and its analogs. / Chiu, Hsien-Tai; Chen, Yi Lin; Chen, Chien Yu; Jin, Chyn; Lee, Meng Na; Lin, Yu Chin.

In: Molecular BioSystems, Vol. 5, No. 10, 28.09.2009, p. 1180-1191.

Research output: Contribution to journalArticle

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