Abstract
The indolocarbazole (ICZ) alkaloids have attracted much attention due to their unique structures and potential therapeutic applications. A series of ICZs were recently isolated and identified from a marine-derived actinomycete strain, Streptomyces sanyensis FMA. To elucidate the biosynthetic machinery associated with ICZs production in S. sanyensis FMA, PCR using degenerate primers was carried out to clone the FAD-dependent monooxygenase gene fragment for ICZ ring formation, which was used as a probe to isolate the 34.6-kb DNA region containing the spc gene cluster. Sequence analysis revealed genes for ICZ ring formation (spcO, D, P, C), sugar unit formation (spcA, B, E, K, J, I), glycosylation (spcN, G), methylation (spcMA, MB), as well as regulation (spcR). Their involvement in ICZ biosynthesis was confirmed by gene inactivation and heterologous expression in Streptomyces coelicolor M1152. This work represents the first cloning and characterization of an ICZ gene cluster isolated from a marine-derived actinomycete strain and would be helpful for thoroughly understanding the biosynthetic mechanism of ICZ glycosides.
Highlights
The firstly reported indolocarbazole (ICZ) was staurosporine (STA, 1), which was isolated from the fermentation culture of Streptomyces staurosporeus AM-2282 (ATCC 55006) in 1977 (Scheme 1) [1].Since more than 130 ICZs have been isolated from various organisms, including bacteria, fungi and invertebrates, during the last 35 years [2,3,4]
The formation of ICZ rings involves four conserved enzymes; a pair of degenerate primers were designed according to the alignment result of RebC (CAC93716), StaC (BAF47693), AtmC
Sequencing results showed that the PCR-amplified product was very similar to known FAD-dependent monooxygenases for ICZ ring biosynthesis, with 59% identity to
Summary
The firstly reported indolocarbazole (ICZ) was staurosporine (STA, 1), which was isolated from the fermentation culture of Streptomyces staurosporeus AM-2282 (ATCC 55006) in 1977 (Scheme 1) [1]. 1,5-carbons of the glycosyl moiety and two indole nitrogens of K252c, the aglycones of streptocarbazoles A and B are linked to the 1,3-carbons of the glycosyl moiety, indicating that a novel enzymatic mechanism might be involved in the C–N bond formation between the C-3′ of deoxysugar and the N-12 of aglycone (Scheme 1). This exceptional cyclic N-glycosidic linkage prompted us to investigate the biosynthetic mechanism of ICZs compounds in the marine-derived S. sanyensis FMA. We report the cloning, characterization and heterologous expression of the spc gene cluster from S. sanyensis FMA
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