| Neocarazostatin A is a microbial origin tricyclic carbazole alkaloid, which possesses strong free radical scavenging activity. It contains a carbazole unit and an unusual pentenylation at C-6 position, without other polycyclic or monocyclic. Previous labelling studies suggested that the biosynthesis mechanism of microbial origin tricyclic carbazole alkaloids is different from that of the reported carbazole alkaloids. In this study, through chemical mining and fermentation analysis, neocarazostatin A was isolated from the metabolites of soil bacterium Streptomyces sp. MA37, and its biosynthetic pathway was identified and characterized in following aspects.First of all, YerE was employed as a probe to mine the genome sequence of MA37 for seeking the biosynthetic gene cluster of neocarazostatin A. Through the gene insertional inactivation, we identified a candidate biosynthetic gene cluster of neocarazostatin A, which was laterdesignated as nzs. The nzs gene cluster possesses 10 ORFs spanning approximately 17.8 kb of the chromosome region. Bioinformatic analysis indicated that, of the ten gene products encoded by nzs, NzsB-F are responsible for the precursor biosynthesis, NzsH-J are responsible for the carbon skeleton assembly, while NzsG and NzsA may be involved in the tailoring steps.Secondly, we have characterized the prenyltransferase NzsG through in vivo and in vitro experiments. Bioinformatic analysis showed that NzsG is ahomologous protein of the phytoene synthase family. The gene nzsG was in-frame deleted, resulting the accumulation of streptoverticillin and precarazostatin in the △nzsG mutant. Mass spectrum and NMR analysis of the structures of the two compounds showed that both of them contain no prenyl moiety at C-6, suggesting that NzsG is actually a kind of prenyltransferase. Further in vitro experiments showed that precarazostatin but not streptoverticillin can be converted into neocarazostatin B, suggesting that precarazostatin is the real intermediate in the biosynthesis of neocarazostatin A, while streptoverticillin is just a by-product. Additionally, biochemical characterization of NzsG with precarazostatin, indole derivatives and FPP as the substrates was performed. The results indicated that NzsG is a metal ion dependent and carbazole ring specific prenyltransferase with strict substrate specificity. To our knowledge, NzsG is the first verified carbazole prenyltransferase so far.We then characterized the function of the putative hydroxylase NzsA. Bioinformatic analysis indicated that nzsA encodes a P450 monooxygenase. In-frame deletion of nzsA resulted the accumulation of neocarazostatin B and 16,17-epoxyneocarazostatin B in the AnzsA mutant. Structural analysis showed that both compounds contain no hydroxyl group at C-10, suggesting that NzsA is a hydroxylase. We then purified NzsA and tested its hydroxylation activity by incubating the enzyme with neocarazostatin B or 16,17-epoxyneocarazostatin B. LC-MS analysis revealed that only neocarazostatin B can be transformed into neocarazostatin A by NzsA, suggesting that 16,17-epoxyneocarazostatin B is a by-product and neocarazostatin A is the real intermediate of the pathway. Following the above experiments, in vitro cascade reaction catalyzed by both NzsA and NzsG with precarazostatin was performed. The result indicated that precarazostatin can be converted into neocarazostatin A via neocarazostatin B, confirming that the prenylation catalyzed by NzsG occurs prior to the hydroxylation catalyzed by NzsA. In this way, NzsA and NzsG together are respossible for the cascade tailoring steps in the biosynthesis of neocarazostatin.We also studied the functions of other genes in nzs cluster. Knockout of the putative precursor biosynthesis related genes nzsB, nzsC or nzsD did not affect the production of neocarazostatin A in the mutants. On the other hand, the inactivation of nzsE, nzsF or the three carbon skeleton assembly genes nzsH, nzsl and nzsJ led to complete abolishment of the production of neocarazostatin A. Together, these results demonstrated that nzsB-D are non-essential genes, and other genes are vital for the biosynthesis of neocarazostatin A.Based on the above results, the biosynthetic pathway of neocarazostatin A was proposed. Tryptophan is converted into indole-3- pyruvic acid through transamination, then the C-2 and C-13 unit arc loaded onto the indole-3-pyruvic acid from pyruvic acid, which condensed with the 3-hydroxy butyryl-ACP to complete the assembly of the carbazole carbon skeleton. Then the generated intermediate undergoes the sequential decarboxylation, cyclization, rearrangement, oxidation, reduction, and methylation to generate precarazostatin, which further undergoes the cascade prenylation and hydroxylation catalyzed by NzsG and NzsA to yield the final product neocarazostatin A.In summary, we revealed the biosynthetic pathway of a bacterial origin tricyclic carbazole alkaloid for the first time. Especially, we have characterized the first carbazole ring specific prenyltransferase NzsG, and demonstrated the special cascade reactions catalyzed by both NzsG and the hydroxylase NzsA in the tailoring steps of the biosynthesis of neocarazostatin A. This study provides not only the important clues for further uncovering of the mechanism of the "ring A" formation, but also the basis for modeling of the biosynthetic pathway of the other members of bacterial tricyclic carbazole alkaloid family. |
