Characterization of the Xiamenmycin Biosynthesis Gene Cluster in Streptomyces xiamenensis 318

Abstract

Xiamenmycin (1) is a prenylated benzopyran derivative with anti-fibrotic activity. To investigate the genetic basis of xiamenmycin biosynthesis, we performed genome mining in the xiamenmycin-producing Streptomyces xiamenensis wild-type strain 318 to identify a candidate gene cluster. The complete gene cluster, consisting of five genes, was confirmed by a series of gene inactivations and heterologous expression. Based on bioinformatics analyses of each gene and feeding experiments, we found that the structure of an intermediate xiamenmycin B (3) accumulated in a ximA inactivation mutant, allowing us to propose a biosynthetic pathway. All five of the genes in the pathway were genetically and biochemically characterized. XimA was biochemically characterized as an ATP-dependent amide synthetase, catalyzing an amide bond formation in the presence of ATP as the final step in Xiamenmycin biosynthesis. The K m value of XimA was determined to be 474.38 µM for the substrate xiamenmycin B. These studies provide opportunities to use genetic and chemo-enzymatic methods to create new benzopyran derivatives as potential therapeutic agents.

Deciphering the streamlined genome of Streptomyces xiamenensis 318 as the producer of the anti-fibrotic drug candidate xiamenmycin

“…All of the five genes that are responsible for xiamenmycin biosynthesis ( ximA-E ) in S. xiamenensis 318 have a high sequence similarity with their counterparts in S. himastatinicus ATCC 53653 17 . Excluding the homologues in S. himastatinicus ATCC 53653, the sequence similarities of the five xim genes obtained through a BlastP search against GenBank were less than 30%.…”

“…The genome-scale metabolic flux maps of S. lividans TK24 growing under various physiological conditions have been obtained 31 . Because the xiamenmycin gene cluster can be expressed heterologously in S. lividans 17 , it is convenient to rapidly evaluate the flux distribution by adopting the metabolic model developed in the model S. lividans strain.…”

“…S. xiamenensis 318 was first isolated from the mangrove soil as a novel species of the Streptomyces genus 13 and produces an anti-fibrotic drug lead, xiamenmycin 14 15 16 . The biosynthetic pathway of xiamenmycin was elucidated 17 , indicating a close connection between its biosynthesis and primary metabolism. In this study, we reported the whole genome sequence of S. xiamenensis 318 in a 5.96 Mb single contig, which is considerably smaller than other reported complete genomes of Streptomyces species.…”

In vitro reconstitution of indolmycin biosynthesis reveals the molecular basis of oxazolinone assembly

“…Thus, Ind3 is functionally distinct from PCLs and more similar to a group of amide ligase or synthetase enzymes, such as NovL, McbA, and XimA, involved in the biosynthesis of novobiocin, marinacarbolines, and xiamenmycin, respectively. All of these enzymes activate carboxylates for nucleophilic attack by donor amino groups to bring together monomers (26)(27)(28). However, phylogenetic analysis reveals that Ind3 is distinct from those enzymes (SI Appendix, Fig.…”

In Vivo Metabolism Study of Xiamenmycin A in Mouse Plasma by UPLC-QTOF-MS and LC-MS/MS

“…The absolute configurations of xiamenmycins were determined by extensive spectroscopic data analyses, Mosher’s method, Marfey’s reagent and quantum mechanical calculations [ 8 ]. The biosynthetic pathway was elucidated, and all of the genes in the pathway were genetically and biochemically characterized [ 12 ]. Spontaneous rifampicin resistance combined with streptomycin resistance by introducing a mutated rpsL gene into S. xiamenensis was performed to increase the production of secondary metabolites [ 9 ].…”