Cloning of the biosynthetic gene cluster for naphthoxanthene antibiotic FD-594 from Streptomyces sp. TA-0256

Abstract

FD-594 is an unique pyrano [4¢,3¢:6,7]naphtho [1,2-b]xanthene polyketide with a trisaccharide of 2,6-dideoxysugars. In this study, we cloned the FD-594 biosynthetic gene cluster from the producer strain Streptomyces sp. TA-0256 to investigate its biosynthesis. The identified pnx gene cluster was 38 143 bp, consisting of 40 open reading frames, including a minimal PKS gene, TDP-olivose biosynthetic genes, two glycosyltransferase genes, two methyltransferase genes and many oxygenase/reductase genes. Most of these enzymes coded in the pnx cluster were reasonably assigned to a plausible biosynthetic pathway for FD-594, in which an unique ring opening process via Baeyer-Villiger-type oxidation catalyzed by a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase, is speculated to lead to the unique xanthene structure. To clarify the involvement of pnx genes in the FD-594 biosynthesis, a glycosyltransferase, PnxGT2, and a methyltransferase, PnxMT2, were characterized enzymatically with the recombinant proteins expressed in Escherichia coli. As a result, PnxGT2 catalyzed the triple olivose transfers to the FD-594 aglycon with TDP-olivose as the glycosyl donor to afford triolivoside. Surprisingly, in the PnxGT2 enzymatic reaction, tetraolivoside and pentaolivoside were significantly detected along with the expected triolivoside. To our knowledge, PnxGT2 is the first contiguous oligosaccharide-forming glycosyltransferase in secondary metabolism. Furthermore, addition of PnxMT2 and S-adenosyl-L-methionine into the PnxGT2 reaction mixture afforded natural FD-594 to confirm that the PnxGT2 reaction product was the expected regiospecifically glycosylated compound. Consequently, the identified pnx gene cluster appears to be involved in FD-594 biosynthesis.

Expanding our Understanding of Sequence-Function Relationships of Type II Polyketide Biosynthetic Gene Clusters: Bioinformatics-Guided Identification of Frankiamicin A from Frankia sp. EAN1pec

“…Biosynthesis of the polyketide core structures of seven of the sixteen pentangular, tetracenomycin, or related unique training set compounds are known or predicted to be initiated by incorporation of a non-acetate starter unit [ 38 , 39 – 42 ]. In each case, a type III ketosynthase or stand-alone adenylation domain is present in the gene cluster.…”

“…It is therefore possible that ABM superfamily members FkmO1 and FkmO2 and their homologues may be involved in pentangular polyketide cyclization. In light of the conservation of homologues of both proteins in the eleven pentangular clusters sequenced thus far [ 5 , 6 , 39 – 42 , 55 – 60 ] but not in tetracenomycin class clusters; and the conserved adjacent co-directional arrangement of their encoding genes, we hypothesize that both FkmO1 and FkmO2 and their homologues are immediate tailoring enzymes that may be involved in B ring oxygenation and/or E ring cyclization and aromatization ( Fig. 8 , Table 2 ).…”

“…A cytochrome P450 monooxygenase PdmJ was shown to introduce a hydroxyl group at the C-5 position in the biosynthesis of pradimicin [ 62 ]. This modification is not conserved in pentangular pathways, but also likely occurs in FD-594 biosynthesis [ 42 ] based on the presence of a C-5 hydroxyl in the structure and a close homologue of PdmJ in the cluster. Surprisingly, a likely candidate for C-5 hydroxylation of G-2A to generate frankiamicin A is absent from both the Frankia sp.…”

Polycyclic xanthone natural products: structure, biological activity and chemical synthesis

“…A similar study on FD-594 ( 5 ) suggested rather an epoxidation followed by a Baeyer-Villiger oxidation was involved in the formation of the xanthone ring. 16 …”

Seamless stitching of biosynthetic gene cluster containing type I polyketide synthases using Red/ET mediated recombination for construction of stably co-existing plasmids

“…Many type I PKS gene clusters and non-ribosomal peptide gene clusters are very large, for example, the biosynthetic gene cluster of type I polyketides for biosynthesis of a 51-membered macrolide complex in Streptomyces ambofaciens can be as large as 150 kb (Laureti et al, 2011b), which makes it difficult to accommodate the gene clusters in one single cosmid due to size limitation (Hu et al, 2005;Kudo et al, 2011;Lohman et al, 2013). Although bacterial artificial chromosome (BAC) vectors are widely used to carry large gene clusters such as type I polyketide gene clusters and non-ribosomal peptide gene clusters (Miao et al, 2005;Perlova et al, 2006a;Molnar et al, 2000), manipulation of very large fragments on single vector remains challenging, and the production titer may be limited by the low copy number of the BAC vector.…”