In contrast, the putative loading module in TsnB1 is unusually composed of only an acyl carrier protein (ACP). The structure of the tailoring domains, ketoreductase and dehydratase, in the PKS genes is reasonable for the biosynthesis of the TSA skeleton. Bioinformatics analysis revealed three PKS genes ( tsnB1– 3) harboring three ketoacyl synthase domains that are responsible for three decarboxylative condensations in the chain elongation process, which utilizes two methylmalonyl-CoA and one malonyl-CoA as building blocks. The inserted DNA had a length of 41 194 bp and contained 26 open reading frames (ORFs), including the genes for type-I PKSs, PabAB, and PabC ( Supplementary Table 1 and Figure 2a). Next, we sequenced and analyzed the inserted DNA of cos10-4. albus::cos10-4 was confirmed using UHPLC and LC/MS, indicating that cos10-4 contains whole genes responsible for the biosynthesis of TSA ( Figure 2b). albus::cos10-4 was cultured for heterologous expression in TSA production medium for Streptomyces sp. A positive cosmid, named cos10-4, was integrated into the genome of Streptomyces albus G153 using the phiC31 integration site. RM72 using the cosmid vector pKU465cos (21) and screened the library for the cosmid containing the putative gene cluster for TSA biosynthesis. We constructed a cosmid library of Streptomyces sp. To clarify the gene cluster responsible for TSA biosynthesis, we performed a heterologous expression experiment. Using these genes as queries, we located a putative gene cluster for TSA biosynthesis spanning over 28.7 kb ( Figure 2a). According to the chemical structure of TSA, we hypothesized that the TSA skeleton is biosynthesized by a type-I polyketide synthase (PKS) and that the pab genes responsible for the biosynthesis of p-aminobenzoic acid (PABA), which is a possible starter unit for the PKS, constitute a biosynthetic gene cluster with the PKS genes. (20) Draft genome sequencing yielded 8.0 Mb of DNA sequences divided into 288 scaffolds. RM72, a producer of TSA and several TSA analogues ( Supplementary Figure 2). To identify the whole gene cluster responsible for TSA biosynthesis, we first performed genome scanning of a draft genome sequence of Streptomyces sp. In this paper, we describe the whole biosynthetic pathway of TSA, especially shedding light on the enzymatic machinery required for the formation of the terminal hydroxamic acid, which involves a nonproteinogenic amino acid, l-glutamic acid γ-monohydroxamate (GluHx), that is unprecedented in natural product biosynthesis. (16−19) However, no biosynthetic studies on TSA have been reported since its discovery.

Because of this property, TSA is generally utilized as a reagent in epigenetics research, and many studies on TSA have referred to biological activities. (15) Furthermore, the aliphatic moiety of TSA is suitable for the placement of the hydroxamic acid group near the active site of HDAC to enable efficient coordination of the zinc(II) ion by the hydroxamic acid moiety. (14) Crystallographic studies have demonstrated that the hydroxamic acid group of TSA is crucial to the mechanism of HDAC inhibition because of its ability to chelate the zinc(II) ion in the active site of HDAC. TSA was first reported as an antifungal antibiotic in 1976 (13) and rediscovered as a potent histone deacetylase (HDAC) inhibitor in 1990. In this context, trichostatin A (TSA, 1) ( Figure 1) is a suitable molecular target for elucidation of an unprecedented biosynthetic mechanism for the terminal hydroxamic acid moiety. (12) However, the biosynthesis of a terminal hydroxamic acid group is unknown. (11,12) Fosmidomycin or FR-900098 biosynthesis exemplifies this pathway involving acetylation or formylation, respectively ( Supplementary Figure 1a). (7−10) The biosynthetic pathway of the hydroxamic acid moiety in siderophores involves the oxidation of a primary amine by a FAD-dependent monooxygenase to give a hydroxylated amine, followed by acetylation or formylation of the resulting hydroxylated amine to give the hydroxamic acid. (5) The hydroxamic acid moiety is often found in the N-hydroxy peptides of siderophores (6) and rarely found in the terminal groups of molecules ( Figure 1). (1−4) Therefore, hydroxamic acid-containing natural products are promising drug lead compounds. Natural products that contain a hydroxamic acid moiety often exhibit inhibitory effects against various enzymes due to the important role of this moiety as an excellent ligand for metal ions.

Kudos origin