Discovery of a family of g -aminobutyrate ureas via rational derepression of a silent bacterial gene cluster †

of a family of γ -aminobutyrate ureas via rational derepression of a silent bacterial gene cluster inactivation a c repressor. Gaburedins, a family of g -aminobutyrate (GABA)-derived ureas, have been discovered by deletion of gbnR , an arpA -like putative transcriptional repressor in Streptomyces venezuelae ATCC 10712. Comparison of metabolite pro ﬁ les in the wild type and mutant strains revealed six metabolites in the mutant that are lacking from the wild type. The structure of gaburedin A was established by HRMS combined with 1- and 2-D NMR spectroscopy and was con ﬁ rmed by total synthesis. The other metabolites were con ﬁ rmed as congeners using HRMS, MS/MS and feeding of putative biosynthetic precursors. Two genes, gbnA and gbnB , are proposed to be involved in gaburedin biosynthesis. Consistent with this hypothesis, deletion of gbnB in the gbnR mutant abolished gaburedin production. This is the ﬁ rst report to disclose the discovery of novel natural products via rational deletion of a putative pathway-speci ﬁ c regulatory gene.

Discovery of a family of g-aminobutyrate ureas via rational derepression of a silent bacterial gene cluster † John D. Sidda, ab Lijiang Song, a Vincent Poon, ab Mahmoud Al-Bassam, c Orestis Lazos, a Mark J. Buttner, c Gregory L. Challis a and Christophe Corre * ab Gaburedins, a family of g-aminobutyrate (GABA)-derived ureas, have been discovered by deletion of gbnR, an arpA-like putative transcriptional repressor in Streptomyces venezuelae ATCC 10712. Comparison of metabolite profiles in the wild type and mutant strains revealed six metabolites in the mutant that are lacking from the wild type. The structure of gaburedin A was established by HRMS combined with 1-and 2-D NMR spectroscopy and was confirmed by total synthesis. The other metabolites were confirmed as congeners using HRMS, MS/MS and feeding of putative biosynthetic precursors. Two genes, gbnA and gbnB, are proposed to be involved in gaburedin biosynthesis. Consistent with this hypothesis, deletion of gbnB in the gbnR mutant abolished gaburedin production. This is the first report to disclose the discovery of novel natural products via rational deletion of a putative pathway-specific regulatory gene.
Streptomyces are soil-dwelling, lamentous bacteria that produce a remarkably diverse range of bioactive natural products. Such molecules nd use in the treatment of infectious diseases, cancer and transplant rejection, as well as in agriculture. Indeed, almost 70% of commercially-available antibiotics originate from the Streptomyces genus. 1 Classical methods for identication of novel natural products have focused on screening cultures for specic bioactivities. However, these approaches oen result in the rediscovery of known compounds. Alternative strategies are therefore required for the discovery of novel bioactive natural products from microbial sources. 2 The recent explosion in the availability of bacterial genome sequences has led to the identication of many cryptic natural product biosynthetic gene clusters. 3 These offer a promising resource for novel natural product discovery. 3 However, many cryptic biosynthetic gene clusters are expressed poorly or not at all under laboratory growth conditions. 4,5 Rational approaches are therefore required to induce the expression of such silent biosynthetic pathways. In addition to biosynthetic and self-resistance genes, single or multiple genes encoding transcriptional regulators (activators or repressors) are typically present in bacterial natural product biosynthetic gene clusters. 5 Recently, it has been reported that the constitutive expression of a putative pathway-specic transcriptional activator results in the production of a novel complex of macrolide antibiotics by S. ambofaciens. 4 A similar strategy has been employed in Aspergillus nidulans to discover the product of a cryptic biosynthetic pathway. 6 Deletion of putative bacterial pathway-specic transcriptional repressors has been investigated as a complementary approach, but has yet to result in the discovery of novel metabolites. 7,8 ArpA-like transcriptional repressors belong to the TetR-family of DNA-binding proteins and are known to control the production of a wide variety of antibiotics. 9,10 ArpA itself controls the production of streptomycin in Streptomyces griseus. 11 The biosynthesis of several other natural products is repressed by ArpA-like proteins and inactivation of arpA-like genes generally boosts production levels, e.g. deletion of mmyR in Streptomyces coelicolor has been shown to result in the overproduction of methylenomycin antibiotics. 12 ArpA-like proteins have been shown to sense and respond to specic ligands such as g-butyrolactones, 2-alkyl-4-hydroxymethylfuran-3-carboxylic acids (AHFCAs) and butenolides. [13][14][15][16][17] In particular, the methylenomycin furans, assembled by the products of the mmfLHP genes, act as methylenomycin production inducers in S. coelicolor. These signaling molecules are thought to interact with the ArpA-like transcriptional repressors MmfR and/ or MmyR. 12,14 Even though MmfR and MmyR share 35% identity and 56% similarity, the phenotypes of mmfR and mmyR mutants are distinct. The strain lacking mmyR overproduces the methylenomycin antibiotics, whereas the strain lacking mmfR exhibits a phenotype similar to the wild type strain. 12 BLAST searches revealed that several Streptomyces genomes contain a cassette of ve genes homologous to mmfR-mmfLHP-mmyR. These are proposed to be involved in the transcriptional regulation of specic natural product biosynthetic pathways. 14 Thus, inactivation of the mmyR homologues in these genomes was predicted to result in induction of the biosynthetic gene clusters under their control and biosynthesis of the corresponding metabolic products.
The genome sequence of Streptomyces venezuelae ATCC 10712 became publicly available in 2012, revealing a cluster of genes that contains the mmyR homologue gbnR (sven_4187), as well as the mmfR homologue sgnR (sven_4182) and the mmfLHP homologues sgnLPH (sven_4183, 4185 and 4188; Fig. 1). This putative regulatory system is located adjacent to gbnABC (sven_4179-4181), which encode potential natural product biosynthetic and export proteins.
In silico analysis of the gbn gene cluster revealed highly conserved DNA sequences in the sgnR-sgnL and gbnR-sgnH intergenic regions as well as in the promoter region of gbnA (Fig. 1). These conserved DNA sequences are proposed to be binding sites for GbnR (Fig. 1). As a consequence, GbnR was predicted to act as a repressor of the gbnABC operon, which should therefore be constitutively expressed if gbnR is deleted.
Here we report that inactivation of gbnR in S. venezuelae induces production of the gaburedins, a novel family of gaminobutyrate ureas. To the best of our knowledge, this is the rst example of the discovery of a novel family of bacterial natural products via rational manipulation of a putative pathway-specic repressor gene.
The S. venezuelae gbnR::apr mutant was constructed using PCR-targeting (see ESI †). 18 The gbnR::apr and wild type strains were grown on a supplemented minimal agar medium for 3 days, aer which the acidied agar medium was extracted with ethyl acetate. The extracts were dried and re-suspended in watermethanol (1:1). Metabolites produced by the wild type and mutant strains were analysed by high resolution LC-MS/MS.
Comparison of the base peak chromatograms revealed six metabolites (1-6), named gaburedins, which were produced by the gbnR::apr mutant strain but not the wild type strain (Fig. 2).
The molecular formulae of the metabolites were deduced to be C 14 H 18 N 2 O 5 for 1, C 11 H 20 N 2 O 5 for 2 and 3, C 10 H 18 N 2 O 5 for 4, C 10 H 16 N 2 O 6 S for 5 and C 10 H 18 N 2 O 5 S for 6. A neutral loss of 129 Da was observed for each parent ion in the MS/MS spectrum, corresponding to loss of a common C 5 H 7 NO 3 fragment (Fig. 3).
Interestingly, the molecular formulae of the resulting daughter ions matched those of the protonated amino acids phenylalanine, isoleucine/leucine, valine and methionine for compounds 1, 2/3, 4 (Fig. 5) and 6 respectively. For compound 5, the molecular formulae for the daughter ion matched that of acetylcysteine (C 5 H 10 NO 3 S). A further loss of a 42 Da fragment from this daughter ion was consistent with the presence of an acetyl group. An analogous fragmentation pattern was observed for the [M + Na] + ions of each gaburedin.
Cultures of the gbnR::apr mutant were scaled up to isolate sufficient quantities of 1 for structure elucidation by NMR spectroscopy (Fig. 4). COSY and HMBC experiments allowed unambiguous elucidation of the planar structure. An authentic standard of S-1 was synthesised via modication of the route reported by Tayaka and co-workers for the synthesis of ureidocontaining derivatives of 5-uorouracil (Fig. 4). 19 LC-MS/MS and NMR analyses conrmed that the compound puried from the gbnR::apr mutant strain was identical to the synthetic standard 1.   Supplementation of the culture medium with specic amino acids was found to inuence gaburedin production. For example, growth of the gbnR::apr mutant in a culture medium enriched in L-phenylalanine (20 mM nal concentration) resulted in overproduction of gaburedin A 1 relative to the other gaburedin congeners. Feeding with D-phenylalanine resulted in the overproduction of a gaburedin with same retention time and fragmentation pattern as 1. However, chiral HPLC analysis indicated that this compound was the enantiomer of 1, con-rming that the absolute conguration of 1 initially isolated from S. venezuelae is S (ESI †).
The gbnR::apr mutant was grown in culture media supplemented with either L-isoleucine or L-leucine (20 mM nal concentration) to distinguish between gaburedin B 2 and gaburedin C 3 which have very similar retention times (Fig. 2). The resulting culture extracts were analysed by LC-MS/MS, revealing that the quantity of 3 increased dramatically when L-isoleucine was fed, whereas the quantity of 2 signicantly increased in the culture extract obtained from the mutant grown in the presence of L-leucine (see ESI †). Specic incorporation of d 10 -leucine into 2 and d 10 -isoleucine into 3 further conrmed the structural identity of these metabolites (ESI †). Similarly the gbnR::apr mutant strain was grown in the presence of N-acetylcysteine, in which case gaburedin E 5 was overproduced. Further precursor-directed biosynthesis experiments were carried out by supplementing the culture medium with various amino acids and gaburedins containing Val, Met, Tyr, Trp, Gly, Ala, Pro, Ser, Thr and Glu were identied by LC-MS analyses (see ESI †). The molecular formulae, fragmentation patterns and retention times were all consistent with the proposed structures (see ESI †). Interestingly supplementation with g-aminobutyrate (GABA) resulted in the production of a GABA dimer (7), in which the two monomers are linked by a ureido bridge (molecular formulae C 9 H 16 N 2 O 5 , Scheme 1). Amino acid analogues lacking the carboxylic acid group were also incorporated into gaburedin analogues, e.g. isobutylamine was found to elicit the production of compound 8 (Scheme 1).
The gbnA gene encodes a putative pyridoxal phosphate (PLP)dependent amino acid decarboxylase (Fig. 1), which is proposed to decarboxylate glutamate to give GABA (Scheme 1). Interestingly, feeding of the S. venezuelae gbnR mutant with glutamate not only yielded the glutamate-derived gaburedin analogue but also analogue 7, consistent with the hypothesis that GABA is derived from decarboxylation of glutamate. The gbnB gene encodes a putative ATP-dependent enzyme belonging to the acyl-CoA synthetase superfamily (Fig. 1). GbnB is proposed to adenylate either the carboxyl or the carbamoyl group of compound 9, which derives from the spontaneous reaction of GABA with CO 2 (Scheme 1). Carboxyl adenylate 10 would need to undergo cyclisation to the N-carboxyanhydride 11, which would then afford the gaburedins via ring opening with amino acids (note, however, that this would involve nucleophilic attack at the less electrophilic carbonyl group of 11, which is chemically unlikely). On the other hand, the carbamoyl adenylate 12 could react with the appropriate amino acids either directly, or via anhydride 11, to form the gaburedins. Deletion of gbnB in the gbnR mutant of S. venezuelae abolished gaburedin production (Fig. 2), consistent with the key role proposed for GbnB in gaburedin biosynthesis. GbnA and GbnB appear to be the only two enzymes required for gaburedin biosynthesis. GbnC is proposed to export either the gaburedins or intermediate 11/12, which could react with amino acids in the extracellular milieu to form the gaburedins.  A handful of other urea-containing natural products are known, such as the syringolins and the pacidamycins. [20][21][22] Interestingly, the adenylation domain of SylC, which has been shown to catalyze urea formation in syringolin biosynthesis, shares 39% similarity and 22% identity with GbnB. 20 The mechanism for SylC-catalyzed urea formation has been proposed to involve a ve-membered cyclic N-carboxyanhydride, or a closely related species. 20 This anhydride is hypothesized to be formed via an analogous mechanism to that suggested for the formation of 11 (Scheme 1). Trapping of the anhydride with an isoleucyl or valinyl acyl carrier protein thioester would lead to formation of the ureido linkage of the syringolins. As noted above, an analogous mechanism for the formation of the gaburedins via intermediate 11 seems unlikely. Thus, further experiments are required to establish whether similar or distinct mechanisms are utilized for urea formation in syringolin and gaburedin biosynthesis.
Interestingly, BLAST searches revealed analogous operons to the gbnABC operon in other bacteria such as Streptococcus mutans, Salmonella enterica and Vibrio vulnicus (see ESI †). Surprisingly, most of these are clinical isolates of opportunistic pathogens. It will be important, therefore, to establish the nature of the metabolites produced by these strains and to investigate the role of gaburedins and related molecules in pathogenicity.

Conclusions
In summary, bioinformatics analyses suggested that expression of the gbnABC gene is controlled by the putative pathway-specic transcriptional repressor GbnR in S. venezuelae. Inactivation of gbnR resulted in derepression of the gbn gene cluster, resulting in the discovery of the gaburedins, a novel class of urea natural products. To the best of our knowledge, this is the rst report of novel natural product discovery via rational manipulation of a pathway specic transcriptional repressor gene, an approach that offers considerable potential for activation of silent biosynthetic pathways in Streptomyces species, which could lead to the discovery of a wide variety of new metabolites.