PMC

Organization of the ssf biosynthetic gene cluster. Genes are categorized according to their proposed role. The gene cluster spans 47.2 kb and contains 40 ORFs. Details of proposed functions are shown in .

In vitro assay of SsfL1 activity and substrate specificity. (A) Synthesis of salicylyl-AMP as indicated by the release of PP_i in the presence of ATP, salicylate 19 and the salicylyl-CoA ligase SsfL1. i) all reaction components; ii) no 19; iii) no ATP; iv) no SsfL1; and v) pyrophosphate reagent only. (B) Utilization of substituted benzoic acids by SsfL1 as indicated by PP_i release assay. The structures of compounds indicated by number here are shown in .

Production of 1 and biosynthetic intermediates by S. sp. SF2575. The data were collected on the Shimadzu LC-MS. (A) LC trace (358 nm) for a mixture of standards 6, 7 and 1. (B) LC trace (358 nm) of the extract after 7 days growth on solid Bennette's media. (C) Selected ion monitoring was used to confirm the identification of 7 ([M+H]⁺ m/z = 696) and 6 ([M+H]⁺ m/z = 576). The UV spectra of these compounds are shown for comparison.

A map of the ssf gene cluster is shown in . The putative boundaries are determined based on sequence analysis and biosynthetic logic. The ssf genes identified along with their proposed function are listed in . Represented within are genes likely responsible for the biosynthesis of the aglycon 8, pendant groups 14, 19 and 25, as well as regulation and self-resistance. Based on the putative functions of these genes, the proposed biosynthetic pathways of 8 and the subsequent tailoring of 8 to 1 are shown in and , respectively.

Reconstitution of tetracycline intermediates using ssf genes expressed in S. lividans K4-114. (A) HPLC analysis (245 nm) of the K4-114/pLP27 extract shows the amidated, reduced polyketide 42 is the major product, confirming the biosynthesis of the polyketide backbone 40 by SsfABCD and the subsequent C-9* reduction by SsfU. (B) HPLC analysis (253 nm) of the K4-114/pLP27/pLP77 extract shows addition of the putative cyclase SsfY1 leads to complete cyclization and aromatization of the D and formation of the shunt benzopyrone 44. (C) HPLC analysis (430 nm) of K4-114/pLP27/pLP126 extract shows 50, the oxidized form of 49, as the dominant product. Biosynthesis of 49 using entirely ssf genes (SsfABCDUY1Y2M4L2) indicates the tetracycline nature of the ssf biosynthetic pathway. (D) HPLC analysis (395 nm) of K4-114/pLP36 extract confirms the function of SsfO2 as the oxygenase that dihydroxylated C-4 and C-12a of 49. The resulting product 51 undergoes spontaneous degradation to afford the observed product 52.

Exploiting the substrate tolerance of SsfX3 and SsfL1 may be useful towards diversifying the C-4 functionality of 1. To probe this, differently substituted benzoic acids () were tested for activation and acyltransfer by SsfL1 and SsfX3, respectively. Since SsfL1 is the initial gatekeeper in these reactions, the different substrates were first examined for recognition by SsfL1 using the PP_i release assay as described above (). Interestingly, nearly all the substrates examined showed levels of PPi release above background (except 33), demonstrating SsfL1 has relaxed substrate specificity. The 2-OH substituent is clearly not essential for binding, as benzoic acid 26, 2-chlorobenzoic acid 31 and 2-methoxy benzoic acid 35 all supported similar rates of pyrophosphate release. Surprisingly, three dihydroxybenzoic acids (27-29), as well as 4-aminosalicylic acid 34 were significantly better substrates than 19, indicating substantial plasticity in the binding pocket of SsfL1.

In vitro assay of SsfX3 activity and substrate specificity. (A) The tandem actions of SsfL1 and SsfX3 transfer 19 to the aglycon substrate 6 to yield 7. The assays are performed in 50 mM HEPES, pH 7.9 and 10 mM MgCl₂. i) the semisynthetic 6 standard; ii) the semisynthetic 7 standard; iii) Complete reaction containing 50 mM HEPES pH 7.9, 10 mM MgCl₂, 2 mM ATP, 2 mM free CoA, 2 mM 19, 20 μM 6, 1.5 μM SsfX3 and 15 μM SsfL1. Control reactions were performed as iii) with the following exclusions: iv) no SsfL1; v) no ATP; vi) no 19; vii) no SsfX3; and viii) no CoA. The reactions were examined with HPLC (358 nm). (B) Synthesis of analogs of 7 using salicylic acid analogs, SsfL1 and SsfX3. All reactions were performed at 25 °C for 30 minutes, extracted with organic solvent and analyzed by LC-MS (358 nm, positive ionization).

Bacterial aromatic polyketide natural products comprise a group of molecules that displays diverse structures and bioactivities yet share common biosynthetic origins. The poly-β-ketone backbone is synthesized by a minimal polyketide synthase (PKS) consisting of a ketosynthase (KS or KS_α), a chain-length factor (CLF or KS_β) and an acyl carrier protein (ACP). Additional tailoring enzymes are required to fix the regioselectivity of sequential cyclization steps, and perform a multitude of decorating reactions to afford the various polycyclic bioactive compounds. A small, yet extremely important group of aromatic polyketides is the tetracycline family. Tetracyclines are characterized by their linearly fused four-ring structure and a heavily oxidized 2-naphthacenecarboxamide carbon skeleton. Other common features include the planar phenyldiketone arrangement and the tricarbonylmethane moiety in the A ring. This family is exemplified by the well-known tetracyclines chlorotetracycline 5 and oxytetracycline 4 (). Discovered in 1948 from Streptomyces aureofaciens and in 1950 from Streptomyces rimosus, respectively, these compounds were commercialized as broad spectrum antibiotics and used successfully until bacterial resistance prompted the need for the creation of analogs to combat drug resistant strains. The natural tetracycline scaffold has since been used to create second and third generation tetracyclines semisynthetically. The third generation tetracycline tigecycline gained FDA approval in 2005, , demonstrating the continued utility of the tetracycline scaffold. Remarkably, since the initial discovery of 4 and 5 over sixty years ago, there have been only a few natural tetracyclines discovered. Among these are SF2575, 1 and related compounds TAN-1518A 2 and TAN-1518B 3, chelocardin and dactylocycline. Each of these compounds contains the tetracycline core and exhibits novel structural features not observed in 4 or 5. Given the rarity of tetracycline natural products, understanding the biosynthesis of these newly discovered compounds may provide a route to the generation of new tetracycline antibiotics through the use of combinatorial biosynthesis and metabolic engineering strategies.

Comparing the core structures of 1 and 4, biosynthesis of the naphthacenecarboxamide carbon frameworks of 1 is predicted to parallel that of the oxytetracycline (oxy) biosynthetic pathway, and may share a number of common intermediates. As shown in , the putative aglycon is likely to be 8, which exhibits the typical features of a tetracycline except replacement of 4-(R)-dimethylamine with 4-(S)-hydroxyl, inversion of stereochemistry at C-6 and O-methylations at C-6 and C-12a. Downstream tailoring of 1 is therefore likely to occur following assembly of 8. In order to decipher the timing of the three unique post-PKS tailoring reactions leading to the biosynthesis of 1: glycosylation with d-olivose 14, O-4′ acylation of angelic acid and C-4 acylation of salicylic acid 19, crude extract of S. sp. SF2575 culture was prepared and analyzed on HPLC and LCMS to try to identify any potential stable intermediates that may be present. S. sp. SF2575 culture was grown on solid Bennette's media for 10 days at 30°C. A sample was extracted each day starting with day 4 to analyze the metabolic profile. Using selected ion monitoring, two potential intermediates identified were 6 ([M+H]⁺ at m/z 576, RT = 18.3 min) and 7 ([M+H]⁺ at m/z 696, RT = 26.8 min), in addition to the parent compound 1 ([M+H]⁺ at m/z 778, RT = 34.2 min) (). The UV spectrum of each compound was similar to that of 1 with a characteristic tetracycline λ_max at 358 nm, and a λ_max at 302 nm for 1 and 7 characteristic of the salicylate. The UV spectrum of 6 lacks this contribution at 302 nm resulting in a smooth peak at 358 nm, and is indicative of the loss of salicylate compared to 7 as suggested by the molecular weight difference. To confirm the identity of these compounds as shown in , authentic standards were prepared from base hydrolysis of purified 1 as described by Hatsu et. al. Treatment of 1 with 0.5 M NaOH led to the hydrolysis of the angelate and afforded 7, while complete conversion to 6 was obtained by treating 1 with 1.0 M NaOH for 15 hours. Following purification of 6 and 7 from the base hydrolysis reactions, proton NMR and HRMS were used to confirm the structures by comparison to published data (). These prepared samples were then used as standards to verify the identities of 6 and 7 in the fermentation extract of S. sp. SF2575 by HPLC retention time, mass fragmentation pattern and UV spectra.