Mei
Zhang‡
a,
Xian-Feng
Hou‡
a,
Li-Hua
Qi
a,
Yue
Yin
a,
Qing
Li
a,
Hai-Xue
Pan
a,
Xin-Ya
Chen
a and
Gong-Li
Tang
*ab
aState Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China. E-mail: gltang@sioc.ac.cn
bShanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai 200237, China
First published on 7th April 2015
Trioxacarcins (TXNs) are highly oxygenated, polycyclic aromatic natural products with remarkable biological activity and structural complexity. Evidence from 13C-labelled precursor feeding studies demonstrated that the scaffold was biosynthesized from one unit of L-isoleucine and nine units of malonyl-CoA, which suggested a different starter unit in the biosynthesis. Genetic analysis of the biosynthetic gene cluster revealed 56 genes encoding a type II polyketide synthase (PKS), combined with a large amount of tailoring enzymes. Inactivation of seven post-PKS modification enzymes resulted in the production of a series of new TXN analogues, intermediates, and shunt products, most of which show high anti-cancer activity. Structural elucidation of these new compounds not only helps us to propose the biosynthetic pathway, featuring a type II PKS using a novel starter unit, but also set the stage for further characterization of the enzymatic reactions and combinatorial biosynthesis.
The high biological activities, especial anti-cancer activity, along with unusual and complex structural features of TXN-A distinguish it from other aromatic polyketides, thus providing an interesting but challenging target for total synthesis. Recently, Myers's group successfully established a multiply convergent, component-based route to chemically synthesize TXN-A and its structural analogues.8,9 However, the biosynthetic studies have never been explored to these structurally complex antibiotics. Herein, we describe (1) incorporation studies with 13C-labelled precursors, which elucidated the biosynthetic origin of the scaffold for the TXN family of natural products; (2) the genetic characterization of txn gene cluster, which afforded four polyketide derivates and seven TXN analogues; and (3) a proposed biosynthetic pathway, involving a different starter unit for priming type II polyketide synthase (PKS) and complex tailoring steps.
A five-carbon unit (C-13 to C-17), most possibly from 2-methylbutyryl-CoA, serving as the starter unit of PKS, is seldom observed in natural product biosynthesis. The only exception is involved in the biosynthesis of avermectin “a” components, which are 16-membered macrocyclic lactones generated by type I PKS through loading 2-methylbutyryl-CoA as the starter unit.12 For the type II PKS, although non-acetate starter units, including propionate, malonamate, polyketide or fatty acid, and even amino acid derivates have also been employed,13 2-methylbutyryl-CoA has never been reported as a starter unit to generate aromatic polyketides. Given the fact that 2-methylbutyryl-CoA is usually derived from L-isoleucine (Ile) through deamination and decarboxylation by transaminase and branched-chain 2-oxo acid dehydrogenase in vivo, we performed the feeding experiment with 13C6-L-Ile to validate this hypothesis (Fig. 2A). Remarkably, ESI-MS showed TXN-A from this feeding experiment was +5 m/z heavier than that without feeding (Fig. 2B), indicating the incorporation of a five-carbon unit which arose from an intact Ile. Further specific and significant signal enrichment at C-13 to C-17 (Fig. 2C) in the 13C-NMR spectra, and all the 13C–13C coupling data (Fig. 2D) are consistent with the same conclusion (JC-16/C-15 = 62 Hz, JC-15/C-14 = 58 Hz, JC-13/C-14 = 54 Hz, and JC-17/C-14 = 32 Hz), which are in agreement with this five-carbon unit originating from Ile via an intact incorporation manner. Thus, these results unambiguously demonstrated that the missing five-carbon unit, C-13 to C-17, is derived from L-Ile, which most likely follows a deamination and decarboxylation process similar to that of the avermectin “a” components biosynthesis.12
To verify that the cloned gene cluster was involved in TXNs biosynthesis, we constructed a mutant strain TG5001 in which the txnA1 gene encoding KS was inactivated by gene disruption (ESI, Fig. S2†). As expected, this mutant strain completely abolished the production of TXN-A (Fig. 4A-II), which proved the essential role of this gene cluster governing TXN biosynthesis. Next inactivation of the genes orf−2 (acyltransferase), orf−1 (unknown), orf+11 (cytochrome P450), and orf+3 (tRNA-synthetase) had no effect on TXN-A production; whereas, inactivation of txnRg1 (regulator) or txnRg6 (regulator) led to obviously decreased the yield of TXN-A (Fig. 4A-III to VIII), which suggested that the txn gene cluster may range from txnRg1 to txnRg6, encompassing 56 ORFs (Fig. 3A and Table 1).
Fig. 4 Genetic characterization of the genes for TXN biosynthesis in vivo. HPLC analysis of TXN-A and analogues production (UV at 271 nm) from S. bottropensis: (I) wild-type NRRL 12051, (A-II) mutant TG5001 (ΔtxnA1), (A-III) TG5002 (Δorf−3), (A-IV) TG5003 (Δorf−1), (A-V) TG5004 (ΔtxnRg1), (A-VI) TG5005 (Δorf+11), (A-VII) TG5006 (Δorf+3), (A-VIII) TG5007 (ΔtxnRg6), (A-VIV) TG5008 (ΔtxnA4); (B-II) TG5009 (ΔtxnC2), (B-III) TG5010 (ΔtxnC4), (B-IV) TG5011 (ΔtxnC3), (B-V) TG5012 (ΔtxnO2), (B-VI) TG5013 (ΔtxnO5), (B-VII) TG5014 (ΔtxnO6), (B-VIII) TG5015 (ΔtxnO12), (B-VIV) TG5016 (ΔtxnB4). (●) TXN-A. The genotypes of all the mutants were confirmed by PCR analysis, and the results were summarized in Fig. S2.† |
Gene | AAa | Protein homolog (accession no.), origin | S/Ib (%) | Proposed function |
---|---|---|---|---|
a Amino acid. b Similarity/identity. | ||||
txnRg1 | 94 | LuxR family regulator (016578673), S. albulus | 65/55 | Regulator |
txnB1 | 330 | ChlC2 (AAZ77689), S. antibioticus | 76/67 | dTDP-glucose 4,6-dehydratase |
txnB2 | 290 | AclY (BAB72036), S. galilaeus | 86/74 | dTDP-glucose synthase |
txnB3 | 327 | KstD7 (AFJ52686), Micromonospora sp. TP-A0468 | 76/66 | Pyruvate dehydrogenase-α |
txnB4 | 345 | KstD8 (AFJ52687), Micromonospora sp. TP-A0468 | 86/79 | Pyruvate dehydrogenase-β |
txnA4 | 561 | OxyP (AAZ78339), S. rimosus | 64/53 | MAT |
txnA3 | 90 | SsfC (ADE34520), S. sp. SF2575 | 76/53 | ACP |
txnA2 | 406 | Snoa2 (CAA12018), S. nogalater | 77/66 | CLF (KSβ) |
txnA1 | 420 | PgaA (AAK57525), S. sp. PGA64 | 84/72 | KSα |
txnRg2 | 263 | DnrI (EFL25867), S. himastatinicus ATCC 53653 | 78/63 | SARP-family regulator |
txnRg3 | 394 | 2-Component kinase (ADO32765), S. vietnamensis | 54/40 | 2-Component kinase |
txnRg4 | 203 | 2-Component regulator (CAA09631), S. violaceoruber | 82/69 | 2-Component regulator |
txnP1 | 579 | RkA (ACZ65474), S. sp. 88-682 | 57/43 | ATP-dependent CoA synthetase |
txnC1 | 318 | ORF27 (AEM44304), e-DNA | 64/50 | Aromatase |
txnA5 | 344 | CosE (ABC00733), S. olindensis | 70/58 | KS-III |
txnP2 | 543 | 2-Isopropylmalate synthase (ACY99077), Thermomonospora curvata DSM 43183 | 70/58 | 2-Isopropylmalate synthase |
txnP3 | 417 | Acyl-CoA transferase/dehydratase (EIE99664), S. glauca K62 | 67/56 | Dehydratase or isomerase |
txnP4 | 260 | Ketoreductase (EDY66493), S. pristinaespiralis ATCC 25486 | 63/46 | Short-chain dehydrogenase |
txnRr1 | 500 | Actinorhodin transporter (EFL40860), S. griseoflavus Tu4000 | 64/48 | Transporter |
txnO1 | 345 | Dehydrogenase (ACZ83978), Streptosporangium roseum DSM43021 | 74/61 | Dehydrogenase |
txnU1 | 126 | Tcur_2795 (ACY98340), Thermomonospora curvata DSM 43183 | 40/33 | Unknown |
txnO2 | 401 | P450 (CBX53644), S. platensis | 66/52 | Cytochrome P450 |
txnU2 | 366 | O3I_28241 (EHY24336), Nocardia brasiliensis ATCC 700358 | 69/53 | Unknown |
txnO3 | 411 | ThcD (AAC45752), Rhodococcus erythropolis | 62/48 | Ferredoxin reductase |
txnO4 | 107 | 2Fe-2S ferredoxin (ZP_09514545), Oceanicola sp. S124 | 68/52 | Ferredoxin |
txnH1 | 494 | Putative tripeptidylaminopeptidase (AAP85358), S. griseoruber | 68/59 | Hydrolase |
txnO5 | 409 | ORF29 (AAP85338), S. griseoruber | 68/53 | Cytochrome P450 |
txnH2 | 373 | Microsomal epoxide hydrolase (EHI80707), Frankia sp. CN3 | 68/56 | Epoxide hydrolase |
txnB5 | 328 | PokS9 (ACN64856), S. diastatochromogenes | 70/60 | dNDP-hexose-4-ketoreductase |
txnB6 | 213 | PokS7 (ACN64855), S. diastatochromogenes | 82/72 | 3,5-Epimerase |
txnM1 | 413 | TylCIII (AAD41823), S. fradiae | 84/73 | dNDP-hexose 3-C-MT |
txnB7 | 488 | SaqS (ACP19377), Micromonospora sp. Tu 6368 | 71/62 | dNDP-hexose 2,3-dehydratase |
txnB8 | 321 | SaqT (ACP19378), Micromonospora sp. Tu 6368 | 70/62 | dNDP-hexose 3-ketoreductase |
txnRr2 | 500 | EmrB/QacA (EGE43895), S. griseus XylebKG1 | 75/59 | Transporter |
txnRg5 | 339 | DeoR regulator (ACZ87003), Streptosporangium roseum DSM43021 | 77/70 | Regulator |
txnO6 | 406 | ORF3 (AAD28449), S. lavendulae | 63/45 | Cytochrome P450 |
txnO7 | 175 | PokC1 (ACN64848), S. diastatochromogenes | 45/35 | Cyclase or hydroxylase |
txnO8 | 371 | AlnT (ACI88867), S. sp. CM020 | 57/43 | Hydroxylase |
txnO9 | 154 | CalC (AAM70338), Micromonospora echinospora | 50/36 | Cyclase or hydroxylase |
txnC2 | 261 | HedA (AAP85364), S. griseoruber | 83/71 | Ketoreductase |
txnO10 | 178 | AsuE2 (ADI58638), S. nodosus subsp. asukaensis | 57/43 | Flavin reductase |
txnC3 | 304 | Gra-ORF33 (ADO32793), S. vietnamensis | 68/56 | 2,3-Cyclase |
txnB9 | 383 | SsfS6 (ADE34512), S. sp. SF2575 | 55/38 | Glycosyl transferase |
txnO11 | 148 | Aln2 (ACI88858), S. sp. CM020 | 52/41 | Cyclase or hydroxylase |
txnU3 | 121 | GrhI (AAM33661), S. sp. JP95 | 46/28 | Unknown |
txnB10 | 424 | UrdGTa1 (AAF00214), S. fradiae | 61/47 | Glycosyl transferase |
txnC4 | 240 | RedLA2 (AAT45284), S. tubercidicus | 82/73 | Ketoreductase |
txnM2 | 340 | MetLA2 (AAT45283), S. tubercidicus | 79/70 | O-Methyltransferase |
txnU4 | 388 | PAI11_01900 (EHN12885), Patulibacter sp. I11 | 82/69 | Unknown |
txnB11 | 397 | Azi15 (ABY83154), S. sahachiroi | 63/50 | O-Acyltransferase |
txnB12 | 427 | UrdGTa1 (AAF00214), S. fradiae | 61/47 | Glycosyl transferase |
txnM3 | 339 | DmpM (AFE08598), Corallococcus coralloides DSM 2259 | 62/45 | O-Methyltransferase |
txnO12 | 407 | FosK (AEC13077), S. pulveraceus | 67/54 | Cytochrome P450 |
txnM4 | 340 | DmpM (AFE08598), Corallococcus coralloides DSM 2259 | 61/44 | O-Methyltransferase |
txnU5 | 182 | RAM_06565 (AEK39805), Amycolatopsis mediterranei S699 | 75/64 | Unknown |
txnRg6 | 286 | SARP regulator (ACU39492), Actinosynnema mirum DSM 43827 | 53/40 | Regulator |
To verify the hypothetical functions of the relative genes in TXN-A biosynthesis, txnC2, txnC3 and txnC4 were inactivated separately by gene replacement with the aac(3)IV apramycin-resistance gene (ESI, Fig. S2†). The resultant mutant strains S. bottropensis TG5009 (ΔtxnC2), TG5010 (ΔtxnC4) and TG5011 (ΔtxnC3) all abolished production of TXN-A; whereas, each of the three mutants accumulated new compounds that are different from TXNs (Fig. 4B-II to IV). Following the optimized fermentation and isolation processes (including silica gel and Sephadex LH-20 column chromatography, preparative HPLC et al.), we obtained 10 mg of 4a and 6 mg of 4b from an 8 L culture of the TG5009 strain; 11 mg of 13a and 2 mg of 13b from a 4 L broth of the TG5010 strain; as well as 40 mg of 6a from a 2 L culture of the TG5011 strain. The chemical structures of these compounds were elucidated by MS, HRMS and 1D, 2D-NMR spectra (Fig. S3–S17, S28–S32 and Tables S4, S5, S8, S11†) and are summarized in Fig. 5. These results strongly support the biological function of the respective gene and the proposed biosynthetic pathway (Fig. 3B). Firstly, the production of compounds 4a and 4b by the TG5009 (ΔtxnC2) mutant verified that TxnC2 reduces the C-9 keto group of the nascent polyketide chain. More importantly, this result indicated that the reduction of C-9 is necessary for the next C7–C12 cyclization and aromatization, and a similar opinion has been widely accepted in type II PKS.19,20 Whereas, the production of a small amount of 4b is unexpected but reasonable, which could be derived from the incorporation of L-valine through deamination and decarboxylation, similar to that of the avermectin “b” components biosynthesis.12,21 Secondly, the TG5011 (ΔtxnC3) mutant affording compound 6a doubly confirmed that TxnC2/C1 catalyzes the C7–C12 first-ring cyclization and aromatization, and a similar cyclized compound SEK4 had been generated by an octaketide minimal PKS, except with a different starter unit and chain length.19,22 Thirdly, the isolation of 13a and 13b from the TG5010 (ΔtxnC4) mutant suggested that this KR catalyzes another ketoreduction, such as 10 into 11 (Fig. 3B), which affords the hydroxyl group for deoxysugar attachment. Together, the two new compounds 4a and 6a further established a different five-carbon starter unit for type-II PKS in TXN biosynthesis. Given the fact that the starter unit has been proven to be an attractive point for engineering aromatic polyketide biosynthetic machinery,21,23 the discovery of the different starter unit in TXN-A biosynthesis will also substantiate the potential for similar efforts.
In a typically bacterial type II PKS system, a MAT sharing with fatty acid biosynthesis loads malonyl-CoA onto the thiol group of the 4′-phosphopantheinyl arm attached to the ACP, which is subsequently decarboxylated to generate an acetate starter unit and also used as extender units catalyzed by a KS-CLF heterodimer.13,15 Meanwhile, non-acetate starter units have been increasingly observed as alternative primers and usually involve an additional KS-III.13 Based on the precursor feeding, bioinformatic analysis and genetic characterization results, we could propose that the biosynthetic pathway of the TXN-A polyketide backbone follows the action of a special type II PKS (TxnA1–A2–A3) as illustrated in Fig. 3B. The enzymes involved in the branched-chain fatty acids catabolism, a transaminase and a branched-chain 2-oxo acid dehydrogenase catalyze the deamination and decarboxylation reactions to generate 2-methylbutyryl-CoA, which might be a direct starter unit for the KS of type II PKS primed by KS-III, TxnA5. Nine units of malonyl-CoA are subsequently incorporated into the PKS biosynthetic system by MAT (TxnA4) to form the full elongated polyketide chain 4. Next, the PKS associated enzymes KR (TxnC2), aromatase (TxnC1), and cyclase (TxnC3) are required to carry out the regioselective folding and cyclization of the nascent chain to yield the aromatic polycyclic backbone 7. Subsequently, a decarboxylation and further cyclization steps should be involved to yield the intermediate 8.
In total, four cytochrome P450 enzymes (P450s) encoded by txnO2, O5, O6 and O12 attracted our attention because this family of oxidative hemoproteins could catalyze many different reactions for structural diversification in natural product biosynthesis.24 Therefore, we constructed the respective gene replacement mutants S. bottropensis TG5012 (ΔtxnO2), TG5013 (ΔtxnO5), TG5014 (ΔtxnO6) and TG5015 (ΔtxnO12), and analyzed the metabolites produced by HPLC and LC-MS. The results showed that each of the four mutants afforded compounds different from the wild type (Fig. 4B-V to VIII). Although attempts to isolate new compounds from the TG5014 (ΔtxnO6) mutant were unsuccessful for the low yield and instability, we finally obtained 40 mg of 14a from a 1 L culture of the TG5012 strain, 15 mg of 9a from a 4 L fermentation broth of the TG5013 mutant, as well as 20 mg of 15a and 4 mg of 15b from a 2 L culture of the TG5015 strain. Evaluation of the MS and NMR spectra and comparison with TXN-A (Fig. S18–S21, S33–S44 and Tables S6, S9–S11†) led to the successful assignment of the chemical structures of all these new compounds (Fig. 5).
Structurally, compound 9a is close to parimycin (9, Fig. 5), which was isolated from another TXN-A producing strain, marine Streptomyces sp. B8652, as a novel 2,3-dihydro-1,4-anthraquinone unrelated to TXNs.25 The isolation of 9a from a ΔtxnO5 mutant not only hinted that this P450 plays a key role in the formation of the highly oxygenated polycyclic skeleton, but also suggested that 9 or 9a should be the intermediate for the bio-generation of TXN-A (Fig. 3B). We believe that TxnO5 (P450), or/and TxnO6 (P450), TxnO4 (ferredoxin), TxnO3 (ferredoxin reductase), TxnH2 (epoxide hydrolase), and TxnM3 or M4 (MT) should be involved in the transformation of 10 from 9 (Fig. 3B and S45†), while this complex process may need more uncharacterized enzymes. In addition, the production of 14a by the ΔtxnO2 mutant and 15a/15b by the ΔtxnO12 mutant showed that the P450s catalyze hydroxylation at the C-16 and C-2 positions, respectively.
To obtain further insight into the deoxysugars pathway, especially the usual γ-branched octose, we inactivated the txnB4 gene, resulting in the mutant strain S. bottropensis TG5016 (ΔtxnB4). This mutation completely abolished TXN-A production, but yielded two new compounds (Fig. 4B-VIV). After fermentation and purification, we isolated 10 mg of 12a and 3 mg of 12b from a 1 L culture, and the structures are shown in Fig. 5 (Fig. S22–S27 and Tables S7, S11†). Compared with TXN-A, the major compound 12a has lost the γ-branched octose moiety at 13-OH, which means that the respective glycosyl transferase bears a relatively strict substrate specificity toward the two-carbon side chain. The production of the minor compound 12b revealed that a sugar C-MT, most likely TxnM1, catalyzes a methylation reaction to form a new sugar donor 23, which partially completed 19 to generate 12b, though it is not the perfect sugar donor for the glycosyl transferase comparable to the native 19 (Fig. 3C and B).
Footnotes |
† Electronic supplementary information (ESI) available: The experimental procedures, strains, plasmids and PCR primers, and compound characterization. See DOI: 10.1039/c5sc00116a |
‡ M. Zhang and X.-F. Hou contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2015 |