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Heterologous Expression Guides Identification of the Biosynthetic Gene Cluster of Chuangxinmycin, an Indole Alkaloid Antibiotic Xiaokun Xu, Haibo Zhou, Yang Liu, Xiaotong Liu, Jun Fu, Aiying Li, Yue-zhong Li, Yuemao Shen, Xiaoying Bian,* and Youming Zhang* Suzhou Institute of Shandong University and Shandong University−Helmholtz Joint Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, People’s Republic of China S Supporting Information *
ABSTRACT: The indole alkaloid antibiotic chuangxinmycin, from Actinobacteria Actinoplanes tsinanensis, containing a unique thiopyrano[4,3,2cd]indole scaffold, is a potent and selective inhibitor of bacterial tryptophanyl-tRNA synthetase. The chuangxinmycin biosynthetic gene cluster was identified by in silico analysis of the genome sequence, then verified by heterologous expression. Systemic gene inactivation and intermediate identification determined the minimum set of genes for unique thiopyrano[4,3,2-cd]indole formation and the concerted action of a radical S-adenosylmethionine protein plus an unknown protein for addition of the 3-methyl group. These findings set a solid foundation for comprehensively investigating the biosynthesis, optimizing yield, and generating new analogues of chuangxinmycin.
M
remains obscure. Structurally it is possible that the biosynthetic origin is Trp. In the 1970s and 1980s, feeding experiments using isotope-labeled precursors suggested that the sulfur atom originated from cysteine and the 3-methyl group came from Sadenosylmethionine (SAM), as demonstrated by a putative indolepyruvate C-methyltransferase from a cell-free extract of A. tsinanensis.20,21 Vitamin B12 promotes CXM biosynthesis.22 Since then, no study has been reported on CXM biosynthesis.23
icrobial products are an important resource for medicine and pesticides.1,2 Investigation of their biosynthesis provides an opportunity to improve fermentation or to generate new analogues by rational engineering. Microbial product biosynthetic pathways have been identified in the native producer by gene inactivation, but for some genetically intractable producers, heterologous expression of putative biosynthetic gene clusters is a feasible alternative.3 Our previous efforts regarding the direct cloning of large gene clusters and seeking a suitable heterologous host realized the heterologous production of multiple products from diverse bacteria.4−11 In this investigation, we used a heterologous expression strategy to identify the biosynthetic gene cluster of the previously reported antibiotic chuangxinmycin from a rare actinomycete. Chuangxinmycin (CXM, 1) is an indole alkaloid antibiotic first isolated from the actinomycetes Actinoplanes tsinanensis 40 years ago.12,13 Due to its structural resemblance to tryptophan (Trp, 2), CXM is a potent and selective inhibitor of bacterial tryptophanyl-tRNA synthetase (TrpRS), which is responsible for its antibacterial activity. It showed antibacterial activity against several Gram-positive and Gram-negative bacteria in vitro and effective inhibition of Escherichia coli and Shigella dysenteriae infections in mice. CXM was also effective against E. coli septicemia, urinary, and biliary infections in a preliminary clinical trial.12 Furthermore, it showed low toxicity in mice. However, now it is almost obsolete presumably on account of the low yield and relatively poor efficacy compared with currently used antibiotics. Besides its biological activity, the unique 3-methyl-3,5dihydro-2H-thiopyrano[4,3,2-cd]indole moiety prompted synthetic chemists to synthesize CXM and its analogues.14−19 However, biosynthesis of the tricyclic indole-S-hetero scaffold © XXXX American Chemical Society and American Society of Pharmacognosy
To understand how CXM is formed and to set the basis for further efforts to improve yield and generate non-natural derivatives, we identified the biosynthetic gene cluster of CXM using heterologous expression of a candidate cluster coupled with systematic in vivo gene inactivation. Received: September 30, 2017
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DOI: 10.1021/acs.jnatprod.7b00835 J. Nat. Prod. XXXX, XXX, XXX−XXX
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The complete genome of A. tsinanensis was sequenced by PacBio technology. Generally, biosynthesis, regulation, and resistance genes for specialized secondary metabolites are clustered in the genome of bacteria. Therefore, to isolate the gene cluster for CXM biosynthesis, we used genome scanning to target the TrpRS gene that confers resistance to CXM or other TrpRS inhibitors such as indolmycin.24,25 Through bioinformatics analysis of the resulting complete genomic sequence, two putative TrpRS genes within the genome were identified. Among them, cxm0 (YDGM004646) attracted our attention, because this gene is located in a putative 10-gene cluster (YDGM004646−YDGM004655) that spans an 11 kb DNA region, and the GC content (64.9%) of this putative gene cluster is significantly lower than the average GC content (70.3%) of the genome, suggesting that it might have been acquired from a foreign source by horizontal gene transfer. Sequence analysis of this gene cluster (the cxm cluster, Figure 1,
Figure 2. (A) HPLC analysis of CXM (1), dmCXM (3), and compound 4 production in S. coelicolor A3(2) recombinant strains with intact cxm gene cluster A3(2)/cxm and deletion mutants Δcxm1−Δcxm9, (B) HPLC analysis of biotransformation from dmCXM (3) to CXM (1) by concerted action of Cxm8 and Cxm9 in S. coelicolor A3(2), and (C) heterologous production of dmCXM (3) by Cxm3456 and production of 4 by Cxm34 in S. coelicolor A3(2).
tive metabolite analysis. All mutants except Δcxm2, Δcxm6, and Δcxm7 had completely abolished chuangxingmycin production (Figure 2A). The cxm2 and cxm7 genes encode a transporter and aminotransferase, respectively, whose function was complemented by their homologues in the heterologous host S. coelicolor A3(2) (Table S5); thus their deletion mutants did not affect the production of CXM. Mutant Δcxm6 produced trace levels of CXM (∼3% yield of intact cxm gene cluster), potentially due to its function not being efficiently complemented by homologues in S. coelicolor. Inactivation of cxm1 resulted in elimination of CXM, demonstrating that cxm1 encodes a positive transcriptional regulator. Both mutants Δcxm8 and Δcxm9 had increased compound 3 (RT 27.3 min, m/z 218.0286 [M − H]−) production, which was purified and characterized as 3-desmethylchuangxinmycin (dmCXM) by using HRMS and NMR (Figures S14 and S15, Table S8). This suggests that both are involved in the 3-methylation during CXM biosynthesis. To verify the concerted action of cxm8 and cxm9, cxm8 and cxm9 were cloned both separately and together under the strong artificial promoter Virolle-a1-14 in S. coelicolor A3(2),30 then fed 3. Only when cxm8 was expressed together with cxm9 was 3 completely converted into CXM (Figure 2B). Generally, a single radical SAM protein is sufficient for one methylation,31 but our in vivo results here show that cxm8 catalyzed 3-methylation only in combination with cxm9. To the best of our knowledge, this is the first example of the concerted action of a radical SAM protein with an unknown protein for a single methylation. Along with compound 3, a new compound 4 (tR 25.7 min, m/z 381.0597 [M − H]−) was found in the heterologous hosts containing the cxm gene cluster and was also present in the Δcxm5 mutant. Compound 4 production was abolished in the cxm3 or cxm4 knockout mutants (Figure 2A), while placing
Figure 1. Gene organization of chuangxinmycin biosynthetic gene cluster (A) and predicted functions of each gene (B).
Table S4) predicts that cxm1 encodes a LysR family transcriptional regulator for regulation of CXM production, cxm2 encodes a MFS (Major Facilitator Superfamily) transporter for efflux of CXM, cxm4 encodes a sulfur carrier protein, cxm5 encodes a cytochrome P450, cxm6 encodes a ketopantoate reductase, cxm7 encodes a pyridoxal 5′-phosphate (PLP)-dependent aminotransferase, cxm8 encodes a vitamin B12-dependent radical SAM protein, and cxm3 and cxm9 encode a hypothetical protein, respectively. Attempts to inactivate genes in A. tsinanensis to verify the biosynthetic pathway were unsuccessful, as has been the case with other rare actinomycetes. To connect this gene cluster with CXM biosynthesis, we directly cloned this DNA region (cxm0−cxm9) into an E. coli plasmid using linear plus linear homologous recombination (Figure S1)4,26 and transferred it into heterologous hosts S. coelicolor A3(2), S. albus J1074, and S. lividans K4-114,27−29 for functional expression. Metabolic profiling showed that CXM was produced in all three strains containing cxm0−cxm9 (Figure 2, Figure S3), which shows that cxm0−cxm9 are sufficient to direct the biosynthesis of CXM in these bacteria. Subsequently, nine mutants with in-frame deletion of cxm1− 9, respectively, were generated in E. coli by Red/ET recombineering and transferred into S. coelicolor for comparaB
DOI: 10.1021/acs.jnatprod.7b00835 J. Nat. Prod. XXXX, XXX, XXX−XXX
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cxm3 and cxm4 under a strong artificial promoter in S. coelicolor A3(2) also resulted in the biosynthesis of 4 (Figure 2C). 4 was purified from the Δcxm5 mutant, and the molecular formula was determined as C16H18N2O5S2 (calcd for 381.0584 [M − H]−) on the basis of HRESIMS analysis. The 1D NMR data were similar to those of 3, indicating that they share a similar indole-3-propanoic acid skeleton, which was further supported by the COSY and HMBC correlations (Figure 3). The
their mutants had eliminated or substantially decreased 3 production. In addition cxm3456 genes under the control of the strong artificial promoter in S. coelicolor A3(2) resulted in the robust production of 3-desmethylchuagnxinmycin 3 (Figure 2C). Thus, we determined the minimal enzymes cxm3−6 are required for production of the unique thiopyrano[4,3,2cd]indole scaffold in vivo. The exact conversion mechanism from indolepyruvate 5 to 3-desmethylchuagnxinmycin 3 is still unclear. Cxm5 is a cytochrome P450 enzyme that would catalyze intermediate thiotryptophan (6) to form 7 (2,3dehydro-3-desmethylchuangxinmycin) in a similar manner to the biosynthesis of cyclobrassinin and thienodolin.33,34 The introduction of the sulfur (first C−S bond formation) involves a mechanism that seems to be similar to that found in thiamine, molybdopterin, cysteine, thioquinolobactin, and BE-7585A biosynthesis.35−39 These sulfur-supplying enzymes generally include common sulfur carrier proteins, cysteine desulfurases, and rhodanese-like proteins.40−42 Cxm4 is a sulfur carrier protein (SCP), but the conserved C-terminal diglycine tail (−GG−COOH) is followed 10 additional amino acids.35−37,43−45 This variation of a sulfur carrier protein is also found in thioquinolobactin biosynthesis or methionine biosynthesis in Wolinella succinogenes, the metal-dependent hydrolase QbsD or HcyD cleaving the final two or one amino acid from SCP generating the diglycyl C-terminus, respectively.36,45 The cxm3 encodes an unknown protein but containing an Mpr1p, Pad1p N-terminal (MPN) domain (Evalue: 6.42 × 10−8) that cleaves isopeptide bonds;46 thus Cxm3 probably catalyzes the Cxm4 processing to remove 10 additional amino acids. The cysteine desulfurase and rhodanese homologues are also found in the genome of A. tsinanensis and S. coelicolor A3(2) (Tables S6 and S7). Cxm6 possibly converts intermediate 7 to 3 to give the final 3,5-dihydro-2Hthiopyrano[4,3,2-cd]indole scaffold. An intermediate 8 was identified from the ΔcxnD (the homologue of cxm5) mutant based on the only HRESIMS/MS analysis in a solid medium culture condition, which suggested a divergent biosynthetic pathway from 5 (Figure 4).23 Both results showed cytochrome P450 enzyme (Cxm5/CxnD) catalyzed the second C−S formation from, probably, different substrates. Based on the above experimental results and bioinformatic analysis, a biosynthetic model for CXM was proposed (Figure 4): Cxm7, or other aminotransferase, converts Trp (2) to indolepyruvate (5), Cxm3 and Cxm4 catalyze sulfur incorporation, Cxm5 catalyzes the S-heterocyclization, Cxm6 catalyzes the reduction of the double bond, and Cxm8 coupled with Cxm9 finally catalyzes C3-methylation. We were able to attribute the function of each enzyme in the CXM biosynthesis, and the timing of each biochemical reaction requires further work. In addition, CXM biosynthesis is not unique, with similar pathways being present in other actinobacteria (Figure S22).
Figure 3. Key COSY and HMBC of compound 4.
additional COSY correlations between H-1′ and H-2′ and between H-2′ and 4′-NH together with the HMBC correction from H-1′ to C-3′ indicated an additional cysteine residue. The acetyl group was located on 4′-N according to the HMBC correlations from 4′-NH to C-1″ and from H-2″ to C-1″. The indole-3-propanoic acid moiety and N-acetylcysteine residue were connected by a disulfide bond. Compound 4 features a disulfide bond connecting N-acetylcysteine and thiotryptophan 6, a putative biosynthetic intermediate (Figure 4). We reasoned
Figure 4. Proposed chuangxinmycin biosynthetic pathway based on this study.
that 4 was formed by reaction between N-acetylcysteine and 6 thiol groups catalyzed by a glutathione-S-transferase;32 thus 4 may be a byproduct of S. coelicolor A3(2)/cxm and 6 was most likely the actual intermediate. Compound 6 contains a C−S bond, indicating the sulfur atom has been inserted into the molecule, and cxm3 and cxm4 are thus necessary for the formation of the first C−S bond (Figure 4). The featured thiopyrano[4,3,2-cd]indole scaffold in compound 3 comprises two unique carbon−sulfur bonds, but its biosynthetic enzymes are yet to be discovered. On the basis of gene inactivation, cxm0−2, cxm7 (converting Trp 2 to indolepyruvate 5), and cxm89 do not participate in the biosynthesis of 3. The remaining cxm3456 genes are likely responsible for the biosynthesis of this tricyclic scaffold because
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EXPERIMENTAL SECTION
General Experimental Procedures. E. coli, S. coelicolor A3(2), S. albus J1074, and S. lividans K4-114 strains were cultivated and manipulated according to standard protocols. Strains and plasmids used in this study are listed in Table S1. A. tsinanensis was purchased from the Sichuan Institute of Antibiotics and was cultivated either on the medium mannitol soy flour (MS) agar or 148G.47 DNA isolation and manipulation in E. coli followed standard methods. Primer synthesis and DNA sequencing were performed at Shanghai Sangon Biotech (China) Co., Ltd. Restriction enzymes were purchased from New England Biolabs (Beijing) Ltd., and DNA polymerases (Taq and PrimeSTAR) were purchased from TaKaRa Biotechnology (China) C
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Overexpression of cxm Gene in S. coelicolor A3(2). For construction of the S. coelicolor A3(2)/cxm8 mutants, the synthetic promoter a1-14 coupled with an antibiotic selectable marker hyg was generated by PCR from plasmid pUC57-Virolle-a1-1430 (Table S2) using primers PS-cxm0-F/PS-cxm7-R. The PCR products together with plasmid p15A-cm-cxm-apra-Nocxm9 were electroporated into a Red/ET recombineering proficient E. coli GB05Red competent cells. Colonies grew on the apramycin, and hygromycin plates were selected for verification. The resulting plasmid PS-cxm8 is confirmed by restriction analysis; then it was transferred into S. coelicolor A3(2) to generate recombinant strain S. coelicolor A3(2)/cxm8. S. coelicolor A3(2)/cxm89, S. coelicolor A3(2)/cxm9, S. coelicolor A3(2)/cxm3456, S. coelicolor A3(2)/cxm34, and S. coelicolor A3(2)/PS were constructed in a similar manner, respectively. The corresponding primers and mutants are listed in Tables S1 and S2. Fermentation and Feeding Experiment. A. tsinanensis or S. coelicolor A3(2) was cultured in 250 mL Erlenmeyer flasks containing 50 mL of M1 medium with agitation at 250 rpm at 28 °C for 7 days. For the feeding experiments, compound 3 from the crude extract of Δcxm8 mutant (25 mL) was dissolved in methanol and added to each test sample after 3 days of inoculation, respectively, and the incubation continued for 4 days. Extraction and Isolation. The cells were separated from culture medium by centrifugation, and the pH of the supernatant was adjusted to 2−3 by HCl (6 M) before being extracted with an equal volume of EtOAc three times. The organic phases were combined and evaporated to dryness under vacuum to afford the crude extract. For dmCXM isolation, the crude extract (0.5 g) from the 5 L culture of S. coelicolor A3(2)/Δcxm8 was fractionated initially on a Sephadex LH-20 column (100 × 2.5 cm) using MeOH as a mobile phase. Fractions containing dmCXM were combined and further purified by a semipreparative RP HPLC (Agilent ZORBAX SB-C18 column, 250 × 9.4 mm, 5 μm, DAD at 226 nm) with gradient elution [solvents A (H2O and 0.1% formic acid) and B (CH3CN and 0.1% formic acid), 0−3 min, 35% B; 3−13 min, 35−43% B; 2.5 mL/min]. A 2.5 mg amount of dmCXM (3) was eluted at tR = 12.3 min. The crude extract of 20 L of broth of S. coelicolor A3(2)/Δcxm5 was subjected to VLC with silica gel using a stepped gradient elution with CH2Cl2/MeOH. Fractions are combined, concentrated, and further separated by ODS CC with a gradient elution step of MeOH/H2O (20% to 100%). The remaining semipreparative RP HPLC separation method for compound 4 is the same as that of dmCXM. Compound 4 (4.1 mg) was eluted at tR = 11.2 min. Compound 3: white solid, [α]20D −32 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 220 (4.3), 280 (3.5), 295 (3.7) nm; 1H and 13C NMR, Table S8; HRESIMS m/z 218.0286 [M − H]− (calcd for C11H9NO2S, 218.0281). Compound 4: white solid, [α]20D −28 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 220 (3.8), 275 (3.2), 300 (3.4) nm; 1H and 13C NMR, Table S9; HRESIMS m/z 381.0597 [M − H]− (calcd for C16H18N2O5S2, 381.0584).
Co., Ltd. 1H and 13C NMR spectra were recorded on an Agilent 500 MHz DD2 using tetramethylsilane as an internal standard. HPLCHRESIMS was carried out on a Thermo Scientific UltiMate 3000 UHPLC system connected to a Bruker ESI-Q-ToF Impact HD operating in negative ionization mode at a scan range of m/z = 100− 1000, auto MSn. Column chromatography (CC) was performed on silica gel (100−400 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20 (Amersham Biosciences), and ODS resin (50 mm, Merck). HPLC separations were performed on an Agilent 1260 instrument (Agilent, USA). A. tsinanensis Genomic DNA Isolation and Genome Sequencing. A. tsinanensis was cultured in 50 mL of medium 148G at 30 °C for 2 days. After centrifugation, the cells were resuspended in 5 mL of SET buffer (75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 7.5). After adding lysozyme to a final concentration of 1 mg/mL and incubating at 37 °C for 0.5−1 h, 500 μL of 10% sodium dodecyl sulfate and 125 μL of 20 mg/mL proteinase K were added, and the mixture was incubated at 55 °C with occasional inversion for 2 h until the solution became clear. The solution was combined with 2 mL of 5 M NaCl and 8 mL of phenol/chloroform/isoamyl alcohol (25:24:1) and incubated at room temperature for 0.5 h with frequent inversion. After centrifugation at 4500g for 15 min, the aqueous phase was transferred to a new tube using a blunt-ended pipet tip, and the DNA was precipitated by adding one volume of 2-propanol and gently inverting the tube. DNA was transferred to a microfuge tube, rinsed with 75% ethanol, dried under vacuum, and dissolved in ddH2O. The genome of A. tsinanensis was sequenced with PacBio technology at the Beijing Novogene Bioinformatics Technology (China) Co., Ltd. NCBI Database Entry. Sequences for the chuangxinmycin gene cluster cxm0−cxm9 have been submitted to GenBank under accession number MG824991. Direct Cloning of cxm Gene Cluster from Genome DNA of A. tsinanensis. The genomic DNA was completely digested with EcoRV and AscI. The linear cloning vector p15A-cm flanked with homology arms to target genes was amplified by PCR using p15A-cm-tetR-ccdBhgy as a template and primers (cxm-1 and cxm-2, Table S2). Then the direct cloning procedure was conducted according to our previous publication.4 Recombinants carrying cxm0−9 were identified by selection for chloramphenicol resistance (cm gene present on the linear cloning vector) and subsequent DNA restriction analysis and sequencing. To construct the plasmid for integration into heterologous hosts, a cassette containing oriT-apra-attP was inserted into p15A-cmcxm to form p15A-cm-cxm-apra by Red/ET recombineering. The primers are cxm-3 and cxm-4 (Table S2), and the template is pSET152. The verified plasmid was transferred into heterologous hosts S. coelicolor A3(2), S. albus J1074, and S. lividans K4-114 by conjugation according to the standard procedure. The exconjugants were verified by colony PCR using three pairs of primers (cxm-ch1/ cxm-ch2, 589bp; attB-L/attP-dn, and attB-R/attP-up). Construction of in-Frame Deletion Mutants. To construct the cxm0 in-frame deletion mutants, the selectable marker bla (the ampicillin-resistance gene) flanked with two PacI sites and homology arms was generated by PCR from plasmid pASK-sylABCDE11 using primers Cxm0-KO-F/R, with plasmid p15A-cm-cxm-apra, was electroporated into the recombinant enzymes, and induced expression of E. coli GB05Red4 competent cells. Colonies from the apramycin and ampicillin plates were selected for preparation of plasmid DNA. The replacement of cxm0 by bla was confirmed by restriction analysis. The resulting plasmid p15A-cm-cxm-apra-Nocxm0-amp was further digested by PacI and self-ligated by T4 ligase to remove the internal bla cassette, generating the recombinant plasmid p15A-cm-cxm-apraNocxm0, in which the 959 bp in-frame coding region of cxm0 was deleted to become a small ORF (15 bp). Plasmid p15A-cm-cxm-apraNocxm0 was transferred into E. coli ET12567/pUZ8002 and then conjugated into S. coelicolor A3(2).9 Colonies of exconjugants were selected and cultured in TSB (50 μg/mL apramycin) for 4 days and verified for integration by PCR screening to obtain the recombinant strains S. coelicolor A3(2)cxm/Δcxm0. Mutants Δcxm1−9 were constructed similarly, respectively. The corresponding primers and mutants are listed in Tables S1 and S2 and Figures S3−S12.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00835. Supporting Tables S1−S9, Supporting Figures S1−S22, NMR spectroscopic data, and copies of 1D and 2D NMR spectra of 3 and 4 (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*(X. Bian) Tel: +86-532-67721928. E-mail: bianxiaoying@sdu. edu.cn. *(Y. Zhang) Tel: +86-532-67720908. E-mail: zhangyouming@ sdu.edu.cn. D
DOI: 10.1021/acs.jnatprod.7b00835 J. Nat. Prod. XXXX, XXX, XXX−XXX
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ORCID
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Yuemao Shen: 0000-0002-3881-0135 Xiaoying Bian: 0000-0002-1356-3211 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Dr. A. Luzhetskyy (Saarland University) for providing the synthetic promoter plasmid and Dr. S. E. Ongley (The University of Newcastle, Australia) and Dr. V. Ravichandran (SDU) for proofreading of the manuscript. We thank the National Natural Science Foundation of China (31500033, 31670098, and 31670097), Natural Science Foundation of Jiangsu Province (BK20150388), National Key R&D Program of China (2017YFD0201405), the Major Basic Program of the Natural Science Foundation of Shandong Province, China (ZR2017ZB0212), Shandong Provincial Natural Science Foundation, China (ZR2017BC059), China Postdoctoral Science Foundation (2017M612261), Recruitment Program of Global Experts (Y.Z.), and Qilu Youth Scholar Startup Funding of SDU (X.B.) for funding support.
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DOI: 10.1021/acs.jnatprod.7b00835 J. Nat. Prod. XXXX, XXX, XXX−XXX
