NRPS Gene Cluster

Oct 11, 2017 - In depth bioinformatic analysis of the genome sequence of S. pactum SCSIO 02999(5b, 10) reveals the presence of the lobophorin BGC (lob...
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Letter Cite This: Org. Lett. 2017, 19, 5697-5700

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Genome Mining and Activation of a Silent PKS/NRPS Gene Cluster Direct the Production of Totopotensamides Ruidong Chen,‡ Qingbo Zhang,‡ Bin Tan, Liujuan Zheng, Huixian Li, Yiguang Zhu, and Changsheng Zhang* Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China S Supporting Information *

ABSTRACT: A 92 kb silent hybrid polyketide and nonribosomal peptide gene cluster in marine-derived Streptomyces pactum SCSIO 02999 was activated by genetically manipulating the regulatory genes, including the knockout of two negative regulators (totR5 and totR3) and overexpression of a positive regulator totR1, to direct the production of the known totopotensamides (TPMs) A (1) and B (3) and a novel sulfonatecontaining analogue TPM C (2). Inactivation of totG led to accumulation of TPM B (3) lacking the glycosyl moiety, which indicated TotG as a dedicated glycosyltransferase in the biosynthesis of 1 and 2.

T

involved in biosynthesizing the nonproteinogenic amino acid (S)-3,5-dihydroxyphenylglycine (Dpg) for their high sequence similarities to DpgA (a type III PKS), DpgB/DpgD (an enoylCoA dehydratase), DpgC (an enoyl-CoA dehydrogenase), and Pgat (a transaminase), respectively, which generated Dpg in glycopeptides.12 The totD1 gene encodes a putative argininosuccinate lyase, and the totD2 gene encodes a fusion protein containing a PLP-dependent cysteine synthase and an argininosuccinate lyase (Table S1). The homologous counterparts of TotD1/TotD2, PacQ/PacS and DabA/DabB, are two key enzymes dedicated for the biosynthesis of 2,3-diaminobutyric acid (DABA) moiety of friulimicin and pacidamycin.13 Both totE1 and totE2 encode scyllo-inosamine-4-phosphate amidinotransferases, similar to streptomycin enzyme StrB1.14 A number of modifying enzyme-encoding genes are present in the tot BGC, including those for monooxygenases (totP1-totP4, and totF), methyltransferase (totM), sulfotransferase (totS), halogenase (totH), and glycosyltransferase (totG). The totI gene encodes an MbtH-like protein. The function of the MbtH-like protein PacJ has been shown to be an activator for the adenylation domain of PacL in the pacidamycin biosynthesis.15 TotK belongs to a conserved family of GHMP kinases. Five regulator encoding genes totR1-totR5 are found in the tot BGC (Table S1), suggesting a complicated regulatory network to govern the biosynthesis of the encoded products. A mining of the sequenced genomes of actinomycetes reveals the presence of similar tot gene clusters in other six Streptomyces strains, including S. olivaceus NRRL B-3009, S. pactum KLBMP 5084, Streptomyces sp. FXJ7.023, NRRL F-5630, PVA 94-07, and CNQ431 (Figure 1, Figure S1 and Table S1). All tot gene

remendous biosynthetic gene cluster (BGC) coding for potential secondary metabolites remains cryptic in actinomycetes.1 This fact prompts the concept of genome mining by developing diverse strategies to awaken these silent gene clusters,2 such as change of cultivation parameters,3 ribosome engineering,4 replacement of promoters,5 overexpression of pathway-specific activators,6 and synthetic biology-guided pathway reconstitution.7 Genome mining of marine actinomycetes was also successful for discovering new bioactive natural products8 and for connecting uncharacterized BGCs with known compounds.9 In our search for novel compounds from marine-derived actinomycetes, Streptomyces pactum SCSIO 02999 was of particular interest due to its ability to produce xiamycin-related indolosesquiterpenes,10 and new polycyclic tetramate macrolactams through BGC activation by promoter engineering.5b Herein we report the activation of a silent hybrid polyketide and nonribosomal peptide BGC (tot) in S. pactum SCSIO 02999 to produce totopotensamide (TPM) A (1) and B (3) and a novel sulfonated derivative TPM C (2) (Figure 1) by manipulating the pathway-specific regulators. In depth bioinformatic analysis of the genome sequence of S. pactum SCSIO 029995b,10 reveals the presence of the lobophorin BGC (lob), identical to that from two deep seaderived Streptomyces strains SCSIO 01127 and FXJ7.023.11 Immediately neighboring the lob BGC, another BGC (tot) is found to span a DNA region of around 92 kb (GenBank accession no. MG012231) and encodes 34 genes (Figure 1, Table S1). The tot BGC putatively codes a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) pathway, for the presence of two modular PKS genes (totA1 and totA2) and three NRPS genes (totB1, totB2 and totB3). The four genes, totC1−totC4, encode proteins that are putatively © 2017 American Chemical Society

Received: September 14, 2017 Published: October 11, 2017 5697

DOI: 10.1021/acs.orglett.7b02878 Org. Lett. 2017, 19, 5697−5700

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Organic Letters

Figure 1. Genetic organization of the tot gene clusters in seven Streptomyces species and the proposed PKS/NRPS assembly line and tailoring modifications directing the production of TPM A (1) and C (3). Nomenclature for catalytic domains: KS, ketosynthase; ACP, acyl carrier protein; AT, acyltransferase; DH, dehydratase; KR, ketoreductase; ER, enoylreductase; C, condensation; A, adenylation; T, thiolation; TE, thioesterase. TPM, totopotensamide. Note: totR5 was previously annotated as lobR1.11a

xiaP gene (essential for XMA biosynthesis10) was inactivated by in-frame deletion in the ΔtotR5i mutant (Figure S3). Interestingly, the titer of 1 was indeed enhanced in the double mutant ΔtotR5i/ΔxiaPi (Figure 2, iii), and was further improved in the triple mutant ΔtotR5i/ΔxiaPi/ΔtotR3 (Figure 2, iv), by insertional mutation of another putative transcriptional repressor gene totR3 (Figure S4). Bioinformatics analysis shows that TotR1, TotR2, and TotR4 belong to putative large ATP-binding LuxR (LAL) family transcriptional regulators,18 which are pathway-specific activators in triggering activation of cryptic BGCs by overexpression.6a In accordance with this prediction, the insertional inactivation of either of totR1, totR2, or totR4 in the double mutant ΔtotR5i/ΔxiaPi, led to a triple mutant (Figure S5) that abolished the production of 1 (Figure 2, v−vii). We then made a plasmid pCSG4810 carrying totR1 under control of the constitutive promoter ermE*p. When pCSG4810 was introduced into the double-mutant ΔtotR5i/ΔxiaPi to overexpress totR1, another two products 2 and 3 were observed (Figure 2, viii). To elucidate structures of 1−3, the engineered strain ΔtotR5i/ΔxiaPi overexpressing totR1 was fermented in a total

clusters display similar genetic organization, except the absence of several ORFs in strains NRRL F-5630, PVA 94−07, and CNQ431 (Figure 1). However, none of these tot gene clusters have been linked with chemically defined natural products. A PCR screening of the SuperCos1-based genomic library of Streptomyces sp. SCSIO 02999 afforded five overlapping cosmids that cover the intact tot gene cluster (Figure S1). Bioinformatics analysis reveals that TotR3 and TotR5 belong to the TetR family transcriptional regulators. This family of regulators are widely distributed in bacteria, and many are repressors to negatively control the antibiotic biosynthesis.16 TotR5 (previously annotated as LobR111a) has been proposed to be involved in lobophorin (LOB) biosynthesis.17 Since no production of LOBs were detected in S. pactum SCSIO 02999, we first attempted to activate the lob BGC by in-frame deletion of the putative repressor gene totR5, yielding the ΔtotR5i mutant (Figure S2). In comparison to the wild type strain (Figure 2, i), the ΔtotR5i mutant could produce a minor amount of an additional product 1 (Figure 2, ii). However, the UV spectrum and molecular mass of 1 did not match those of LOBs. Since that the major products of WT and the ΔtotR5 mutant are xiamycin (XMA)-related indolosesquiterpenes, the 5698

DOI: 10.1021/acs.orglett.7b02878 Org. Lett. 2017, 19, 5697−5700

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Bioinformatics analysis provides insights into necessary enzyme machineries that direct the biosynthesis of 1 and 2 (Figure 1). The aglycone (4) is biosynthesized by two multifunctional modular type I PKSs TotA1/TotA2 (to generate the linear polyketide chain) and three NRPS enzymes TotB1/TotB2/TotB3 (to sequentially assemble six amino acids into the polyketide chain). The assembly line matches well with the colinearity rule of PKS/NRPSs.20 The only exception is the presence of an incomplete module lacking a requisite A domain, between modules 8 and 9 in TotB1. A KSQ domain was present in the loading module of TotA1, and thus methylmalonyl-CoA was proposed to be the starter unit. The three AT domains in modules 1−3 of TotA1 and the first AT domain in module 4 of TotA2 were predicted to have the substrate specificity for methylmalonyl-CoA, while the AT domain in module 5 of TotA2 preferred malonyl-CoA (Figure S10). Unlike other 4 KR domains in modules 1−4, the KR domain in module 5 was likely inactive for lacking the catalytic residue Y (Figure S10). The DH domains of modules 3 (TotA1) and 4 (TotA2) displayed the conserved active site motif of H(x)3G(x)4PG. However, the corresponding motif in the DH domain of module 1 (TotA1) was H(x)2G(x)5PG (Figure S10). As such, this DH domain is likely inactive. According to these bioinformatics analyses, the PKSs TotA1 and TotA2 would generate a linear polyketide chain that matches exactly with that of 1−3 (Figure 1). The nonproteinogenic amino acid ClPhg should be biosynthesized through a pathway analogous to those established in glycopeptides (Figure 1), which involved a type III PKS TotC1, an enoyl-CoA dehydratase TotC2, an enoyl-CoA dehydrogenase TotC3, and a transaminase TotC4. The modifications leading to ClPhg, including a methylation by the methyltransferase TotM and a chlorination by the halogenase TotH, could occur prior to loading into in the NRPS assembly line, or happen as tailoring steps. We suppose that D-allo-Thr (probably generated by D-Thr aldolases21) is the precursor for the unusual amino acid (2R,3R)-DABA (Figure 1). A β-replacement of D-allo-Thr hydroxy group of the αamino of aspartate by TotD2, and a subsequent breakdown to release fumarate by the argininosuccinate lyase TotD1 would generate (2R,3R)-DABA in analogy to DABA biosynthesis in friulimicin and pacidamycin.13 The involvement of a βreplacement reaction in DABA biosynthesis was previously confirmed in mureidomycin A.22 Subsequently, a C-12 hydroxylation by oxygenase TiaP1−TiaP4 on 4 would generate 2, which was glycosylated by TotG to yield 1. Finally, the 7-Osulfonation by the sulfotransferase TotS afforded 2 (our unpublished observations). In summary, a cryptic PKS/NRPS gene cluster (tot) was activated in marine-derived S. pactum SCSIO 02999 to direct the production of TPMs by manipulating the regulator genes and confirmed the involvement of the tot gene cluster in the biosynthesis of TPMs by inactivating a dedicated glycosyltransferase. Our results indicate that the tot gene clusters in six other Streptomyces strains should encode products similar to TPMs. This work highlights the potential of marine actinomycetes as a source for novel natural products.

Figure 2. HPLC analysis of production profile of S. patum SCSIO 02999 wild type and mutant strains: (i) wild type; (ii) the ΔtotR5i mutant; (iii) the ΔtotR5i/ΔxiaPi double mutant; (iv) the ΔtotR5i/ ΔxiaPi/ΔtotR3 triple mutant; (v) the ΔtotR5i/ΔxiaPi/ΔtotR1 triple mutant; (vi) the ΔtotR5i/ΔxiaPi/ΔtotR2 triple mutant; (vii) the ΔtotR5i/ΔxiaPi/ΔtotR4 triple mutant; (viii) the ΔtotR5i/ΔxiaPi double mutant overexpressing totR1; (ix) the ΔtotR5i/ΔxiaPi/ ΔtotA2 triple mutant; (x) the ΔtotR5i/ΔxiaPi/ΔtotG triple mutant. The filled circles denote xiamycin-related indolosesquiterpenes.

culture volume of 12 L. Upon multiple chromatographic purification steps, 1 (28.3 mg), 2 (10.0 mg), and 3 (2.0 mg) were isolated. The molecular formula of 1 was established as C52H84ClN7O19 (m/z 1168.5423 [M + Na]+, calcd for 1168.5402) by HRESIMS. Inspection of 1H and 13C NMR spectroscopic data suggested the structural identity of 1 and totopotensamide (TPM) A (Table S2 and Figure S6), which was previously isolated from Streptomyces sp. 1053U.I.1a.1b.19 The presence of L-Ile, L-Ala, D-allo-Thr, and Gly was confirmed (Figure S7); however, the absolute configurations of D-2R,3RDABA and D-4-chloro-5,7-dihydroxy-6-methylphenylglycine (DClPhg) were assigned according to a previous study,19 without confirmation due to lack of standards. The molecular formula of 2 (designated TPM C) was determined to be C52H84ClN7O22S (m/z 1224.5025 [M − H]−, calcd for 1224.5005) by HRESIMS, indicating 2 to be a sulfonate group substituted analogue of 1. A comparison of the 1H and 13 C NMR spectroscopic data of 1 and 2 (Table S2 and Figure S8) revealed chemical shifts in the ClPhg moiety of 2. The values of high-field shift changes of C3 (0.4 ppm) and C5 (0.3 ppm) were comparable but largely different from that of C7 (3.5 ppm). It indicated the presence of a 7-O-sulfonate group in the ClPhg moiety of 2 (Figure 1), which was supported by the downfield shifts of ortho carbons (C6 7.7, C8 7.0 ppm) and para carbon (C4 5.0 ppm) of C7. Comparison of our and literature spectroscopic data determined 3 to be TPM B (Figure S9).19 To test if the tot BGC was involved in TPMs biosynthesis, the PKS gene totA2 and the glycosyltransferase gene totG were inactivated in the double mutant ΔtotR5i/ΔxiaPi (Figure S5). As a result, the triple mutant ΔtotR5i/ΔxiaPi/ΔtotA2 abolished the production of 1 (Figure 2, ix), while the other triple mutant ΔtotR5i/ΔxiaPi/ΔtotG produced only TPM B (3) (Figure 2, x). These experiments unequivocally confirmed that the tot BGC is responsible for the biosynthesis of TPMs, and furthermore, TotG was indicated as a tailoring glycosyltransferase. However, no antibacterial and antitumor activities were found for TPMs A−C (1−3), similar to a previous report.19 5699

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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.orglett.7b02878. Experimental details and compound characterization data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. ORCID

Changsheng Zhang: 0000-0003-2349-3138 Author Contributions ‡

R.C. and Q.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from NSFC (21472203, 41406183, 31290233), CAS (XDA11030403 and QYZDJ-SSW-DQC004), and Guangdong Province (GD2012D01-002). We are grateful to the analytical facilities in SCSIO.

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DOI: 10.1021/acs.orglett.7b02878 Org. Lett. 2017, 19, 5697−5700