Heterologous Expression of a Cryptic Giant Type I PKS Gene Cluster

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Heterologous Expression of a Cryptic Giant Type I PKS Gene Cluster Leads to the Production of Ansaseomycin Shuang He Liu,†,§ Wen Wang,†,§ Kai Biao Wang,† Bo Zhang,† Wei Li,† Jing Shi,† Rui Hua Jiao,† Ren Xiang Tan,†,‡ and Hui Ming Ge*,† †

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State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, Nanjing 210023, China ‡ State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, China S Supporting Information *

ABSTRACT: Genome mining of the marine Streptomyces seoulensis A01 enabled the identification of a giant type I polyketide synthase gene cluster (asm). Heterologous expression of the cryptic asm cluster using a bacterial artificial chromosome vector in heterologous host led to the production of ansaseomycins A (1) and B (2). A plausible biosynthetic pathway was also proposed. Additionally, compounds 1 and 2 are active against K562 cell lines with IC50 values of 13.3 and 18.1 μM, respectively.

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S3). Through CluterBlast analysis (a module in antiSMASH),9 the asm BGC is similar (45−80% identities) to BGCs of rifamycin, divergolide, or hygrocin,10−12 all of which are ansamycin-type compounds. Six modular type I PKSs, AsmC1−AsmC6, arranged in colinearity, were predicted assembly of the polyketide backbone. In particular, a sub gene cluster consisting of asmA1, A2, A3, A4, A5, and A6 is homologous to divN, divJ, divK, divL, divM, and divG, respectively, which account for the assembly of the starter unit 3-amino-5-hydroxybenzoic acid (AHBA) in divergolide biosynthetic pathway.11 Besides this, six gene products, AsmB1−AsmB6, showed sequence homology (57−76% identities) with enzymes Orf5−Orf10 in polyoxypeptin pathway, which are predicted to be responsible for the biosynthesis of an unusual extender unit, 2-(2-methylbutyl)malonyl-CoA.12 Thus, we speculate the asm gene cluster could be responsible for the biosynthesis of a new ansamycin-type compound with an unusual branch chain, which is somehow similar to divergolide or hygrocin.12,13 However, attempts to obtain its production of S. seoulensis A01 strain under various cultivation conditions proved unsuccessful, implying the asm cluster in its native host is silent. Previously, we constructed a bacterial artificial chromosome (BAC) library for S. seoulensis A01 strain to investigate the biosynthesis of streptoseomycin.7 The average DNA insert length was ∼120 kb, which is long enough to cover the intact asm cluster, as determined by analysis of 30 random clones.7 To isolate the BACs that contain the intact asm cluster, the BAC library was screened using the putative upstream

he recent explosion in genome sequencing clearly indicated that the biosynthetic potential of microorganisms, even the well-studied actinobacteria, is underappreciated.1,2 However, due to most of the gene clusters being transcriptionally silent under laboratory cultivation conditions, only a fraction of secondary metabolites have been isolated and characterized so far.3 Diverse synthetic biological efforts have been made to bypass the endogenous regulatory control and activate these silent pathways.4 In particular, heterologous expression of an intact biosynthetic gene cluster (BGC) in surrogate host has resulted in the production of many interesting natural products.5,6 Streptomyces seoulensis A01 is a marine actinobacterium derived from the gut of prawn collected from the Yellow Sea of China. Previously, a novel macrobilactone biosynthesized by a type I polyketide synthase (PKS) named streptoseomycin with potent antibacterial activities was isolated from A01 strain.7 In addition, this strain has the ability to produce ansamycin and limazepines G and H when cultivated in various fermentation conditions.8 Genome sequencing and annotation of S. seoulensis A01 strain indicated the presence of 33 BGCs. Besides the BGCs responsible for the biosynthesis of streptoseomycin and ansamycin,7,8 another giant type I PKS gene cluster (asm) putatively encoding ansamycin-type compounds was revealed by antiSMASH analysis.9 Herein, we report the heterologous expression of the cryptic asm gene cluster as well as the isolation, structure elucidation, and biosynthesis of two novel ansamycin-type compounds, ansaseomycins A and B (1 and 2). Bioinformatic analysis indicated the asm gene cluster (GenBank Accession no. MK599163) spans a ∼77-kb DNA fragment consisting of 36 open reading frames (orfs) (Table © XXXX American Chemical Society

Received: April 9, 2019

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DOI: 10.1021/acs.orglett.9b01237 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters (asmA1) and downstream (asmB6) genes as two probes, which lead to the identification of two positive hits. Among them, one positive BAC, pHG6001, spanning the intact asm gene cluster, was further confirmed by end-sequencing. We thus transformed pHG6001 into three model Streptomyces hosts, including S. lividans SBT18, S. coelicolor M1146, and S. albus J1074, through conjugation according to the standard procedure.7 The corresponding exconjugants with apramycin resistance were picked, purified, and named S. lividans SBT18/pHG6001, S. coelicolor M1146/pHG6001, and S. albus J1074/pHG6001, respectively. The genotypes of these recombinant strains carrying the asm gene cluster were further verified by diagnostic PCR. The three recombinant strains together with S. lividans SBT18, S. coelicolor M1146, and S. albus J1074 as negative controls were fermented to examine if the production of the final product can be observed in heterologous host. Gratifyingly, two major peaks both having the molecular weight of 561 (m/z, 584.2 [M + Na]+) were observed when S. lividans SBT18/pHG6001 was fermented in ISP3 liquid medium, whereas these peaks were absent in its corresponding negative control, S. lividans SBT18. In addition, the same peaks were also present in S. coelicor M1146/ pHG6001, while no products were observed in the recombinant strain S. albus J1074/pHG6001 (Figure 1). Thus, a large scale (20 L) fermentation was then carried out, which led to the isolation of compounds 1 and 2.

Figure 2. Key 2D NMR correlations of 1.

and third spin systems through quaternary olefinic carbon C-8. Similarly, the HMBC correlations from H-7, H-10, H2-29, and H3-28 to C-9 (δC 206.5) enabled the assignment of a linkage between C-8 and C-10 via the ketone carbonyl carbon C-9. Further HMBC correlations from H-19 (δH 3.61) to C-3 (δC 42.8), C-5 (δC 127.4), C-6 (δC 36.7), C-7 (δC 138.2), and from H-2 to a carbonyl carbon C-1 (δC 177.7) and C-20 (δC 67.6) suggested the presence of a cyclohexene ring fused to a γlactamide ring via C-3 and C-20, which was supported by HMBC correlations of NH (δH 8.31) with C-1, C-2, C-3, and C-20. Inspection of HMBC cross peaks from H-3 and H-19 to C-21 (δC 200.6) and from H-19 (δH 3.61) to C-18 (δC 194.4), C-21 and C-17 (δC 130.8) as well as a weak 4J HMBC correlation between H-19 and C-22 (δC 110.5) indicated the presence of a cyclohexene-1,4-dione moiety, which is fused to the established 5/6 bicyclic ring through C-19 and C-20. Moreover, the HMBC correlations from H3-26 (δH 2.22) to C23 (δC 162.7) and C-25 (δC 159.4) established a fully substituted phenyl ring, which was further reinforced by 4J HMBC correlations of H3-26 and C-22 and C-16 (δC 126.3). Finally, the diagnostic HMBC correlations of H3-27 (δH 1.28) and C-15 (δC 208.3) and H-14 and C-16 (δC 126.3) indicated the remaining carbonyl carbon C-15 was placed between C-14 and C-16 to complete the planar structure of 1. The double bond geometries of 1 were determined as 7E and 12E by NOESY correlations of H-6/H-29 and H-11/H-13. In addition, the clear NOE correlations of H-6/H-19, H-19/H3, H-3/NH, and NH/H-19 indicated the cis configuration of these four protons. However, due to lack the key NOE correlation, the relative configurations of remaining chiral carbons in 1 are difficult to be assigned. Fortunately, a high quality single crystal of 1 was obtained from MeOH-H2O (50:1) after several attempts. The X-ray diffraction analysis of 1 (Figure 3) measured using Cu Kα radiation at low temperature confirms the elucidated planar structure and further established the absolute stereochemistry of 1 as shown in Scheme 1. It is notable that 1 is indeed an ansamycin-type compound with an unprecedented 5/6/6/6/14-pentacyclic ring system. Compound 2 was obtained as a pale-yellow powder with the same molecular formula, C32H35NO8, as 1. The 1H and 13C NMR spectral data (Table S5) resembled those of 1. However, the 1H−1H COSY correlations from H-13 (δH 5.97) to H3-28 (δH 0.99) indicated that the Δ12,13 alkene in 1 was shifted to Δ13,14 position in 2. This assumption was confirmed by HMBC correlations of H3-27 (δH 1.82) to C-13 (δC 135.9), C-14 (δC 141.5), C-15 (δC 197.0), and H-13 to C-15 (Figure 4). Detailed interpretation of 1D and 2D NMR established the structure of 2.

Figure 1. Expression of asm gene cluster in heterologous hosts.

Compound 1 was isolated as a pale-yellow powder. The molecular formula of C32H35NO8 was determined by its HRESIMS data (m/z 584.2254 [M + Na]+, calcd for 584.2255), indicating 16 degrees of unsaturation. The 13C NMR data (Table S4) indicates 32 carbon signals corresponding to five carbonyl carbons, 12 aromatic/olefinic carbons, five methyls, as well as two sp3-hybridized methylenes, seven sp3hydridized methines, and one sp3-hydridized quaternary carbon as distinguished by HSQC spectrum. Analysis of the 1H−1H COSY NMR data reveled three discrete spin systems (Figure 2). The spin system spanning from H3-27 (δH 1.28) to H3-28 (δH 1.08) through H-14 (δH 3.43), H-13 (δH 5.69), H-12 (δH 5.70), H-11 (δH 4.06), and H-10 (δH 3.45) assigned according to COSY signals established the connectivity from C-27−C-28. The COSY correlations from H2-29 (δH 2.36) to H3-31 (δH 0.81) and H332 (δH 0.99) through H-30 (δH 1.55) indicated the presence of an isobutyl subunit. The third spin system from H-2 (δH 2.39, 2.90) to H-7 (δH 5.93) was also assigned based on the COSY correlations (Figure 2). The HMBC correlations from H2-29 to C-7 (δC 138.2) and from H-6 and H2-29 to C-8 (δC 140.7) connected the second B

DOI: 10.1021/acs.orglett.9b01237 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

Figure 4. Key 2D NMR correlations of 2.

Figure 3. X-ray crystal structure of 1.

With structures of 1 and 2 in hand, we thus reanalyzed the asm gene cluster in detail. Indeed, as we hypothesized previously, the gene products from two subclusters, asmA1− A7 and asmB1−B6, account for the biosynthesis of AHBA and isobutyryl side chain units in ansaseomycin, respectively. Six PKS genes transcribed in the same direction encode one loading module and eight chain extender modules and were suggested to assemble the polyketide backbone. The DH domains in modules 1 and 2 are relatively small and lack the key catalytic H residue (Figure S2), and the KR domain in module 3 lacks the key conserved Y residue (Figure S2),

The geometries of double bonds in 2 were deduced as 4Z, 7E, and 13E on the basis of coupling constant (JH‑4/H‑5 9.3 Hz) and NOE correlations of H-27/H-12 and H-6/H-29. The relative configurations of C-3, C-6, C-19, and C-20 are identical to those of 1, as confirmed by the analysis of NOE correlations and coupling constant data. In addition, on the basis of the same biosynthetic origin, we speculated that the configurations for chiral centers for 2 could also be the same to those in 1. Thus, the absolute configuration of 2 was defined as shown in Scheme 1. Scheme 1. Biosynthesis of Ansaseomycins A (1) and B (2)a

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(A) The gene cluster for biosynthesis of 1 and 2 in S. seoulensis A01. (B) Proposed biosynthetic pathway for 1 and 2. KS, ketosynthase; ACP, acyl carrier protein; AT, acyltransferase; KR, ketoreductase; DH, dehydratase; domains in grey circle are predicted to be inactive. C

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Organic Letters suggesting these three domains are inactive.14 In addition, although the DH domain in module 5 seems functional (Figure S2), no dehydration step is required according to the structure of 1 and 2. AsmD, a FAD-dependent monooxygenase, showing 61% amino acid sequence identity with Hgc2 was proposed to be involved in the formation of naphthalene ring in hygrocin biosynthesis.15 Knocking out asmD completely abolished the production of 1 and 2 (Figure 1), confirming its necessity in the biosynthesis of ansaseomycin. The fully extended polyketide chain was finally cyclized and released from the PKS to afford intermediate 3 by an amide synthase AsmE.13,15 The cyclohexene ring in 1 and 2, which has not been observed in other ansamycin-type compounds, is expected to be formed via an enzyme-catalyzed or spontaneous intramolecular Diels−Alder reaction using 4 as the substrate.16−18 Thus, 3 could undergo the double bond shift to afford 4, followed by a Diels−Alder reaction to give 2. The Δ13,14 alkene in 2 shift to Δ12,13 position gives 1. Finally, 1 and 2 were evaluated for their antibacterial activity against several pathogenic strains, including Micrococcus luteus CMCC 28001, Staphylococcus aureus CMCC 26003, Bacillus subtilis CICC 10283, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa CICC 10351. Both were almost inactive to the tested strains (MIC > 64 μM). In addition, compounds 1 and 2 exhibited moderate activities against K562 (leukemia) cell lines with IC50 values of 13.3 and 18.1 μM, respectively. In summary, awakening of a cryptic type I PKS biosynthetic gene cluster from S. seoulensis A01 using a BAC vector in a surrogate host was successfully achieved. Two structurally novel ansamycin-type compounds, featuring a unique pentacyclic 5/6/6/6/14 ring system, were isolated and characterized. The biosynthetic pathway involving a [4 + 2]cycloaddition step was proposed. The successful heterologous expression of asm gene cluster not only paves the way for future biosynthetic study of 1 and 2 but also indicates the feasibility of heterologous expression for activating cryptic clusters and excavating the great biosynthetic potential from thousands of sequenced strains.

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Hui Ming Ge: 0000-0002-0468-808X Author Contributions §

S.H.L. and W.W. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by NSFC (Grants 21861142005, 21572100, 81773591, 81530089, 81673333, 81803380, 21761142001, and 21661140001), MOST (Grant 2018YFC1706205), and the Fundamental Research Funds for the Central Universities (Grants 020814380092 and 020814380113).

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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.9b01237. Detailed experimental procedures and NMR data for 1 and 2 (PDF) Accession Codes

CCDC 1888294 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, by emailing data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ren Xiang Tan: 0000-0001-6532-6261 D

DOI: 10.1021/acs.orglett.9b01237 Org. Lett. XXXX, XXX, XXX−XXX