Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
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Targeted Discovery and Combinatorial Biosynthesis of Polycyclic Tetramate Macrolactam Combamides A−E Yan Liu,† Haoxin Wang,‡ Rentai Song,‡ Jining Chen,† Tianhong Li,† Yaoyao Li,*,† Liangcheng Du,‡,§ and Yuemao Shen†,‡ †
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, P. R. China ‡ State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, P. R. China § Department of Chemistry, University of Nebraska Lincoln, Lincoln, Nebraska 68588, United States S Supporting Information *
ABSTRACT: Polycyclic tetramate macrolactams (PoTeMs) are a growing class of natural products with distinct structure and diverse biological activities. By promoter engineering and heterologous expression of the cryptic cbm gene cluster, four new PoTeMs, combamides A−E (1−4), were identified. Additionally, two new derivatives, combamides E (5) and F (6), were generated via combinatorial biosynthesis. Together, our findings provide a sound base for expanding the structure diversities of PoTeMs through genome mining and combinatorial biosynthesis.
P
olycyclic tetramate macrolactams (PoTeMs) are a family of tetramic acid containing macrolactams with a fused carbocyclic system, and most of them exhibit pronounced biological activities including antimicrobial and antiprotozal activities, cytotoxicity, and inhibition of superoxide generation.1−5 According to the polycyclic system, PoTeMs can be classified into three different types, with alteramide A (5,5),6 HSAF (5,5,6),7 and ikarugamycin (5,6,5)8 being typical examples (Figure 1). PoTeMs are synthesized by an unusual hybrid iterative polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway, which only contains a single-module PKS-NRPS coupled with remarkably low numbers of redox enzymes.7−12 Owing to the complex structures and diverse biological activities of PoTeMs, and the simplicity of their biosynthetic loci, it has generated a significant degree of interest in genome mining of novel PoTeMs.13−16 However, the combinatorial biosynthesis of new PoTeM products was barely reported. Herein, we report the activation of a cryptic PoTeM gene cluster (cbm) in Streptomyces sp. S10 and the identification of four new PoTeMs, combamides A−D (1−4). In addition, two new derivatives, combamides E (5) and F (6), were generated by combinatorial biosynthesis. The results demonstrated that adding or swapping redox genes, involved in the formation of the polycylic system, between pathways would be a desirable approach to generate novel PoTeMs. © XXXX American Chemical Society
Figure 1. Chemical structure of representative PoTeMs.
Streptomyces sp. S10 was isolated from the soil collected at Nanjing Botanical Garden, Nanjing, China. By mining its recently sequenced genome for cryptic gene clusters, we identified a putative PoTeM gene cluster (cbm, Figure 2a and Table S1, GenBank accession no. MH167394). This cluster Received: April 23, 2018
A
DOI: 10.1021/acs.orglett.8b01285 Org. Lett. XXXX, XXX, XXX−XXX
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Since several cryptic PoTeM gene clusters have been activated by a promoter engineering approach,13−15 we decided to introduce the strong constitutive promoter kasO* upstream of the cbmA-D operon.19 However, after several trials no desired recombination strain was obtained. Previously, we have successfully expressed the HSAF biosynthetic genes in Streptomyces sp. LZ35 and showed the production of HSAF analogues;9 we thus decided to use a similar approach to activate the cbm gene cluster. First, we isolated a fosmid clone XIV-12H, from the genomic library of Streptomyces sp. S10, which contains the entire cbm gene cluster. The gene cluster (without cbmE) was then transferred into pSET152-kasOp* to give pSET152-PoTeMS10 for expression in Streptomyces (Figure S3). Subsequently, we introduced pSET152-PoTeMS10 into strain SR111, which is derived from Streptomyces sp. LZ35 through deletion of seven native PKS gene clusters and one native NRPS gene cluster, to generate strain SR111-PoTeMS10. HPLC analysis revealed that several extra peaks were detected in strain SR111-PoTeMS10 compared with the control strain SR111 (Figures 2b, S5, S6). The large-scale cultivation of SR111-PoTeMS10 led to the isolation of combamides A−C (1−3) (Figure 3). All of them
Figure 2. (a) Genetic organization of cbm gene cluster in Streptomyces sp. S10; (b) HPLC profiles of the metabolites of SR111, SR111PoTeMS10, SR111-PoTeMS10ΔcbmD, and SR111-PoTeMS10OX4. The “o” symbol denotes uncharacterized one-ring-containing combamides (details see Figure S6). The “*” symbol denotes uncharacterized combamide analogues. The “Δ” symbol denotes an uncharacterized compound produced by SR111 (details see Figures S5b, S6).
encodes one PKS-NRPS protein (CbmA) containing nine domains (KS-AT-DH-KR-ACP-C-A-PCP-TE), which shares 64.3% sequence identity with the PKS-NRPS from the HSAF biosynthetic gene cluster.15 Flanking the PKS-NRPS are four genes that encode a cascade of three tightly clustered redox enzymes (CbmB, CbmC, and CbmD) on one side and a sterol desaturase (CbmE) on the other side. The cbmB and cbmC genes encode enzymes with sequence similarity to NAD(P)/ FAD-dependent oxidoreductase, which is proposed to be involved in the formation of the polycyclic system of PoTeMs. To gain insight into the functions of CbmB and CbmC, we performed a phylogenetic analysis of currently available CbmBC homologues. The cladogram revealed that CbmC is clustered with SGR812 (77.6% identity) and PtmB2 (77.2% identity), which are proposed to be involved in the formation of the first ring of compound a and pactamides, respectively, and CbmB is clustered with SGR813 (66.2% identity) and PtmB1 (63.6% identity), which are assumed to be responsible for closure of the second five-member ring (Figures S1, S2).13,15 Sequence analysis revealed that CbmD is homologous to cytochrome P450 enzymes, but exhibits limited sequence identity with IkaD (47.5%) and CftA (53.8%), both of which perform oxidative reactions on the backbone of ikarugamycins.17,18 It indicated that CbmD may have distinct oxidative activities or regioselectivity. CbmE is similar to sterol desaturase (SD, 56.6% identity) in the HSAF gene cluster, and FtdA (60.7% identity) in the frontalamides gene cluster, which catalyze the C3 hydroxylation.11,12 Overall, the cbm gene cluster is quite different from the known PoTeM gene clusters (Figure S2). On the basis of the bioinformatics analysis, the predicted products of the cbm cluster would be 5,5-dicyclic PoTeMs with oxidative modifications. However, no PoTeMs was detected or isolated from Streptomyces sp. S10 under our culture conditions.
Figure 3. Chemical structure of combamides A−F (1−6).
were fully characterized by the analysis of HRESIMS and NMR data (Tables S3−S5, Figures S7−S9, S13, S15−S35). Compounds 1 (∼3.0 mg/L titer) and 2 (∼3.0 mg/L titer) are new PoTeMs containing the desired 5,5 bicyclic unit with oxidations at C16 and C30. The hexacyclic compound 3 (∼1.5 mg/L titer) is probably derived from the hypothetical compound IV via an intramolecular photochemical [4 + 4] cycloaddition as reported for alteramide A (Scheme 1).6 Compounds 1−3 lack the 3-hydroxy group due to the absence of the cbmE gene in strain SR111-PoTeMS10. The production of compounds 1−3 in strain SR111PoTeMS10 indicated that the IkaD and CftA homologue, CbmD, is probably responsible for the oxidation of C16 and C30 in combamides. To verify the function of CbmD, we B
DOI: 10.1021/acs.orglett.8b01285 Org. Lett. XXXX, XXX, XXX−XXX
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deleted the cbmD gene from pSET152-PoTeM S10 and constructed another recombination mutant SR111-PoTeMS10ΔcbmD. HPLC analysis showed that the strain produced three new peaks that were absent in SR111PoTeMS10 and eliminated the production of compounds 1−3 (Figure 2b). In addition, the “Δ” labeled peak was increased due to an unknown reason. The UV−vis spectrum of it is similar to that of a class of uncharacterized metabolites of Streptomyces sp. LZ35 (Figures S5b, S6). For the three new peaks, the two “o” labeled ones are identified as the one-ring-
containing combamides according to their UV spectra, while the remaining one (4) exhibits a similar UV−vis spectrum to that of compound 1, indicating compound 4 may be the new intermediate (Figure S6).21 To determine the chemical structure of the accumulated intermediate, we set out a preparative solid culture and obtained the purified compound 4 (∼7 mg/L titer). The structure of 4 was established by comparison of its NMR data (Table S6, Figures S10, S13, S36− S42) with those of 1. The relative configuration of C16 in 4 is C
DOI: 10.1021/acs.orglett.8b01285 Org. Lett. XXXX, XXX, XXX−XXX
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polycyclic systems through the combinatorial biosynthesis approach. Taken together, a biosynthetic route for combamides was proposed (Scheme 1). CbmA is responsible for the formation of the polyene-tetramate-polyene precursor as reported for other PoTeMs. 8−10,15 Then, CbmC catalyzes the first cyclization reaction with loose stereospecificity leading to the production of compounds I and II. Next, CbmB performs the second reductive cyclization to convert II to 4, or I to III and IV, respectively. Subsequently, CbmD installs the C30 ketone on compound IV, which may be further converted into the hexacyclic derivative 3 spontaneously as reported for alteramide A.6 Meanwhile, CbmD hydroxylates C30 of compound III to give compound V, which was further oxidized by CbmD alone or coupled with OX4 to generate 1, 2, 5, and 6. Although CbmD only shares less than 54% amino acid sequence identity with IkaD and CftA, it can also oxidize C30 as IkaD and CftA, but on varied PoTeM scaffold. One thing to be noted is that the order of the oxidation and the third ring formation might be reversed. The variety of stereochemistry in the polycyclic system of combamides is probably due to the relaxed selectivity of redox enzymes during the cyclization reactions as reported for the biosynthesis of HSAFs.20,21 Activities against Hela, HCT116, and SW480 cell lines of 1− 6 were determined using the SRB assay (Table S9). Compounds 1, 2, 4, and 5 were not active (IC50 > 100 μM) against the cell lines, and 3 showed weak activity (IC50 > 20 μM) against Hela and HCT116 cell lines. Interestingly, compound 6 lacking the hydroxyl group at C16 but containing a ketone group at C30 showed activity. Additionally, compound 6 strongly (MIC < 50 μM) blocked the secretion of Salmonella pathogenicity island 1 (SPI-1)-associated effector proteins, while 1−5 were inactive (Figure S14). The antimicrobial activities of 1−6 against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa PA01, Salmonella enterica serovar Typhimurium UK-1 χ8956, Candida albicans 5314, and Rhizoctonia solani were also detected by the filter paper method, and all of them were inactive at 50 μg/disk. In conclusion, we have activated a cryptic PoTeM gene cluster (cbm) by promoter engineering and heterologous expression, and identified four new PoTeMs, combamides A−E (1−4), from the recombination strains. In addition, we demonstrated a combination of redox enzymes from homologue pathways to generate new combamide analogs with a varied polycyclic system. Interestingly, the new analog 6 exhibited strong inhibitory effects on the secretion of SPI-1 effectors, which have not been reported for other PoTeMs. Considering the huge number of cryptic PoTeM gene clusters in the databases,12 our work present herein will prompt the future exploitation of new PoTeM products and generation of new derivatives with various polycyclic systems, oxidation positions, and configurations of the rings.
different from those of 1−3. Similar results have been reported for HSAFs produced by Lysobater enzymogenes.20,21 The structure of 4 provides evidence for the oxidation function of the cbmD gene in combamides biosynthesis. The data also establish the intermediate status of combamide D in combamides biosynthesis and provide a clue for the timing of the oxidative modifications of combamides in the biosynthetic pathway. The oxidations likely occurred after the formation of the 5,5 dicyclic unit, rather than an event prior to the closure of the second ring. To date, only two cytochrome P450 enzymes IkaD and CftA, which catalyze epoxidation of the double bond at C13/C14 and oxidation at C30 of ikarugamycins, have been identified.17,18 The PoTeMs can contain oxidative modifications at the C12, C13, C14, C15, C16, C20, C21, and C30 positions, in addition to the 3-hydroxy group catalyzed by SD.22 The oxidations at various positions suggest the presence of multiple enzymes, mainly P450s, capable of catalyzing specific modifications on the PoTeM backbones. Therefore, it will be interesting to further exploit the P450 enzymes involved in these post-PKS oxidative processes. The OX4 gene in the HSAF gene cluster and the ptmC gene in the ptm gene cluster have been proven to catalyze the formation of the six-membered ring of the 5,5,6 tricyclic system in HSAFs and pactamides, respectively.13,21 We set out to generate new combamide derivatives containing a 5,5,6 tricyclic unit by combining the OX4 gene with the cbm gene cluster in strain SR111. The OX4 gene, under the control of the ermE* promoter, was inserted downstream of cbmD in pSET152PoTeMS10 to give pSET152-PoTeMS10OX4. We introduced pSET152-PoTeMS10OX4 into strain SR111 to generate strain SR111-PoTeMS10OX4 and analyzed the metabolites in the transformant by using HPLC. Strain SR111-PoTeMS10OX4 produced two major peaks (5 and 6) that were absent in strain SR111-PoTeMS10 (Figure 2b). Their UV−vis spectra are similar to that of 3 de-OH HSAF, indicating compounds 5 and 6 probably contain three rings (Figure S6). Combamide E (5, ∼2.8 mg/L titer) and combamide F (6, ∼2.5 mg/L titer) were purified from strain SR111-PoTeMS10OX4 upon large-scale fermentation. Their corresponding chemical structures (Figure 3) were elucidated by extensive 1D- and 2D-NMR spectroscopy and MS experiments (Tables S7−S8, Figures S11−S13, S43−S56). The structural analysis showed that both of the new combamide derivatives (5 and 6) had the desired 5,5,6 tricyclic ring system, and the oxidative modifications at C16 and C30. However, only a fraction of available combamides seem to be converted. Several reasons may account for this observation, such as the suboptimal expression of the OX4 gene (Lysobacter) in a heterologous host (Streptomyces) or the substrate tolerance of OX4. To date, three different types of PoTeM gene cluster have been successfully expressed in a heterologous host, but the low yield of target compounds hindered the indentification of minor components, such as the “*” labeled ones in Figure 2.9,13,15,21 The challenge may be addressed by the development of a more suitable heterologous expression system, and the integration of the PoTeM resistant genes. To date, only two known PoTeMs (butremycin and clifednamide A) and a new one (clifednamide C, not fully characterized) were obtained by the combined expression of the ikarugamycin biosynthetic genes with the SD gene or the P450 gene (cf tA), respectively.17,23 Hence, our work presented the first example to generate new “unnatural” PoTeMs by varying their
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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.8b01285. Details of experimental procedures, metabolite production and isolation, and spectroscopic data description (PDF) D
DOI: 10.1021/acs.orglett.8b01285 Org. Lett. XXXX, XXX, XXX−XXX
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(21) Li, Y.; Wang, H.; Liu, Y.; Jiao, Y.; Li, S.; Shen, Y.; Du, L. Angew. Chem., Int. Ed. 2018, accepted, DOI: DOI: 10.1002/anie.201802488. (22) Zhang, G.; Zhang, W.; Saha, S.; Zhang, C. Curr. Top. Med. Chem. 2016, 16, 1727−1739. (23) Greunke, C.; Antosch, J.; Gulder, T. A. M. Chem. Commun. 2015, 51, 5334−5336.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yaoyao Li: 0000-0002-8762-9615 Liangcheng Du: 0000-0003-4048-1008 Yuemao Shen: 0000-0002-3881-0135 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by the NSFC (81573311, 81773598), the Young Scholars Program of Shandong University (2016WLJH31), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_17R68). L.D. was partly supported by Taishan Scholars Program of Shandong Province.
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DOI: 10.1021/acs.orglett.8b01285 Org. Lett. XXXX, XXX, XXX−XXX
