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Dec 7, 2017 - Chinese Academy of Sciences, Beijing, 100101, People's Republic of ... State Key Laboratory of Plant Genomics, Institute of Genetics and...
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Identification and Characterization of a Membrane-Bound Sesterterpene Cyclase from Streptomyces somaliensis Yanlong Yang,†,# Yuting Zhang,†,‡,# Shasha Zhang,§ Qingwen Chen,∥ Ke Ma,†,‡ Li Bao,†,‡ Yong Tao,§ Wenbing Yin,†,‡ Guodong Wang,∥ and Hongwei Liu*,†,‡ †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China ‡ Savaid Medical School, University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China § Chinese Academy of Science, Key Laboratory of Microbial Physiology and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China ∥ State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China S Supporting Information *

ABSTRACT: Sesterterpenes are usually found in plants and fungi, but are rare in bacteria. Here, we present the identification of StsC from Streptomyces somaliensis, a member of the UbiA superfamily, as a membrane-bound sesterterpene cyclase in bacteria. The cyclized products for StsC, somaliensenes A (1) and B (2), were identified by expressing the corresponding gene in an engineered Escherichia coli strain. The structures of 1 and 2 were determined by analysis of the NMR and MS spectroscopic data.

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known linear sesterterpenoids from bacteria.6 Recently, SpS from Streptomyces xinghaiensis was identified as a terpene synthase with sesqui-, di-, and sesterterpene synthase activity.17 However, no specific sesterterpene cyclase has been reported from bacteria. Herein, we report the discovery and characterization of a sesterTPS that catalyzes the formation of new cyclic sesterterpenes 1 and 2 from GFPP in bacteria. Streptomyces species are well known as a valuable source for bioactive secondary metabolites, especially antibiotics.18 However, terpenes are rarely isolated from this class of microbe. In general, the UbiA superfamily proteins catalyze the attachment of a prenyl chain or its derivatives in the production of ubiquinones, menaquinones, plastoquinones, hemes, chlorophylls, vitamin E, structural lipids, prenylated flavonoids, coumarin, and meroterpenoids.19 In a previous study, we identified a family of UbiA proteins (EriG) responsible for the biosynthesis of terpenes with different carbon skeletons in bacteria and fungi.20 The family EriG constitutes 990 homologues, including 28 homologues from Streptomyces species. To explore terpenes from the genus Streptomyces, we tried to express these potential terpene cyclases in the GGPPengineered E. coli BW25113. As a result, lydicene, a new

erpenes represent the largest group of natural products widely distributed in plants, marine organism, and microorganism.1,2 Terpenes are biosynthesized from linear polyprenyl diphosphate substrates, such as geranyl pyrophosphate (GPP, C10), farnesyl pyrophosphate (FPP, C15), geranylgeranyl pyrophosphate (GGPP, C20), and geranylfarnesyl pyrophosphate (GFPP, C25).3 In contrast to monoterpenes, sesquiterpenes, diterpenes, and triterpenes, the sesterterpenoids derived from GFPP are seldom reported from nature. Approximately 1000 sesterterpenes have been identified with only one isolated from bacteria.4−6 Sesterterpenoids have been shown to possess various bioactivities including cytotoxicity, anticancer, antimicrobial, antitubercular, and antiparasitic activities.4 In the terpenoid biosynthetic pathway, terpene cyclases play an important role in catalyzing the formation of diverse chemical scaffolds.7,8 Some sesterterpene synthases (sesterTPSs) including AcOS from Aspergillus clavatus, NfSS from Neosartorya f ischeri, EvSS, EvAS, and EvQS from Emericella variecolor, AtsesterTPS1, AtsesterTPS2, AtTPS17, -18, -19, -25, and -30 from Arabidopsis thaliana, Cr237 from Capsella rubella, Bo250 from Brassica oleracea, and Bcl-TS from Bacillus clausii have been reported so far.6,9−16 Bcl-TS is characterized as the sesterterpene synthase responsible for the biosynthesis of linear sesterterpenoid β-geranylfarnesene and β-hexaprene, which are © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 7, 2017

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DOI: 10.1021/acs.jnatprod.7b01033 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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corresponding product of StsC at 21.80 min failed because it was unstable. Compound 1 was isolated as a colorless oil. Its molecular formula was determined to be C25H40 (6 degrees of unsaturation) by HRESIMS. The 1D NMR data (Table 1) of

diterpene with a novel skeleton, has been identified as the metabolite produced by StlTC from Streptomyces lydicus.20 To further discover new diterpenes from Streptomyces, homologue genes of EriG in Streptomyces and their flank region were analyzed. As a result, a gene cluster (named sts, Figure 1A and Table S3), including one GGPPS (stsB), a

Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data for 1 (acetone-d6) and 2 (CDCl3) 1

Figure 1. Functional analysis of StsC. (A) Organization of the sts gene cluster in S. somaliensis. PPDPS: polyprenyl diphosphate synthase; GGPPS: geranylgeranyl pyrophosphates synthase; UbiA: UbiA prenyltransferase; ABC: ABC transporter; SHC: squalene-hopene cyclase. (B) GC-MS detection of products formed by expression of stsC in E. coli. a: compound 1. b: compound 2. c: cellular extracts from E. coli BW25113 transformant expressing stsC. d: cellular extracts from E. coli BW25113. (C) Structures of somaliensene A (1) and somaliensene B (2).

a

position

δC

1 2 3 4 5

145.1 46.2 32.1 39.8 31.8

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

117.2 41.8 39.4 24.4 126.1 135.0 40.5 27.2 125.1 135.4 40.5 27.5 125.2 131.6 25.8 17.7 16.0 16.1 17.8 23.2

2

δH (J in Hz) 2.04, 2.35, 2.17, 2.17, 2.23, 5.21,

oa o o o o o

1.66, o 2.02, o 5.21, o 1.99, o 2.09, o 5.11, o 1.99, o 2.09, o 5.14, o 1.66, 1.59, 1.61, 1.63, 0.86, 1.65,

s s s s s o

δC 133.9 121.0 31.6 39.8 28.5 30.9 154.5 35.1 26.9 124.4 135.3 39.9 26.9 124.4 135.3 39.9 26.9 124.6 131.4 25.9 17.8 16.2 16.2 107.3 23.6

δH (J in Hz) 5.40, 2.07, 2.09, 1.81, 1.47, 1.98,

s o o o o 2.06, o

2.06, o 2.06, o 5.11, o 1.98, o 2.06, o 5.11, o 1.98, o 2.06, o 5.11, o 1.68, 1.60, 1.61, 1.61, 4.76, 1.65,

s s s s d (6.5) o

“o” signals overlapped.

1 combined with HSQC experiment indicated that 1 possessed four trisubstituted double bonds [δH 5.21 (2H, H-6 and H-4′); 5.14 (H-12′); 5.11 (1H, H-8′); δC 145.1 (C-1), 117.2 (C-6), 126.1 (C-4′), 135.0 (C-5′), 125.1 (C-8′), 135.4 (C-9′), 125.2 (C-12′), and 131.6 (C-12′)], six singlet methyls including five vinylic methyls, eight sp3 methylenes, two sp3 methines, and one sp3 quaternary carbon. Compound 1 was deduced to be a bicyclic sesquiterpene to meet the requirement of unsaturation degrees on the basis of the above analysis. Further analysis of the 1H−1H COSY and HMBC correlations (Figure 2A), as well as the NMR data comparison between 1 and (−)-α-transbergamotene, assigned the planar structure of 1. NOE correlations of H-3 with H-2′, H-3′ with H-16′, and H-7′ with H-17′ confirmed the relative configuration at C-1′ and the trans configurations for double bonds between C-4′ and C-5′ and between C-8′ and C-9′ (Figure 2B). The absolute configuration in 1 was confirmed as 2S, 4S, 1′R by comparison of the optical rotation sign of 1 ([α]25D −109.9 (c 0.4, CHCl3)) with that of (−)-α-trans-bergamotene ([α] 20 D −31.0 (CHCl3)).21 Compound 2 was obtained as a colorless oil. Its molecular formula of C25H40 was established on the basis of HRESIMS. The 1H and 13C NMR data of 2 (Table 1) showed similarity to those of (−) axinyssene, except for the presence of an extra

polyprenyl diphosphate synthase (stsA), a squalene-hopene cyclase (stsE), and a UbiA-type diterpene cyclase (stsC), was found in the genome of Streptomyces somaliensis. The potential UbiA-type diterpene cyclase gene stsC from S. somaliensis was synthesized and expressed in E. coli BW25113 (Table S1). GCMS analysis of the extract from the resultant E. coli showed no expected diterpene hydrocarbons, but three major products at 21.30, 21.75, and 21.80 min with the same molecular ion peaks at m/z 340 [M]+ (Figures 1B, S1) corresponding to those of sesterterpene hydrocarbons were detected. To determine their structures, a 10 L scale fermentation of the E. coli strain expressing stsC was extracted with acetone and concentrated in vacuo. The acetone extract was suspended in MeCN and partitioned with hexane. The hexane-soluble portion was subject to silica gel column chromatography (CC), followed by semipreparative HPLC to afford sesterterpenes 1 and 2 (Figure 1C). The efforts to isolate and characterize the B

DOI: 10.1021/acs.jnatprod.7b01033 J. Nat. Prod. XXXX, XXX, XXX−XXX

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StsC shows 27−42% identity (Table S2) over 309 amino acids with other bacterial UbiA-type diterpene cyclases and shared two common highly conserved motifs, NXXX(G/ A)XXXD and DXXXD (Figure S6), which suggests that StsC is a class I terpene cyclase.20 Therefore, we propose an ionizationdependent cyclization reaction mechanism for the formation of sesterterpenes 1 and 2 by StsC (Scheme 1). The mechanism Scheme 1. Proposed Mechanism for the Formation of 1 and 2 from GFPP As Catalyzed by StsC Figure 2. (A) Selected key 1H−1H COSY and HMBC correlations for 1. (B) Key NOE correlations for 1.

trisubstituted double bond [δH 5.11 (H-8′); δC 124.4 (C-8′), 135.3 (C-9′)], two sp3 methylenes [δH 1.98 (H-6′), 2.06 (H7′); δC 39.9 (C-6′), 26.9 (C-7′)], and one vinylic methyl group [δH 1.61 (H-17′); δC 16.2 (C-17′)] in 2.22 Analysis of its 1 H−1H COSY, HSQC, and HMBC data of 2 (Figure S5) assigned the planar structure of 2 as shown in Figure 1C. The optical rotation sign of 2 {[α]25D −93.0 (c 0.5, CHCl3)} was in good accordance with that of (−) axinyssene {[α]D −34.2 (c 0.2, CHCl3)}.22 Therefore, we reasoned that 2 has the same absolute configuration (4S) as (−)-axinyssene. To further confirm the function of stsC, the coding region of stsC was cloned into a pET-28a expression vector. The resulting plasmid was expressed in E. coli BL21 (DE3), and a membrane fraction was prepared by ultracentrifugation at 100000g. Given that there is no commercial GFPP available, the catalytic activity of StsC was assayed in combination with a GFPP synthase, AtGFPPS2, from A. thaliana.23 When incubated, the membrane fraction of the E. coli BL21 (DE3) expressed stsC with AtGFPPS2, FPP, IPP, and Mg2+, and three product peaks at 21.30, 21.75, and 21.80 min (retention time) were observed for StsC in the GC-MS spectrum (Figures 3 and S2). In

for the reaction catalyzed by StsC presumably involves ionization of GFPP and formation of carbocation intermediate 4 by 1,6-cyclization. Deprotonation of 4 at C-15′ yields 2. 1 is formed via the intermediate 5, which is produced via pathway a. Finally, to determine whether sesterterpenes 1 and 2 were produced by S. somaliensis in vivo, the strain was fermented in four different types of media, and chemical extracts were evaluated by GC-MS with selected ion monitoring for 1 and 2; compounds 1 and 2 were not detected in any of the mycelia extracts by GC-MS. These data suggest that the genes encoding the StsC enzyme are silent in S. somaliensis under these standard laboratory culture conditions or that sesterterpenes 1 and 2 are converted into as-yet unidentified products by S. somaliensi. In the sts gene cluster, StsB shows 55% identity with CotB1, a GGPP synthase from Streptomyces melanosporofaciens MI614-43F2;24 StsA showed 21% identity to E-IDS2-S and 23% identity to E-IDS2-L, which can form GFPP from GGPP;25 StsE showed 24% identity with SqhC, a tetraprenylβ-curcumene cyclase from Bacillus subtilis JCM2499.26 Therefore, we proposed sts may be responsible for the biosynthesis of a more complex sesterterpene. In summary, we identified a new bacterial sesterterpene cyclase, StsC, in the genome of S. somaliensis, which converts GFPP into two unprecedented sesterterpenes, 1 and 2. StsC is a membrane-bound protein and belongs to the UbiA superfamily, which distinguishes it from other sesterTPSs. StsC represents the first sesterTPS in the UbiA superfamily. Some UbiA-related proteins have been identified as sesquiterpene cyclases (FmaTC from Aspergillus f umigatus)27 and diterpene cyclases including EriG from Hericium erinaceum, CyaTC from Cyathus striatus, SapTC1 from Saprospira grandis,

Figure 3. GC-MS results from the in vitro assay with FPP, IPP, AtGFPPS2, and the membrane fraction containing StsC.

contrast, no products were detected from the pET-28a vector. Enzyme products at 21.30 and 21.75 min were identified as sesterterpenes 1 and 2 by comparison to the retention time and dominant mass fragments with authentic 1 and 2. When GPP, FPP, or GGPP was used as substrates, no enzymatic product was detected. These results confirmed that StsC is indeed a sesterTPS responsible for the biosynthesis of sesterterpenes 1 and 2. C

DOI: 10.1021/acs.jnatprod.7b01033 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and ChlTC2 from Chlorof lexus sp.20 This study further expands the terpenoid cyclization function of UbiA proteins. Compared to fungi and plants, sesterterpenes are rarely isolated from bacteria. Our study provides opportunities for the discovery of new bacterial sesterTPSs and sesterterpenoids with novel scaffords.

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Author Contributions #

Y.-L. Yang and Y.-T. Zhang contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Natural Science Foundation of China (81673334 and 21472233).

EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectroscopic data were recorded on a Bruker Avance 500 spectrometer equipped with a cryogenic probe for 1H and 13C measurements (Bruker). Mass spectra were recorded on an Agilent Accurate-Mass-Q-TOF LC/MS 6520 spectrometer. GC-MS detection of products formed by expression of stsC in E. coli was performed on a GCMS-QP2010 Ultra with an autosampler (Shimadzu) and an Rtx-wax capillary column (60 m, 0.25 mm i.d., 0.25 μm film thickness; Shimadzu). GC-MS detection for products of StsC produced by in vitro assay was carried out using a Thermo Trace GC ultra-ISQ spectrometer with a DB-5 ms glass capillary column (0.25 mm × 30 m, 0.25 μm film thickness). Optical rotations were obtained on a polarimeter with sodium light (589 nm) by using a PerkinElmer 241 polarimeter. HPLC separation was conducted on an Agilent 1200 HPLC system equipped with an Agilent G1315D detector, using a Kromosil-ODS C8 column (5 μm; 9.4 × 250 mm). Methods Summary. The coding regions of stsC were synthesized by Sangon Biotech (Shanghai) and subcloned into the vector pRC1kPagE-idi. The resulting constructs were cotransformed with plasmids pYESKs and pSKPMIc into the E. coli BW25113 strain. Enzymatic products were analyzed by GC-MS of organic extracts from the relevant recombinant cultures. Details for the expression and in vitro assays of stsC and the fermentation of transformed strains are provided in the Supporting Information. The cultures were scaled up to enable production and purification of larger quantities for structural characterization by NMR. The cells in a 10 L culture of transformed E. coli were harvested by centrifugation (8000 rpm, 5 min, 4 °C). The cell pellet was extracted by acetone (200 mL) at room temperature. The acetone extract was suspended in MeCN (100 mL) and partitioned with hexane (100 mL × 2). Compounds 1 (0.4 mg) and 2 (0.8 mg) were obtained from the hexane extract by silica gel column chromatography eluted with hexane followed by semipreparative reversed-phase HPLC (98% MeCN in H2O for 40 min; 1: tR = 17.5 min; 2: tR = 14.6 min). Somaliensene A (1): colorless oil; [α]25D −109.9 (c 0.4, CHCl3); IR (KBr) νmax 2961, 2924, 2854, 1458, 1376, 1261, 1096, 1022, 801 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 341.3209 [M + H]+ (calcd for C25H40, 341.3203). Somaliensene B (2): colorless oil; [α]25D −93.0 (c 0.5, CHCl3); IR (KBr) νmax 3376, 2960, 2925, 1719, 1458, 1376, 1261, 1032, 802 cm−1; 1 H and 13C NMR data, see Table 1; HRESIMS m/z 341.3209 [M + H]+ (calcd for C25H40, 341.3203).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b01033. Full details of experimental methods; 1H and 13C NMR spectra of 1 and 2 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +86 10 64806074. E-mail: [email protected] (H.-W. Liu). ORCID

Wenbing Yin: 0000-0002-9184-3198 Guodong Wang: 0000-0001-9917-0656 Hongwei Liu: 0000-0001-6471-131X D

DOI: 10.1021/acs.jnatprod.7b01033 J. Nat. Prod. XXXX, XXX, XXX−XXX