微生物学报

脱叶链霉菌中白菖烯合酶基因的鉴定

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高彦微 ¹ , 张梦倩 ¹ , 陈珂 ¹ , 李静 ¹^,² , 刘道占 ¹ , 蔡由生 ¹ , 邓子新 ¹ , 朱冬青 ¹^,^*

微生物学报 | 研究报告 2025,65(12): 5294-5308

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微生物学报 | 研究报告 2025, 65(12): 5294-5308

脱叶链霉菌中白菖烯合酶基因的鉴定

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高彦微¹, 张梦倩¹, 陈珂¹, 李静¹^,², 刘道占¹, 蔡由生¹, 邓子新¹, 朱冬青¹^,^*

作者信息

^1.武汉大学药学院，组合生物合成与新药发现教育部重点实验室，湖北武汉

^2.广州医科大学附属第三医院，生物医学研究中心，广东广州

Identification of a calarene synthase gene from Streptomyces exfoliatus

Yanwei GAO¹, Mengqian ZHANG¹, Ke CHEN¹, Jing LI¹^,², Daozhan LIU¹, Yousheng CAI¹, Zixin DENG¹, Dongqing ZHU¹^,^*

Affiliations

^1.Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan, Hubei, China

^2.Biomedicine Research Center, the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China

出版时间: 2025-12-04 doi: 10.13343/j.cnki.wsxb.20250245

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摘要

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目的确认脱叶链霉菌UC5319中法尼烯焦磷酸(farnesyl diphosphate, FPP)环化酶基因orf2064的功能。方法采用大肠杆菌蛋白表达系统对orf2064进行表达，纯化重组蛋白，以FPP为底物开展体外反应，利用气相色谱质谱仪(GC-MS)检测产物。构建可高效合成FPP的大肠杆菌基因工程菌，对orf2064进行异源表达，通过GC-MS检测发酵产物，并从发酵产物中分离纯化目标产物，利用核磁共振(nuclear magnetic resonance spectroscopy, NMR)确定其结构。以链霉菌为宿主对orf2064进行异源表达，用GC-MS检测发酵产物。结果 ORF2064重组蛋白体外反应产物、orf2064大肠杆菌异源表达产物和链霉菌异源表达产物在GC-MS分析中均显示产生了1种保留时间相同、分子量为204的产物，分离纯化后经NMR分析确认其为白菖烯。结论脱叶链霉菌中基因orf2064所编码的FPP环化酶为白菖烯合酶。

关键词

白菖烯合酶 / 倍半萜 / 链霉菌

Abstract

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Objective To confirm the function of the farnesyl diphosphate (FPP) cyclase encoded by orf2064 in Streptomyces exfoliatus UC5319. Methods orf2064 was expressed in Escherichia coli, and the recombinant protein was purified and assayed with FPP as the substrate. The reaction products were detected by GC-MS. An FPP-overproducing E. coli strain was engineered for heterologous expression of orf2064. The fermentation products were analyzed by GC-MS, and the target compound was isolated and structurally characterized by nuclear magnetic resonance spectroscopy (NMR). In addition, orf2064 was heterologously expressed in Streptomyces, and the fermentation products were analyzed by GC-MS. Results GC-MS revealed that both the in vitro reaction of the recombinant protein ORF2064 and the heterologous expression products in E. coli and Streptomyces consistently produced a compound with identical retention time and [M⁺] of m/z 204. Subsequent isolation, purification, and NMR analysis confirmed this compound as calarene. Conclusion The FPP cyclase encoded by orf2064 in S. exfoliatus is identified as an calarene synthase.

Key words

calarene synthase / sesquiterpene / Streptomyces

引用本文

高彦微, 张梦倩, 陈珂, 李静, 刘道占, 蔡由生, 邓子新, 朱冬青. 脱叶链霉菌中白菖烯合酶基因的鉴定. 微生物学报, 2025 , 65 (12) : 5294 -5308 . DOI: 10.13343/j.cnki.wsxb.20250245

Yanwei GAO, Mengqian ZHANG, Ke CHEN, Jing LI, Daozhan LIU, Yousheng CAI, Zixin DENG, Dongqing ZHU. Identification of a calarene synthase gene from Streptomyces exfoliatus[J]. Acta Microbiologica Sinica, 2025 , 65 (12) : 5294 -5308 . DOI: 10.13343/j.cnki.wsxb.20250245

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Sesquiterpenes are a family of natural products with a variety of biological activities that are widely distributed in microbes and plants^[1-5]. Calarene (2) and its derivatives are sesquiterpenes typically found in plants, including those used in traditional Chinese medicine or as spices, such as Nardostachys spp.^[6-10], Schisandra chinensis^[11], Kadsura heteroclite^[12], Aquilaria spp.^[13], Panax spp.^[14-16], Acorus spp.^[17-18], Piper cordoncillo^[19], Valeriana spp.^[20-21], Humulus lupulus^[22], Trichosanthes kirilowii^[23], and Patrinia scabiosaefolia^[24].

Calarene appears effective against the larvae of the malaria vector Anopheles stephensi, the dengue vector Aedes aegypti, and the filariasis vector Culex spp.^[12]. Calarene was also shown to have a sedative effect^[13]. Calarene derivatives kanshone C and nardosinone are proven serotonin transporter (SERT) inhibitors^[7]. However, some calarene derivatives have the property of SERT activators, including 1(10)-aristolen-9β-ol, kanshone H, nardostachone, nardochalaristolones C, and nardoflavaristolone A^[7,25]. SERT is a classic drug target for neuropsychiatric and digestive disorders. The study of active molecules acting on SERT could contribute to drug discovery for mood disorders such as depression, anxiety, or obsessive-compulsive disorder, and for digestive disorders such as irritable bowel syndrome, slow transit constipation, and functional abdominal bloating^[7]. Nardosinone and kanshone B, the peroxide derivatives of calarene, inhibit NF-κB- and MAPK-mediated inflammatory pathways, demonstrating their potential in the treatment of neuroinflammation^[26].

Although certain sesquiterpene synthases produce calarene in trace amounts as a byproduct, no genuine calarene synthase has been identified to date. Here, we focused on a putative terpene synthase gene orf2064 in Streptomyces exfoliatus UC5319. Biochemical assays and heterologous expressions in this work proved that orf2064 is a calarene synthase gene.

1 Materials and methods

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1.1 Main reagents

Reagents and solvents were purchased and were of the highest quality available, and were used without further purification. Restriction enzymes, T4 DNA ligase, and DNA polymerase were obtained from New England Biolabs and Beijing Tsingke Biotech Co., Ltd., and were used according to the manufacturer’s specifications. Ni-NTA affinity columns were sourced from GE Healthcare and Beyotime Biotech Co., Ltd., Shanghai, China. Amicon Ultra Centrifugal Filters were acquired from Millipore. DNA primers were synthesized by Beijing Tsingke Biotech Co., Ltd. and Sangon Biotech (Shanghai) Co., Ltd..

1.2 Strain culture conditions and product detection methods

The growth media and conditions for Escherichia coli and Streptomyces strains, as well as the standard methods for handling the strains, were previously described. Bacillus subtilis was cultivated in SR medium (25 g/L yeast extract, 15 g/L tryptone, 3 g/L K₂HPO₄, pH 7.2) at 37 ℃. Enterococcus faecium, Enterococcus faecalis, and Streptococcus pneumoniae were cultivated in TSYE medium (30 g/L trypticase soy broth, 3 g/L yeast extract) at 37 ℃. Pantoea ananatis was cultivated in TSB medium (30 g/L trypticase soy broth) at 30 ℃. DNA sequencing was performed by Tsingke Biotech Co., Ltd. or Sangon Biotech (Shanghai) Co., Ltd. Protein concentrations were determined using the Bradford method with bovine serum albumin as the standard. Protein purity was assessed by SDS-PAGE. An Agilent 7890A/5975C-GC/MSD at 70 eV electron impact (positive ion mode) and an HP5MS capillary column (30 m×0.25 mm, 0.25 μm) were used for the GC-MS analysis of the terpenes. The conditions were as follows: a solvent delay of 3 min, a temperature program starting at 60 ℃ for 2 min, a temperature gradient from 60-280 ℃ for 11 min at 20 ℃/min, and a hold at 280 ℃ for 2 min. Samples were also analyzed using HPLC (Shimadzu SPD-M20A/LC-20AT) with a ThermoFisher Scientific C18 reversed-phase HPLC column (250 mm×4.6 mm, 5 μm). The HPLC conditions for terpenes (mobile phase A: water; mobile phase B: acetonitrile; UV detection λ: 210 nm) were as follows: 80% B for 30 min at a flow rate of 0.8 mL/min. The HPLC conditions for lycopene (mobile phase A: methanol; mobile phase B: acetonitrile; UV detection λ: 471 nm) were as follows: 30% B for 30 min at a flow rate of 1.0 mL/min.

1.3 Expression and purification of recombinant ORF2064 protein

A 1 044 bp DNA fragment containing orf2064 was inserted into Nde I and Not I sites of pET28a to generate plasmid pCK11. E. coli BL21(DE3) harboring the plasmid pCK11 was cultured with LB medium containing 50 μg/mL kanamycin at 37 ℃. IPTG (isopropyl β-d-1-thiogalactopyranoside) was added to a final concentration of 0.1 mmol/L when the OD₆₀₀ of the culture reached 0.6-0.8. The culture was further incubated at 18 ℃ overnight and centrifuged at 5 000×g for 15 min. The cells were harvested and resuspended in lysis buffer (300 mmol/L NaCl, 50 mmol/L Tris-HCl, 10 mmol/L imidazole, pH 7.4). The cells were disrupted by sonication and then centrifuged at 20 000×g for 60 min. The supernatant was transferred to a Ni-NTA column. The column was washed with wash buffer (20 mmol/L imidazole in lysis buffer). Protein was eluted with elution buffer (200 mmol/L imidazole in lysis buffer). Fractions were analyzed by SDS-PAGE. The purified recombinant ORF2064 protein was concentrated and buffer-exchanged into storage buffer (PBS buffer, pH 7.4, containing 10% glycerol) using an Amicon Ultra centrifugal filter (10 kDa MWCO).

1.4 Incubation of recombinant ORF2064 with farnesyl diphosphate

Purified recombinant protein ORF2064 (1 µmol/L) was incubated with 20 µmol/L FPP in 1 mL of assay buffer (50 mmol/L PIPES, 100 mmol/L NaCl, 15 mmol/L MgSO₄, 5 mmol/L β-mercaptoethanol, 20% glycerol, pH 6.8) overlaid with 1 mL of n-hexane at 30 ℃ for 16 h. The reaction mixture was quenched with 25 μL of 500 mmol/L EDTA and extracted three times with n-hexane. The organic extracts were dried over anhydrous Na₂SO₄, concentrated, and analyzed by GC-MS.

1.5 Construction of the plasmids harboring lycopene biosynthetic genes, MEP pathway genes or MVA pathway genes

The bacterial strains and plasmids utilized in this study have had their relevant data deposited in the National Microbiology Data Center (NMDC) under accession number NMDCX0002140. The primer sequences are also available under this same NMDC accession number.

pACYCDuet-1 and pCDFDuet-1 were used to construct the plasmids. To avoid burdening the E. coli host with too much protein expression, one of the T7 promoters of pACYCDuet-1 was replaced by the P _tac with a weaker transcriptional level to generate a new vector named pLJ74. Then the remaining T7 promoter of pLJ74 was also replaced by the P _tac to generate a new vector named pQQ21. One of the T7 promoters of pCDFDuet-1 was replaced by the P _tac to generate a new vector named pLJ81. Then the remaining T7 promoters of pLJ81 were replaced by the P _tac to generate a new vector named pLJ83.

DNA fragments harboring geranylgeranyl diphosphate synthase gene crtE, 15-cis-phytoene synthase gene crtB, and phytoene desaturase gene crtI were cloned from P. ananatis CGMCC 1.15633 and sequenced. According to the sequences, new primer pairs were synthesized to amplify crtE, ribosome binding site (RBS)-crtB, and RBS-crtI, respectively, which were inserted downstream of P _tac of pQQ21 successively to generate plasmid pQQ26.

DNA fragments harboring type II isopentenyl-diphosphate delta-isomerase gene idi, 1-deoxy-d-xylulose-5-phosphate synthase gene dxs, and polyprenyl synthetase gene ispA were cloned from B. subtilis R-179 and sequenced. New primer pairs were synthesized according to the sequences to amplify idi, RBS-dxs, and RBS-ispA, respectively, which were inserted downstream of P _tac of pLJ74 successively to generate plasmid pLJ79. The DNA fragment harboring P _tac, idi, dxs, and ispA from pLJ79 was also inserted into the corresponding sites of pQQ26 harboring crtE-crtB-crtI to generate plasmid pQQ27.

DNA fragments harboring hydroxymethylglutaryl- CoA reductase gene mvaE, hydroxymethylglutaryl-CoA synthase gene mvaS, mevalonate kinase gene mvaK1, diphosphomevalonate decarboxylase gene mvaD, phosphomevalonate kinase gene mvaK2 and type II isopentenyl-diphosphate delta-isomerase idi were cloned from Enterococcus faecium R-026 and sequenced. The mvaE, RBS-mvaS, mvaK1, RBS-mvaD, RBS-mvaK2, and RBS-idi were amplified respectively, which were inserted in the downstream of two tac promoters of pLJ83 successively to generate plasmid pLJ91. DNA fragments harboring mvaE and mvaS were cloned from Enterococcus faecalis FA2-2 and sequenced. DNA fragments harboring mvaK1, mvaD, mvaK2, and idi were cloned from Streptococcus pneumoniae CGMCC 1.8722 and sequenced^.Then the primer pairs were synthetized according to the sequences. Gene mvaE and RBS-mvaS were amplified and inserted downstream of one tac promoter of pLJ83, and gene mvaK1, RBS-mvaD, RBS-mvaK2, and RBS-idi were amplified and inserted downstream of another tac promoter to generate plasmid pYW7. Finally, RBS-ispA was amplified and inserted downstream of mvaS of pYW7 to generate plasmid pYW14.

1.6 Fermentation, extraction, and quantitative analysis of lycopene

E. coli strains carrying the crtE-crtB-crtI plasmids were cultured in LB medium with antibiotics at 37 ℃ overnight. Then 1% (V/V) of the culture was transferred to 50 mL of fresh 2×YT medium (1% yeast extract, 1.6% tryptone, 0.5% NaCl, pH 7.0) and cultured at 37 ℃. IPTG was added to a final concentration of 0.1 mmol/L when the OD₆₀₀ reached 0.4-0.6. The culture was further incubated at 28 ℃ for 24 h. Cells were harvested, freeze-dried, resuspended in acetone, and then disrupted by sonication. Cell debris was removed by centrifugation at 20 000×g for 5 min. The supernatant was filtered through a microporous membrane (0.22 μm, nylon) and used for HPLC analysis. The presence of lycopene in the samples was confirmed by the comparison of retention time and mass spectra with the pure authentic lycopene standard. The production of lycopene was measured using the external standard method.

1.7 Fermentation and product analysis of E. coli strains harboring orf2064

The orf2064 gene was inserted into pET21a to generate plasmid pCK10. E. coli strains carrying pCK10 were cultured overnight at 37 ℃ in LB media with antibiotics. Then, 1% (V/V) of the culture was transferred to fresh 2×YT media and incubated at 37 ℃ until the OD₆₀₀reached 0.4-0.6. IPTG was added to a final concentration of 0.1 mmol/L, and the culture was further incubated at 28 ℃ for 3-4 h. The culture was then overlaid with n-hexane at 30 ℃ for overnight and extracted three times with n-hexane. The combined organic extracts were dried over anhydrous Na₂SO₄ and concentrated for GC-MS analysis. To obtain the target compound, the crude extract of approximately 250 mg from the 5 L culture was dissolved using n-hexane, preliminarily separated by using SiO₂ column chromatography, and further purified by HPLC. HPLC (Shimadzu SPD-M20A/LC-20AT) and ThermoFisher Scientific C18 reversed-phase HPLC column (250 mm×9.6 mm, 5 μm) were used in purification. The HPLC conditions (mobile phase A: water; mobile phase B: acetonitrile; UV detection λ: 210 nm) were as follows: 80% B for 30 min at a flow rate of 3 mL/min. 4 mg of the target compound was recovered and analyzed by NMR. Carbon-13 and proton spectra were recorded on a Bruker AVANCE 400 MHz NMR spectrometer operating at 400 MHz and 100 MHz for ¹H and ¹³C respectively, equipped with a 5 mm BBFO Smart Probe. All experiments were carried out at 303 K in CDCl₃. The chemical shifts were reported in ppm and referenced to the solvent resonance signal (CDCl₃: ¹H δ_ppm 7.26, ¹³C δ_ppm 77.16).

1.8 Fermentation and product analysis of Streptomyces strains harboring orf2064

The orf2064 gene was inserted downstream of ermEp* of pIB139 to generate plasmid pCK12. Streptomyces strains carrying pCK12 were cultured overnight at 28 ℃ in TSBY medium (0.5% yeast extract, 3% tryptic soy broth, 10.3% sucrose) containing 50 μg/mL aparmycin. Then, 1% (V/V) of the culture was transferred to the fresh SFM medium (2% soy flour and 2% mannitol) and incubated at 28 ℃ for 7 days. The culture was then extracted three times with n-hexane. The organic extracts were dried over anhydrous Na₂SO₄ and concentrated for GC-MS analysis.

2 Results and discussion

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2.1 Terpene biosynthetic gene cluster orf2064 - 2065 of S. exfoliatus

Pentalenolactone (3) is the first typical sesquiterpene studied in detail^[27-28]. The pentalenene synthase gene penA (GenBank accession number ADO85594.1) from S. exfoliatus UC5319 encodes a 38 kDa monomer consisting of a single domain with a class I α-helical terpene cyclase fold and contains the conserved Asp-rich (DDXXD) and NSE/DTE motifs for binding the diphosphate moiety of the substrate along with three Mg^2+[28-30]. Pentalenene synthase catalyzes the cyclization of FPP to pentalenene (4) (Figure 1A)^[30-31]. Pentalenene (4) undergoes multi-step oxidoreductase catalysis to yield the final product, pentalenolactone (3)^[32-36].

To find new terpene synthase genes, the genome of the pentalenolactone-producing strain S. exfoliatus UC5319 was scanned. We found a gene cluster containing two open reading frames, orf2064 and orf2065 (GenBank accession number PQ468278). Gene orf2064 (Figure 2A, red arrow) encodes a 347 aa terpene synthase family protein with 22% identity and 37% similarity to pentalenene synthase PenA. ORF2064 displays the two highly conserved Mg²⁺-binding motifs of terpene synthases, the aspartate-rich ¹⁰⁹DDGVCD motif and the downstream ²⁵¹NDLFSYLKE motif. Similar to all other terpene synthases, the conserved motifs are separated by 142 amino acids. Gene orf2065 (Figure 2A, blue arrow) downstream of orf2064 encodes a 441 aa cytochrome P450 monooxygenase with 41% identity and 57% similarity to PenI (GenBank accession number ADO85595.1), which catalyzes the oxidative conversion of pentalenene (4) to 1-deoxypentalenic acid (5) (Figure 1A). The upstream gene orf2063 encodes a RluA family pseudouridine synthase, and the downstream gene orf2066 encodes a putative amino acid permease.

Homologs of the gene orf2063-orf2066 are present in some Streptomyces spp. strains (Figure 2B-2E). However, the homologs of orf2064 and orf2065 are replaced by a GNAT family N-acetyltransferase gene SAVERM_RS34460 in S. avermitilis MA-4680 (Figure 2F) and a terpene cyclase gene SCAB_RS35290 of unknown function (with 24% identity and 37% similarity to ORF2064 at the amino acid level) in S. scabiei 87.22 (Figure 2G) between the homologs of orf2063 and orf2066.

2.2 Biochemical characterization of recombinant ORF2064

To identify the function of ORF2064, gene orf2064 was amplified and inserted into the E. coli expression vector pET28a to generate plasmid pCK11. pCK11 was transferred into E. coli BL21(DE3) to express recombinant ORF2064. The recombinant ORF2064 protein was purified by Ni-NTA chromatography (Figure 3A). The purified recombinant ORF2064 protein was incubated with 20 μmol/L FPP in the presence of 15 mmol/L Mg²⁺ at 30 ℃ for 16 h. The mixture was quenched with EDTA and then extracted with n-hexane. The organic extracts were dried, concentrated, and tested by using GC-MS. GC-MS analysis showed the formation of a single sesquiterpene with an [M⁺] of m/z 204 and a base peak of 161.1 (Figure 3B, 3C). Comparison of the spectrum in the National Institute of Standards and Technology MS database allowed us to identify the compound as calarene (2) (Figure 3D).

2.3 Engineered E. coli host for the efficient in vivo synthesis of terpenes

The pure authentic standard of calarene could not be purchased; therefore, the product of FPP catalyzed by ORF2064 had to be isolated and purified for structural identification. To obtain sufficient quantities of the target compound, we used an engineered E. coli host BL21(DE3) (pMH1, pFZ81) designed for the efficient in vivo synthesis of terpenoid metabolites^[37]. Gene orf2064 was inserted into pET21a to generate plasmid pCK10, which was transferred into E. coli BL21(DE3) (pMH1, pFZ81). The GC-MS analysis revealed a sesquiterpene with an [M⁺] of m/z 204 (not shown), consistent with the product of FPP cyclization catalyzed by recombinant ORF2064 protein in vitro. According to GC-MS analysis, the production in this work was lower than 0.05 mg/L. A host cell with higher FPP production was therefore needed.

We need a reporter system to compare the FPP production of engineered E. coli hosts intuitively. FPP is the precursor for lycopene biosynthesis, so the red tetraterpenoid lycopene was chosen as a reporter. The plasmid pQQ26 was constructed (Figure 4A), carrying three lycopene biosynthesis genes amplified from Pantoea ananatis (geranylgeranyl diphosphate synthase gene crtE, 15-cis-phytoene synthase gene crtB, and phytoene desaturase gene crtI, GenBank accession numbers were PQ468284 and PQ468285). The resulting strain E. coli BL21(DE3) (pQQ26) turned a pale red, indicating that the reporter system was working.

Next, the plasmids pYW7, pYW14, pYW13, and pLJ91, which carry the genes of the mevalonate (MVA) pathway from different sources, were constructed separately. Plasmid pYW7 (Figure 4A) was constructed using MVA genes amplified from Enterococcus faecalis (hydroxymethylglutaryl-CoA reductase gene mvaE and hydroxymethylglutaryl-CoA synthase gene mvaS, GenBank accession number PQ468287 and PQ468286) and Streptococcus pneumoniae (mevalonate kinase gene mvaK1, diphosphomevalonate decarboxylase gene mvaD, phosphomevalonate kinase gene mvaK2, and type 2 isopentenyl-diphosphate delta-isomerase gene idi, GenBank accession number PQ468288). Gene ispA from B. subtilis (GenBank accession number PQ468280) was inserted into pYW7 to generate the plasmid pYW14 (Figure 4A). pYW13 is derived from a plasmid pJBEI-6410^[38] carrying MVA genes, a GPP synthase gene, and a limonene synthase gene from different sources. The GPP synthase gene of pJEBI-6410 was replaced with the FPP synthase gene ispA amplified from B. subtilis, and the limonene synthase gene of pJEBI-6410 was deleted to generate plasmid pYW13. Plasmid pLJ91 contains MVA genes and ispA amplified from Enterococcus faecium (GenBank accession numbers were PQ468281, PQ468282, and PQ468283).

The plasmids were separately transferred into E. coli BL21(DE3) (pQQ26) and the resulting transformants were cultured and induced with IPTG. BL21(DE3) (pQQ26, pYW7) and BL21(DE3) (pQQ26, pYW14) showed deeper red comparing with the control strain BL21(DE3) (pQQ26, pLJ83). BL21(DE3) (pQQ26, pLJ91) and BL21(DE3) (pQQ26, pYW13) didn’t deepen (not shown). HPLC and the pure authentic standard of lycopene were used to analyze the production of lycopene (Figure 4B, 4C). The result showed that the lycopene production of the control strain BL21(DE3) (pQQ26, pLJ83) was 0.09 mg/L in this work and that the lycopene production of BL21(DE3) (pQQ26, pYW7) increased 42-fold (about 3.93 mg/L) (Figure 4C, red columns). The lycopene production of BL21(DE3) (pQQ26, pYW14) (about 4.26 mg/L) didn’t increase significantly compared to BL21(DE3) (pQQ26, pYW7).

In summary, we constructed an E. coli host BL21(DE3) (pYW7) for the efficient in vivo synthesis of terpenes, which can be used to obtain sufficient quantities of the sesquiterpene product in the next work.

2.4 Heterologous expression of orf2064 in E. coli BL21(DE3) (pYW7)

The plasmid pCK10 carrying orf2064 was transformed into E. coli BL21(DE3) (pYW7). The GC-MS analysis of the culture of BL21(DE3) (pYW7, pCK10) gave a main product with the same characteristics as the product of ORF2064-catalyzed cyclization of FPP (Figure 5A, 5B). The culture containing the target compound was isolated by using SiO₂ column chromatography and semi-preparative HPLC, and tested by using GC-MS (NMDCX0002140). The target compound purified was analyzed by NMR (NMDCX0002140): ¹³C NMR (100 MHz, CDCl₃) δ 144.3, 120.5, 36.9, 36.8, 33.6, 30.0, 29.9, 27.4, 25.9, 23.1, 21.0, 19.7, 18.7, 16.7, 16.2 ppm; ¹H NMR (400 MHz, CDCl₃) δ 5.24 (m, 1H), 2.23 (m, 1H), 1.99-1.92 (m, 3H), 1.76 (m, 1H), 1.73 (m, 1H), 1.42 (m, 2H), 1.39 (m, 1H), 1.07 (s, 3H), 1.02 (s, 3H), 0.97 (d, J=7.7 Hz, 6H), 0.74 (m, 1H), 0.56 (d, J=9.2 Hz, 1H). The results showed that ¹H and ¹³C NMR spectra of the compound were consistent with the published NMR spectra of calarene^[6](NMDCX0002140). In summary, in vivo heterologous expression of orf2064 in E coli showed that ORF2064 is a calarene synthase.

2.5 Heterologous expression of orf2064 in Streptomyces

Several media were used to culture S. exfoliatus UC5319 and no calarene or its derivatives were found (data not shown). Gene cluster orf2064-2065 may be silent in S. exfoliatus UC5319. S. avermitilis was selected to express orf2064. Gene orf2064 was inserted into the Streptomyces expression vector pIB139 to generate plasmid pCK12. pCK12 was transferred into S. avermitilis NRRL 8165. The resultant strain S. avermitilis NRRL 8165::pCK12 was cultured. GC-MS analysis showed a sesquiterpene product with the same characteristics as the product of the E. coli strain harboring orf2064 (Figure 5C, 5D). In summary, in vivo heterologous expression of orf2064 in Streptomyces showed that ORF2064 is a calarene synthase.

3 Discussion

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3.1 Proposed biosynthetic mechanism of calarene

Sesquiterpenes are generated from FPP, and their structural diversity results from the first step catalyzed by sesquiterpene synthases. Sesquiterpene synthases convert FPP and generate and control the reactivity of high-energy carbenium ion intermediates in reactions involving carbon skeleton rearrangement, and methyl and hydride migration^[31,39-40]. According to other sesquiterpene synthase cyclization mechanisms^{[28,31,41-43]}, we presumed that the cyclization mechanism catalyzed by calarene synthase is initiated by ionization of the allylic diphosphate ester to the corresponding allylic cation-pyrophosphate ion pair, and subsequent electrophilic attack at C10 of the distal double bond, resulting in the formation of the intermediate germacradienyl cation (6) (Figure 1B). Insertion of the 2-propyl cation into the C-H bond with loss of the original proton would result in the formation of bicyclogermacrene (7). The intermediate bicyclogermacrene (7) would be further cyclized by protonation at C6 and intramolecular attack of the resulting carbocation on the C-2,3 bond to form the maaliane cation (8). Subsequent methyl migration and hydride shift, followed by deprotonation of C6, would then lead to the formation of calarene. The exact mechanism of cyclization needs to be investigated in future work.

3.2 Cytochrome P450 monooxygenase gene orf2065

We also focused on the cytochrome P450 monooxygenase gene orf2065. According to the structure of calarene, we presumed three possible oxidations catalyzed by ORF2065 (Figure 1C). First, ORF2065 may catalyze the conversion of C5 to the compound 10 (also known as 1(10)-aristolen-2-one) by stepwise oxidation via the compound 9. Second, ORF2065 may catalyze the epoxidation of the C6-C7 double bond to the compound 11, followed by the ring-opening reaction to produce the compound 12, or the compounds 13 and 14. Third, ORF2065 may catalyze the carboxylation of C12 (C13, C14, or C15) to the compound 17 (or 20, 23) by stepwise oxidation via the compound 15 (or 18, 21) and 16 (or 19, 22).

To confirm the function of the ORF2065 protein, orf2065 was amplified and inserted into pET28a to generate plasmid pCK14, which was transferred into E. coli BL21(DE3) to express. However, soluble recombinant protein of ORF2065 was not obtained. We tried feeding assays in E. coli BL21(DE3) (pCK14) by using calarene as the substrate. GC-MS analysis of the organic extract of the cultures. We wanted to find the new peak with an [M⁺] of m/z 220 (the compound 9, 11, 14, 15, 18, or 21), 218 (the compound 10, 16, 19, or 22), or 222 (the compound 12 or 13). While we didn’t find any new peaks. The organic extract was also methylated with TMS-CHN₂ and analyzed by GC-MS. The new peak with an [M⁺] of m/z 248 (methyl esters of the compound 17, 20, or 24) was not observed. Typical bacterial P450s require electron transport proteins for catalysis. We presumed that there are no auxiliary proteins (the ferredoxin reductase and ferredoxin) of ORF2065 in E. coli so Streptomyces may be a better host for ORF2065 expression. The orf2064 and orf2065 genes were amplified and inserted into pIB139 to generate plasmid pCK17, which was transferred into S. avermitilis NRRL 8165. NRRL 8165::pCK17 was cultured. GC-MS analysis of the organic extract. No new peak with an [M⁺] of m/z 220, 218, 222, or 248 was observed.

A BLASTp search of the NCBI protein database with the ORF2065 sequence revealed 11 sequence records of Streptomyces proteins with high degrees of sequence similarity and identity. Sequence alignment of ORF2065 and its homologs revealed that ORF2065 has 10 residues that differ from those at homologous sites found in its homologs, including Y37, R120, L135, T203, C267, R274, A330, S405, A409, and the absence of a glutamic acid residue between A376 and R377 (NMDCX0002140). We speculate that a mutation in one or more of the key residues may have rendered ORF2065 inactive.

3.3 E. coli host for the efficient in vivo synthesis of terpenes

An efficient host for FPP biosynthesis is useful in functional studies of sesquiterpene synthase. Here, we constructed E. coli BL21(DE3) (pYW7) for the efficient in vivo synthesis of terpenes. Unlike pJEBI-6410, pMH1, and pFZ81 carrying MVA genes from Saccharomyces cerevisiae and E. coli, pYW7 with a higher plasmid copy number and stronger promoters contains MVA genes from Streptococcus pneumoniae and Enterococcus faecalis. These differences may have contributed to pYW7 being more effective in this study.

To further enhance the production, the gene ispA and two key enzyme genes of the methylerythritol 4-phosphate (MEP) pathway in B. subtilis including type II isopentenyl-diphosphate delta-isomerase gene idi (GenBank accession number PQ468279) and 1-deoxy-d-xylulose-5-phosphate synthase gene dxs (GenBank accession number PQ468280), were inserted into pQQ26 to generate plasmid pQQ27, which was transferred into BL21(DE3) (pYW7), BL21(DE3) (pYW14) and the control strain BL21(DE3) (pLJ83). HPLC analysis showed that the lycopene production of the strains containing pQQ27 decreased by 22%-24% compared to the strains containing pQQ26, which was unexpected (Figure 4C, pink columns).

E. coli JM109(DE3) was also used in this study. The results showed that lycopene production of JM109(DE3) strains decreased by 55%-72% compared to BL21(DE3) strains (Figure 4C, orange columns and light orange columns).

4 Conclusion

收起

We cloned a terpene synthase gene orf2064 from S. exfoliatus UC5319. Incubation of purified recombinant protein ORF2064 with FPP in the presence of Mg²⁺ generated a sesquiterpene product. An engineered E. coli host BL21(DE3) (pYW7) was constructed for the efficient in vivo synthesis of FPP. Gene orf2064 was transferred into BL21(DE3) (pYW7) for heterologous expression, and the same product was observed. NMR analysis of the isolated product identified the sesquiterpene as calarene. Heterologous expression of orf2064 using S. avermitilis NRRL 8165 as the host also yielded the product calarene. The calarene synthase identified in this study provided a valuable tool for the biosynthesis of active compounds with a calarene skeleton.

Credit authorship contribution statement

收起

GAO Yanwei: Investigation, Writing-original draft; ZHANG Mengqian: Investigation, Methodology; CHEN Ke: Investigation; LI Jing: Writing-original draft; LIU Daozhan: Methodology; CAI Yousheng: Supervision; DENG Zixin: Resources; ZHU Dongqing: Conceptualization, Funding acquisition, Writing-review & editing.

Declaration of competing interest

收起

The authors declare no competing financial interest.

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doi: 10.13343/j.cnki.wsxb.20250245

接收时间：2025-03-26
首发时间：2025-12-08
出版时间：2025-12-04

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收稿日期：2025-03-26
录用日期：2025-05-22

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国家重点研发计划(2021YFA0909500)

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^1.武汉大学药学院，组合生物合成与新药发现教育部重点实验室，湖北武汉

^2.广州医科大学附属第三医院，生物医学研究中心，广东广州

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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