Difunctional terpene synthase mutant and sesterterpenoids generated by catalysis of difunctional terpene synthase mutant

Abstract

The invention relates to a difunctional terpene synthase mutant and a sesterterpenoid compound generated by catalysis of the difunctional terpene synthase mutant, the difunctional terpene synthase mutant is a protein composed of a new amino acid sequence formed by mutating 195th valine of an amino acid sequence as shown in SEQ ID NO.1 into alanine, and the amino acid sequence of the difunctional terpene synthase mutant is as shown in SEQ ID NO.3. The invention also relates to a preparation method of the difunctional terpene synthase mutant. Compared with the prior art, the method has the advantages that the difunctional terpene synthase FoFS is subjected to semi-rational transformation, so that the mutant FoFS-V195A of the difunctional terpene synthase FoFS can catalyze the generation of novel skeleton sesterterpenoids with different stereo chirality. The strategy not only is beneficial to expanding the structural diversity of terpenoid natural products, but also provides important molecular resources for developing novel terpenoid compounds with potential drug value, and has important scientific significance and application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering, and in particular to a bifunctional terpene synthase mutant and a sesquiterpenoid compound catalyzed by the mutant. BACKGROUND

[0002] Terpene synthase is a key catalytic enzyme in the biosynthetic pathway of terpenoids, which precisely regulates the cyclization of linear precursors to form terpenoid skeletons with different ring systems and enantiomeric centers. Bifunctional terpene synthase has both prenyltransferase (PT) domain and terpene cyclase (TC) domain, which can independently complete the extension of isoprene chain and subsequent cyclization reaction to produce diterpene and sesquiterpene skeletons with different ring systems. The diversity of terpenoid skeletons is due to the precise regulation of the cyclization process of carbonium ion intermediates by bifunctional terpene synthase, while the instability of high-energy carbonium ion intermediates, the transient nature of the cyclization process, makes it difficult to capture, which brings difficulties to the study of the catalytic mechanism of bifunctional terpene synthase.

[0003] Protein engineering methods include directed evolution, rational design and semi-rational design. Due to the difficulty in obtaining protein crystals of bifunctional terpene synthase, the relationship between its spatial structure and function is still a great challenge, which limits the feasibility of rational design. In addition, due to the lack of detailed structural information, rational design cannot accurately locate the key sites, resulting in low success rate, so it is not suitable. Directed evolution is an effective means commonly used for enzyme protein modification and screening. This method generates a mutant library through random mutation, and combines high-throughput screening to optimize the function of the enzyme. However, due to the randomness of mutation, it often leads to a large scale of mutation library, which increases the screening cost and is accompanied by low efficiency and long cycle.

[0004] In recent years, semi-rational design has gradually become a more efficient protein modification strategy. This method uses bioinformatics tools, combines homologous protein sequence alignment, protein spatial structure analysis and catalytic mechanism research progress, and rationally selects the modification sites to construct a more simplified and efficient mutant library. This not only improves the success rate of protein modification, but also significantly reduces experimental cost and time. Therefore, based on the limited structure and catalytic function information of bifunctional terpene synthase, reasonable design and construction of high-efficiency mutant library not only helps to analyze the structure-activity relationship of terpene synthase, but also expands the diversity of terpenoid natural products, providing important support for the development of new terpenoid compounds with potential medicinal value. SUMMARY

[0005] The purpose of the present application is to modify bifunctional terpene synthase to catalyze the production of novel carbon skeleton sesquiterpenoid compounds, thereby providing a bifunctional terpene synthase mutant and a sesquiterpenoid compound catalyzed by the mutant.

[0006] This invention utilizes semi-rational design to perform molecular modification and identify mutant products of terpene synthases, thereby further understanding the relationship between the structure and function of terpene synthases, expanding the library of natural terpene products, and adding more potential pharmaceutical terpene compounds to the pipeline.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] One of the technical solutions of the present invention is to provide a bifunctional terpene synthase mutant, which is a protein composed of a new amino acid sequence formed by mutating valine at position 195 of the amino acid sequence shown in SEQ ID NO.1 to alanine, and its amino acid sequence is shown in SEQ ID NO.3.

[0009] The second technical solution of the present invention is to provide a nucleic acid encoding a mutant of a bifunctional terpene synthase as described in one of the above technical solutions, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0010] The third technical solution of the present invention is to provide a recombinant expression vector comprising the nucleic acid as described in the second technical solution above.

[0011] The fourth technical solution of the present invention is to provide a recombinant expression transformant, comprising the recombinant expression vector as described in the third technical solution above.

[0012] The fifth technical solution of the present invention is to provide an application of the bifunctional terpene synthase mutant as described in one of the above technical solutions in the synthesis of compounds containing a disesquiterpene skeleton.

[0013] The sixth technical solution of the present invention provides a catalytic product of a bifunctional terpene synthase mutant as described in one of the above technical solutions, comprising: catalytic product 1 of the bifunctional terpene synthase mutant is a compound containing a 5 / 3 / 7 / 6 / 5 pentacyclic sesquiterpene skeleton, with the molecular formula C 25 H 40 The structural formula is shown in Formula 1, and it is named: fusoxypene A;

[0014] The catalytic product 5 of the bifunctional terpene synthase mutant is a compound containing a 5 / 15 bicyclic disesquiterpene skeleton, with the molecular formula C5. 25 H 40 The structural formula is shown in Equation 5, and it is named: fictetraene A;

[0015] The catalytic product 6 of the bifunctional terpene synthase mutant is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C. 25 H 40 The structural formula is shown in Equation 6, and it is named: fusoxypene H;

[0016] The catalytic product 7 of the bifunctional terpene synthase mutant is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C. 25 H 40 The structural formula is shown in Equation 7, and it is named: fusoxypene G;

[0017] The catalytic product 8 of the bifunctional terpene synthase mutant is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C. 25 H 40 The structural formula is shown in Equation 8, and it is named: fusoxypene I;

[0018] The catalytic product 9 of the bifunctional terpene synthase mutant is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C. 25 H 40 The structural formula is shown in Equation 9, and it is named: fusoxypene J;

[0019]

[0020] The seventh technical solution of the present invention is to provide a method for preparing the catalytic product as described in the sixth technical solution above, comprising the following steps:

[0021] S1. Recombinant Escherichia coli containing the gene encoding a bifunctional terpene synthase mutant was inoculated into LB medium containing resistance elements, shaken and cultured, then transferred to self-induction medium supplemented with the same resistance elements, shaken and cultured to obtain fermented wet Escherichia coli cells.

[0022] S2. Acetone ultrasonic lysis and extraction of Escherichia coli fermentation wet cells, collection of supernatant, extraction with ethyl acetate to obtain crude extract of fermentation culture cells;

[0023] S3. The crude extract of the fermented cells was purified and prepared by silica gel column chromatography and semi-preparative liquid chromatography to obtain catalytic product 1 and catalytic products 5–9.

[0024] In some specific embodiments, step S1, more specifically, involves inoculating recombinant *E. coli* DH10b containing the gene encoding a bifunctional terpene synthase mutant into LB medium supplemented with 50 μg / mL kanamycin, and incubating at 37°C with shaking at 220 rpm for 8–10 h. Then, at a 2% inoculum size, the culture is transferred to self-induction medium supplemented with 50 μg / mL kanamycin and incubated at 37°C with shaking at 220 rpm until OD... 600 When the pH is 0.6–1.0, the culture conditions are changed to 17℃, 220rpm, and shaken for 60–72h to obtain Escherichia coli fermented wet cells.

[0025] In some specific embodiments, the self-inducing culture medium comprises: 0.5% (w / v) yeast extract, 1% (w / v) tryptone, 5% (v / v) 20× inorganic salt buffer, 5% (v / v) 20× carbon source, 0.2% (v / v) 500× MgSO4, pH 7.5–8.0; wherein the 20× inorganic salt buffer comprises: 1M Na2HPO4, 1M KH2PO4, 1M NH4Cl, and 0.1M Na2SO4. The 20× carbon source comprises: 2% (w / v) anhydrous glucose, 10% (w / v) glycerol, and 20% (w / v) α-lactose. The 500× MgSO4 comprises: 1M anhydrous MgSO4.

[0026] In some specific embodiments, step S3, more specifically, involves: preliminarily separating the crude extract of the fermented bacterial cells using silica gel column chromatography, with n-hexane as the elution mobile phase. TLC monitoring yields multiple components containing compounds 1, 5–9. The target components eluted from the silica gel column are then further separated and purified using a semi-preparative liquid chromatography column: using a ZORBAX SB-C18 semi-preparative liquid chromatography column (9.4 × 250 mm, 5 μm), component D2 containing compounds 1 and 8 is separated under isocratic elution conditions of 100% acetonitrile at a flow rate of 4 mL / min to obtain compounds 1 and 8; using a ZORBAX SB-C18 semi-preparative liquid chromatography column (9.4 × 250 mm, 5 μm), component D3 containing compounds 7 and 9 is separated under isocratic elution conditions of 100% acetonitrile at a flow rate of 4 mL / min to obtain compounds 7 and 9; using a ZORBAX semi-preparative liquid chromatography column... SB-C18 (9.4×250mm, 5μm) was used to separate fraction D4 containing compounds 5 and 6. Compounds 5 and 6 were obtained by isocratic elution with 100% acetonitrile at a flow rate of 4 mL / min.

[0027] This invention utilizes semi-rational modification of FoFS to enable its mutant, FoFS-V195A, to catalyze the formation of novel disesquiterpenoid compounds with different stereochiral structures 1,5–9. This strategy not only contributes to expanding the structural diversity of terpenoid natural products but also provides important molecular resources for developing novel terpenoid compounds with potential pharmaceutical value, demonstrating significant scientific importance and application prospects. Attached Figure Description

[0028] Figure 1 The catalytic product and GC-MS chromatogram of the bifunctional terpene synthase mutant FoFS-V195A.

[0029] Figure 2This is a schematic diagram of HR-EI-MS of compound 6 in methanol provided in an embodiment of the present invention.

[0030] Figure 3 – Figure 8 Compound 6 provided in the embodiments of the present invention in Benzene-d6 1 H NMR, 13 C NMR, HSQC, 1 H- 1 Schematic diagram of H COSY, HMBC, NOSEY.

[0031] Figure 9 This is a schematic diagram of HR-EI-MS of compound 7 in methanol provided in an embodiment of the present invention.

[0032] Figure 10 – Figure 15 Compound 7 provided in the embodiments of the present invention in Benzene-d6 1 H NMR, 13 CNMR, HSQC 1 H- 1 Schematic diagram of H COSY, HMBC, NOSEY.

[0033] Figure 16 This is a schematic diagram of HR-EI-MS of compound 8 in methanol provided in an embodiment of the present invention.

[0034] Figure 17 – Figure 22 Compound 8 provided in the embodiments of the present invention in Benzene-d6 1 H NMR, 13 C NMR, HSQC, 1 H- 1 Schematic diagram of H COSY, HMBC, NOSEY.

[0035] Figure 23 This is a schematic diagram of HR-EI-MS of compound 9 in methanol provided in an embodiment of the present invention.

[0036] Figure 24 – Figure 29 Compound 9 provided in the embodiments of the present invention in Benzene-d6 1 H NMR, 13 C NMR, HSQC, 1 H- 1 Schematic diagram of H COSY, HMBC, NOSEY.

[0037] Figure 30This is a schematic diagram illustrating the actual CD measurement and ECD calculation of compound 6 provided in an embodiment of the present invention.

[0038] Figure 31 A schematic diagram illustrating the actual CD measurement and ECD calculation of compound 7 provided in an embodiment of the present invention.

[0039] Figure 32 This is a schematic diagram illustrating the actual CD measurement and ECD calculation of compound 8 provided in an embodiment of the present invention.

[0040] Figure 33 A schematic diagram illustrating the actual CD measurement and ECD calculation of compound 9 provided in the embodiments of the present invention.

[0041] Figure 34 The relative configuration of compound 6 provided in the embodiments of the present invention and its key two-dimensional NMR correlation.

[0042] Figure 35 The relative configuration of compound 7 provided in the embodiments of the present invention and its key two-dimensional NMR correlation.

[0043] Figure 36 The relative configuration of compound 8 provided in the embodiments of the present invention and its key two-dimensional NMR correlation.

[0044] Figure 37 The relative configuration of compound 9 provided in the embodiments of the present invention and its key two-dimensional NMR correlation. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. In the following embodiments, unless otherwise specified, the raw materials or processing techniques are all conventional commercially available raw materials or conventional processing techniques in the art.

[0046] Example 1

[0047] In this embodiment, the wild-type bifunctional terpene synthase is a bifunctional terpene synthase screened from *Fusarium oxysporum* F. oxysporum 14005, and the amino acid sequence of the wild-type bifunctional terpene synthase is shown in SEQ ID NO.1. In this invention, the wild-type bifunctional terpene synthase is abbreviated as FoFS.

[0048] Fusarium oxysporum F. oxysporum 14005 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21067, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 31, 2020. Information regarding the deposit of Fusarium oxysporum F. oxysporum 14005 has been disclosed in patent CN113046332A.

[0049] The nucleotide sequence (also referred to as wild-type nucleotide, abbreviated as FoFS) of this wild-type bifunctional terpene synthase is shown in SEQ ID NO.2. The nucleotide sequence of this wild-type bifunctional terpene synthase was obtained by extracting RNA from Fusarium oxysporum (accession number: CGMCC No. 21067), obtaining cDNA through reverse transcription, and then sequencing.

[0050] This embodiment provides a bifunctional terpene synthase mutant, which is a protein composed of a new amino acid sequence formed by mutating valine at position 195 of the amino acid sequence shown in SEQ ID NO.1 to alanine, also known as FoFS-V195A.

[0051] Specifically, the new amino acid sequence formed by mutating valine at position 195 of the amino acid sequence shown in SEQ ID NO.1 to alanine is shown in SEQ ID NO.3.

[0052] This embodiment also provides an isolated nucleic acid encoding the bifunctional terpene synthase mutant. The nucleotide sequence of the isolated nucleic acid is shown in SEQ ID NO.4, abbreviated as FoFS-V195A.

[0053] The nucleotide sequence shown in SEQ ID NO.4 was obtained by PCR by mutating bases 582–584 of SEQ ID NO.2 from GTC to GCA.

[0054] The bifunctional terpene synthase and its mutants provided in this embodiment are sesquiterpene synthases that simultaneously possess both an isopentenyltransferase domain and a terpene cyclase domain.

[0055] This embodiment further provides a method for preparing a bifunctional terpene synthase, comprising the following steps:

[0056] The nucleotide sequence (SEQ ID NO. 2) and protein sequence (SEQ ID NO. 1) of the FoFS encoding gene were extracted from the bifunctional terpene synthase gene of *Fusarium oxysporum* F. oxysporum 14005. A protein model of the TC domain of FoFS was constructed using AlphaFold2. Based on the conserved motifs of the bifunctional terpene synthases and referring to the resolved crystal structures of the TC domains of fungal terpene synthases NfSS (PDB:8YLA) and PbSS (PDB:8YL9), a protein model consisting of a PPi group and three Mg groups was reconstructed in the TC protein model of FoFS-V195A. 2+ Mg composed of ions 2+ Coordination shell.

[0057] The active pocket of FoFS-TC was calculated using POVME, and in Seven key amino acid residues near the bottom of the active pocket were identified within the range, including two residues located on the G2 helix. Conserved kinks on the G1 / 2 helix are crucial features in the catalytic process of terpene synthases, playing a significant role in regulating the specificity of catalytic products. Among these, the kinks on the G1 / 2 helix have been identified as mutation hotspots, while the two key amino acid residues on the G2 helix have side chains facing the active cavity and jointly participate in constructing the pocket profile. Therefore, it is hypothesized that these two residues may regulate the cyclization reaction of carbocation intermediates during catalysis. Based on this, this study focuses on these two amino acid residues on the G2 helix to further explore their potential roles in the catalytic mechanism.

[0058] The coding gene for the wild-type bifunctional terpene synthase FoFS was recombined with the linearized plasmid pET28a fragment, and the recombinant product was transformed into *Escherichia coli* BL21(DE3). The mutation site was then designed into primers for whole-plasmid PCR. The PCR product was digested with DpnI to remove the plasmid template, and 2 μL of the digested product was transformed into *E. coli* BL21(DE3). After selection using antibiotic plates, single clones were picked and sequenced. The correctly sequenced clones were then induced for expression in *E. coli* BL21(DE3), and the bifunctional terpene synthase mutant FoFS-V195A was obtained.

[0059] This embodiment provides disesquiterpenoid compounds, the catalytic products of the bifunctional terpene synthase mutant FoFS-V195A, including:

[0060] The catalytic product 1 of the bifunctional terpene synthase mutant FoFS-V195A is a compound containing a 5 / 3 / 7 / 6 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C1. 25 H 40The structural formula is shown in Formula 1, and it is named fusoxypene A. The catalytic product 5 of the bifunctional terpene synthase mutant FoFS-V195A is a compound containing a 5 / 15 bicyclic disesquiterpene skeleton, and its molecular formula is C. 25 H 40 The structural formula is shown in Formula 5, and it is named fictetraene A; the catalytic product 6 of the bifunctional terpene synthase mutant FoFS-V195A is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, and its molecular formula is C 25 H 40 The structural formula is shown in Formula 6, and it is named fusoxypene H. The catalytic product 7 of the bifunctional terpene synthase mutant FoFS-V195A is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, and its molecular formula is C. 25 H 40 The structural formula is shown in Formula 7, and it is named fusoxypene G; the catalytic product 8 of the bifunctional terpene synthase mutant FoFS-V195A is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, and its molecular formula is C 25 H 40 The structural formula is shown in Formula 8, and it is named fusoxypene I. The catalytic product 9 of the bifunctional terpene synthase mutant FoFS-V195A is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, and its molecular formula is C. 25 H 40 The structural formula is shown in Equation 9, and it is named: fusoxypene J;

[0061]

[0062] This embodiment provides a method for obtaining disesquiterpenoid compounds by heterologous expression of the bifunctional terpene synthase mutant FoFS-V195A in Escherichia coli Escherichiacoli BL21(DE3), comprising the following steps:

[0063] The host bacterium was Escherichia coli BL21(DE3): using a bifunctional terpene synthase mutant encoded by a genetically engineered bacterium obtained by fermentation culture, the bifunctional terpene synthase mutant in the bacterial cell was used as a catalyst to directly extract terpenoid products from the bacterial cell and detect them by GC-MS.

[0064] (1) Recombinant *E. coli* DH10b containing the gene encoding the bifunctional terpene synthase mutant FoFS-V195A was inoculated into LB medium (0.5% (w / v) yeast extract, 1% (w / v) tryptone, and 1% (w / v) sodium chloride) supplemented with 50 μg / mL kanamycin and cultured at 37°C with shaking at 220 rpm for 8–10 h. Then, 2% of the inoculum was transferred to self-induction medium supplemented with 50 μg / mL kanamycin and cultured at 37°C with shaking at 220 rpm. When OD... 600 When the pH is 0.6–1.0, the culture conditions are changed to 17℃, 220rpm, and shaken for 60–72h to obtain Escherichia coli fermented wet cells.

[0065] Self-induction medium: yeast extract 0.5% (w / v), tryptone 1% (w / v), 20× inorganic salt buffer 5% (v / v), 20× carbon source 5% (v / v), 500× MgSO4 0.2% (v / v), pH 7.5–8.0. The 20× inorganic salt buffer consists of: Na2HPO4 1M, KH2PO4 1M, NH4Cl 1M, and Na2SO4 0.1M. The 20× carbon source consists of: anhydrous glucose 2% (w / v), glycerol 10% (w / v), and α-lactose 20% (w / v). The 500× MgSO4 consists of: anhydrous MgSO4 1M.

[0066] (2) Acetone was used to lyse and extract the fermented wet cells of Escherichia coli by ultrasonication (130W, 30min) for 30min. After centrifugation, the supernatant was collected and the lyse and extract were repeated 3 times. The 3 acetone extractions were combined and extracted 3 times with an equal volume of ethyl acetate to obtain the crude extract of the fermented cells.

[0067] (3) The crude extract of fermented bacterial cells was purified and prepared using silica gel column chromatography and semi-preparative liquid chromatography (HPLC), including the following steps: 2.7 g of the crude extract of fermented bacterial cells was initially separated by silica gel column chromatography. Hexane was used as the elution mobile phase for silica gel column chromatography, and TLC monitoring revealed multiple components containing compounds 1,5–9.

[0068] The target components eluted from the silica gel column were then separated and purified using a semi-preparative liquid chromatography (HPLC) column. Using a ZORBAX SB-C18 semi-preparative HPLC column (9.4 × 250 mm, 5 μm), fraction D2 containing compounds 1 and 8 was separated under isocratic elution conditions of 100% acetonitrile at a flow rate of 4 mL / min, yielding compound 1 (1.4 mg) and compound 8 (2.0 mg). Using the same ZORBAX SB-C18 semi-preparative HPLC column (9.4 × 250 mm, 5 μm), fraction D3 containing compounds 7 and 9 was separated under isocratic elution conditions of 100% acetonitrile at a flow rate of 4 mL / min, yielding compound 7 (1.0 mg) and compound 9 (0.8 mg). The fraction D4 containing compounds 5 and 6 was separated using a semi-preparative liquid chromatography column, ZORBAX SB-C18 (9.4 × 250 mm, 5 μm). Compound 5 (5.0 mg) and compound 6 (1.1 mg) were obtained under isocratic elution conditions of 100% acetonitrile at a flow rate of 4 mL / min.

[0069] (4) The prepared compounds 1,5–9 were detected by gas chromatography-mass spectrometry (GC-MS) under the following conditions: pulse split injection mode, injection port temperature of 250 °C, helium as carrier gas, and column flow rate of 1.77 mL / min. The initial temperature was 60 °C, then increased to 280 °C at a rate of 25 °C / min, and then increased to 310 °C at a rate of 10 °C / min, and held at 310 °C for 6 min.

[0070] The catalytic product and GC-MS chromatogram of the bifunctional terpene synthase mutant FoFS-V195A are shown below. Figure 1 As shown. By comparing with GC-MS spectra reported in the literature, 1 H and 13 By comparing the C NMR spectra, compounds 1 and 5 were identified as fusoxypenes A and fictetraene A, respectively.

[0071] Compound 6 is a colorless oil. +106.8 (c 1.0, CH3OH), the molecular formula was determined to be C using HR-EI-MS data. 25 H 40 (m / z 340.3132[M] + (C 25 H 40 The calculated value is 340.3130), and the degree of unsaturation is 6. Figure 2 ). 13C NMR of compound 6 1 H NMR, 13Analysis of C10 NMR and HSQC NMR spectra revealed that this compound possesses 5 methyl groups, 10 methylene groups, 4 aliphatic methine groups, 1 allyl methine group, and 4 sp groups. 2 and 1 sp 3 The presence of quaternary carbon atoms indicates a tricyclic structure. Compound 6 shows two new sp atoms. 2 It has quaternary carbon atoms (C-10 and C-3) and contains an external methyl double bond (H-20, δ 4.83, 4.87). The planar structure of compound 6 can be determined by... 1 D and 2 D NMR experiments determined ( Figure 3 – Figure 8 , Figure 34 (Table 1). The correlation between H3-22 and H-12 in NOESY indicates that Δ 11,12 The double bond has a Z configuration. The NOESY correlation between H-2 and H-7 / H3-23, and between H-13a and H-19 / H3-23, indicates that H-7, H3-23, and H-19 have the same orientation. Finally, electron circular dichroism (ECD) spectroscopy calculated based on density functional theory (TD-DFT) confirmed that the absolute configuration of compound 6 is 7S,14R,15S,18S,11Z. Figure 30 The compound was named fusoxypene H.

[0072] Compound 7 is a colorless needle-like crystal. +84.4 (c 0.05, CH3OH), its molecular formula is C 25 H 40 The value was determined using HR-EI-MS data (m / z 340.3131 [M]). + C 25 H 40 The calculated value is 340.3130, indicating that it has six degrees of unsaturation. Figure 9 ). 13 The C10 NMR and DEPT 135° spectra revealed 25 carbon signals, attributed to 6 methyl groups, 8 methylene groups, 6 aliphatic methine groups, and 5 quaternary carbon atoms, including two sp2 carbons. 2 Carbon atoms (C-2 and C-12) and three sp atoms 3 Carbon atom. The presence of a double bond indicates that compound 7 has a pentacyclic structure. The compound 7... 1 H and 13 Comparing the C NMR spectrum with the previously reported spectrum of quiannulatene, it was found that compound 7 showed a high ΔC NMR spectrum. 2,12There is a tetrasubstituted double bond at this position, which distinguishes it from quiannulatene. Two-dimensional NMR data analysis confirmed that the planar structure of 7 is a quiannulatene-type sesquiterpene with a 5 / 6 / 5 / 5 / 5 pentacyclic framework. Figure 10 -15, Figure 35 (Table 2). The correlations between H-9 and H3-20 / H3-21 / H-13b in NOESY, and between H-13b and H-14, indicate that H-9, H3-20, H3-21, and H-14 are on the same side. Similarly, the correlations between H-13a and H3-23 / H-19 / H-6, and between H-6 and H-7, indicate that H-6, H-7, H3-23, and H-19 are also on the same side. These NOESY interactions determine the relative configuration of 7 as 3R*, 6S*, 7S*, 10S*, 11S*, 14R*, 15S*, 18S*. Single-crystal X-ray diffraction experiments were successfully performed on the crystals of compound 7 obtained by multiple recrystallizations with methanol at -20°C. The analysis ultimately determined its configuration to be 3R*, 6S*, 7S*, 10S*, 11S*, 14R*, 15S*, 18S* [Flack parameter = 0.2(3)]. To further confirm the stereochemical assignment, time-dependent density functional theory (TD-DFT) calculations were performed at the B3LYP / 6-31G(d,p) level, simulating theoretical electronic circular dichroism (ECD) spectra. Figure 31 The simulated ECD spectrum showed a high degree of agreement with the experimental data, confirming its absolute configuration as 3R,6S,7S,10S,11S,14R,15S,18S. Therefore, compound 7 was identified as a novel sesquiterpene and named fusoxypene G.

[0073] Compound 8 is a colorless amorphous solid. +93.7 (c 0.05, CH3OH), as Figure 16 As shown, its molecular formula was determined to be C using HR-ES-MS data. 25 H 40 (m / z 340.3133; C) 25 H 40 The calculated value is 340.3130). Similar to compound 7, compound 8... 1 H and 13 The C10 NMR spectrum shows that it contains 6 methyl groups, 8 methylene groups, 6 aliphatic methine groups, and 5 quaternary carbon atoms (including two sp2 carbon atoms). 2 Carbon atoms C-2 and C-12 and three sp atoms 3 Carbon atoms). However, unlike compound 7, compound 8 exhibits one-dimensional nuclear magnetic resonance (NMR). 1The 3D NMR spectrum revealed a tetrasubstituted double bond at the C-2 and C-3 positions, matching the structure of quiannulatene. This similarity indicates that compound 8 has the same planar structure as quiannulatene. Figure 17 -twenty two, Figure 36 (Table 3). Further analysis revealed that the signals of C-4, C-5, C-6, C-7, and C-22 on the right side of the structure differed from those of quiannulatene, indicating a stereochemical difference between C-6 and C-7. The correlations between H-12 and H-6 / H3-23 in NOESY, and between H3-23 and H-19, suggest that H-6, H-12, H3-23, and H-19 are located on the same side. Furthermore, the correlation between H-9 and H3-21 / H3-22 indicates that H3-21, H-9, and H3-22 are also located on the same side. These data determined the relative configuration of compound 8 to be 6S*, 7S*, 10R*, 11R*, 12S*, 14R*, 15S*, 18S*. The absolute configuration of compound 8 was confirmed by comparing the predicted electronic circular dichroism (ECD) spectra of two possible enantiomers of compound 8 (6S,7S,10R,11R,12S,14R,15S,18S-8 and 6R,7R,10S,11S,12R,14S,15R,18R-8) with experimental spectra. Figure 32 The simulated ECD spectra of 6S, 7S, 10R, 11R, 12S, 14R, 15S, and 18S-8 were consistent with experimental data, thus determining the absolute configuration of compound 8. This compound was identified as a novel sesquiterpene and named fusoxypene I.

[0074] Compound 9 is a colorless needle-like crystal. +121.1 (c 0.05, CH3OH), as Figure 23 As shown, its molecular formula was determined to be C by HR-EI-MS data. 25 H 40 (m / z 340.3127; C) 25 H 40 The calculated value is 340.3130). Compound 9's... 1 H and 13 The C10 NMR spectrum showed 6 methyl groups, 8 methylene groups, 6 aliphatic methine groups, and 5 quaternary carbon atoms (including two sp2 carbon atoms). 2 Carbon atoms C-2 and C-12 and three sp atoms 3 The signals corresponding to carbon atoms indicate that it has a pentacyclic sesquiterpene core skeleton. NOESY further revealed its stereochemical correlations. Figure 24 -29, Figure 37(See Table 4). The NOESY correlation signals between H-6 and H-7 indicate that they are on the same side, as do the correlation signals between H-9 and H3-20. Furthermore, the NOESY correlation signals between H-13b and H-19 / H3-23, and between H3-23 and H-2, indicate that H-2, H-19, and H3-23 are on the same side. Based on these data, the relative configuration of compound 9 was determined to be 2R*, 3S*, 6S*, 7S*, 10S*, 14R*, 15S*, 18S*. To verify this hypothesis, single-crystal X-ray diffraction experiments were performed on compound 9 crystals obtained after multiple recrystallizations (at -20°C using a 1:1 mixture of methanol and chloroform). The experimental results clearly determined the relative configuration of compound 9 to be 2R*,3S*,6S*,7S*,10S*,14R*,15S*,18S*, with a Flack parameter of 0.2 (3). To further verify this result, time-dependent density functional theory (TD-DFT) calculations were performed at the B3LYP / 6-31G(d,p) level, simulating the theoretical electronic circular dichroism (ECD) spectrum. Figure 33 The simulated ECD spectrum showed a high degree of agreement with the experimental data, thus confirming its stereochemical inference. Therefore, compound 9 was named fusoxypene J.

[0075] Table 1. 1D and 2D NMR data of compound 6

[0076]

[0077] Table 2. 1D and 2D NMR data of compound 7

[0078]

[0079]

[0080] Table 3. 1D and 2D NMR data of compound 8

[0081]

[0082]

[0083] Table 4. 1D and 2D NMR data of compound 9

[0084]

[0085] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A bifunctional terpene synthase mutant, characterized in that, It is a protein composed of a new amino acid sequence formed by mutating valine at position 195 of the amino acid sequence shown in SEQ ID NO.1 to alanine, and its amino acid sequence is shown in SEQ ID NO.

3.

2. A nucleic acid encoding a mutant of the bifunctional terpene synthase as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A recombinant expression vector, characterized in that, It contains the nucleic acid as described in claim 2.

4. A recombinant expression transformant, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. The use of a bifunctional terpene synthase mutant as described in claim 1 in the synthesis of compounds containing a sesquiterpene skeleton.

6. A catalytic product of a bifunctional terpene synthase mutant as described in claim 1, characterized in that, include: The catalytic product 1 of the bifunctional terpene synthase mutant is a compound containing a 5 / 3 / 7 / 6 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C5. 25 H 40 The structural formula is shown in Formula 1, and it is named: fusoxypene A; The catalytic product 5 of the bifunctional terpene synthase mutant is a compound containing a 5 / 15 bicyclic disesquiterpene skeleton, with the molecular formula C5. 25 H 40 The structural formula is shown in Equation 5, and it is named: fictetraene A; The catalytic product 6 of the bifunctional terpene synthase mutant is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C. 25 H 40 The structural formula is shown in Equation 6, and it is named: fusoxypene H; The catalytic product 7 of the bifunctional terpene synthase mutant is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C. 25 H 40 The structural formula is shown in Equation 7, and it is named: fusoxypene G; The catalytic product 8 of the bifunctional terpene synthase mutant is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C. 25 H 40 The structural formula is shown in Equation 8, and it is named: fusoxypene I; The catalytic product 9 of the bifunctional terpene synthase mutant is a compound containing a 5 / 6 / 5 / 5 / 5 pentacyclic disesquiterpene skeleton, with the molecular formula C. 25 H 40 The structural formula is shown in Equation 9, and it is named: fusoxypene J; 7. A method for preparing the catalytic product as described in claim 6, characterized in that, Includes the following steps: S1. Recombinant Escherichia coli containing the gene encoding a bifunctional terpene synthase mutant was inoculated into LB medium containing resistance elements, shaken and cultured, then transferred to self-induction medium supplemented with the same resistance elements, shaken and cultured to obtain fermented wet Escherichia coli cells. S2. Acetone ultrasonic lysis and extraction of Escherichia coli fermentation wet cells, collection of supernatant, extraction with ethyl acetate to obtain crude extract of fermentation culture cells; S3. The crude extract of the fermented cells was purified and prepared by silica gel column chromatography and semi-preparative liquid chromatography to obtain catalytic product 1 and catalytic products 5–9.

8. The preparation method according to claim 7, characterized in that, In step S1, the more specific steps are as follows: Recombinant *E. coli* DH10b containing the gene encoding a bifunctional terpene synthase mutant was inoculated into LB medium supplemented with 50 μg / mL kanamycin and cultured at 37°C with shaking at 220 rpm for 8–10 h. Then, at a 2% inoculum size, the culture was transferred to self-induction medium supplemented with 50 μg / mL kanamycin and cultured at 37°C with shaking at 220 rpm. When OD... 600 When the pH is 0.6–1.0, the culture conditions are changed to 17℃, 220rpm, and shaken for 60–72h to obtain Escherichia coli fermented wet cells.

9. The preparation method according to claim 8, characterized in that, The self-inducing culture medium consists of: 0.5% (w / v) yeast extract, 1% (w / v) tryptone, 5% (v / v) 20× inorganic salt buffer, 5% (v / v) 20× carbon source, 0.2% (v / v) 500× MgSO4, pH 7.5–8.

0. The 20× inorganic salt buffer solution consists of: Na2HPO4 1M, KH2PO4 1M, NH4Cl 1M, and Na2SO4 0.1M. The composition of the 20× carbon source is: 2% (w / v) anhydrous glucose, 10% (w / v) glycerol, and 20% (w / v) α-lactose. The composition of 500×MgSO4 is: 1M anhydrous MgSO4.

10. The preparation method according to claim 7, characterized in that, For step S3, the more specific steps are as follows: The crude extract of fermented cells was initially separated using silica gel column chromatography, with n-hexane as the elution mobile phase. By TLC monitoring, multiple components containing compounds 1,5–9 were obtained. The target components eluted from the silica gel column were then separated and purified using a semi-preparative liquid chromatography column: using a ZORBAX SB-C18 semi-preparative liquid chromatography column (9.4×250mm, 5μm), the component D2 containing compounds 1 and 8 was separated under isocratic elution conditions of 100% acetonitrile and a flow rate of 4 mL / min to obtain compounds 1 and 8. Using a semi-preparative liquid chromatography column ZORBAX SB-C18 (9.4×250mm, 5μm), fraction D3 containing compounds 7 and 9 was eluted isocratically with 100% acetonitrile at a flow rate of 4 mL / min to obtain compounds 7 and 9. The fraction D4 containing compounds 5 and 6 was separated using a semi-preparative liquid chromatography column ZORBAX SB-C18 (9.4×250mm, 5μm). Compounds 5 and 6 were obtained by isocratic elution with 100% acetonitrile at a flow rate of 4 mL / min.

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