Bifunctional terpene synthase, its mutants and catalytic products

Abstract

The present invention relates to a bifunctional terpene synthase and its mutants, as well as the catalytic products. The amino acid sequence of the bifunctional terpene synthase is shown in SEQ ID NO.1. The bifunctional terpene synthase mutant is a protein composed of a new amino acid sequence formed by mutating the leucine at the 89th position of the amino acid sequence shown in SEQ ID NO.1 to glutamine or asparagine. The bifunctional terpene synthase FoFS to be modified in the present invention simultaneously has an isopentenyl transferase domain and a terpene cyclase domain, and has the dual functions of catalyzing the head-to-tail connection and cyclization of the terpene synthesis precursors DMAPP and IPP. FoFS has sesquiterpene products with multiple different ring systems. By modifying FoFS, the mutant protein can catalyze sesquiterpene compounds with other complex ring systems, which is of great significance for expanding the library of terpene natural products and adding more potential drug-value terpene compounds to the reserve force.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to bifunctional terpene synthases and their mutants, as well as catalytic products. Background Art

[0002] Terpene synthases are a class of important enzymes in the biosynthetic pathway of terpenoids, responsible for cyclizing linear terpenoid precursors to form structures with different ring systems and enantiomeric stereocenters. The complex and precise catalysis of terpene synthases determines the rich and diverse structural skeletons of terpenoids. Terpenoids are the most numerous, diverse, and structurally rich secondary metabolite natural products in nature. The complexity and diversity of the structures of terpenoids endow them with a wide range of biological activities. There are many well-known drug molecules among terpenoids, such as artemisinin for anti-malaria, paclitaxel for anti-tumor, and glycyrrhizic acid for anti-hepatitis drugs, etc.

[0003] Bifunctional terpene synthases mainly come from fungi and have both a prenyltransferase (PT) domain and a terpene cyclase (TC) domain. Therefore, they have the dual functions of head-to-tail connection and cyclization of the terpene synthesis precursors dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). So far, the catalytic products of bifunctional terpene synthases from fungi are all diterpenes and sesterterpenes, and most of them are tricyclic or tetracyclic. According to the catalytic mechanism, the PT domain determines the carbon chain length of the catalytically synthesized terpenoids, while the TC domain plays a key role in the ring system skeleton and stereoconfiguration of the product. And with the migration of the carbocation on the cyclization reaction intermediate, the ring system structure of the product will gradually become more complex. However, due to the existence of high-energy carbocation intermediates, the cyclization process is rapid and difficult to capture, which also brings difficulties to the study of the catalytic mechanism of bifunctional terpene synthases. At the same time, due to the existence of the double domains and multiple flexible regions of bifunctional terpene synthases, it is very difficult to obtain and analyze the protein crystal structure. So far, there is still no crystal structure of the whole bifunctional terpene synthase enzyme. Even for the truncated TC domain crystal, there is only one case. Therefore, the understanding of the relationship between the structure and function of enzymes in this field is relatively limited. However, recently, David W. Christianson et al. analyzed the structure of the bifunctional terpene synthase PaFS by cryo-electron microscopy, providing a new solution for the analysis of the structure of bifunctional terpene synthases.

[0004] Protein engineering methods include directed evolution, rational design and semi-rational design. Due to the lack of bifunctional terpene synthase protein crystals, there is still a lack of in-depth understanding of its spatial structure and functional relationship. Rational design has no way to start, and the success rate is low, so it is not applicable. Directed evolution is a common and effective means to transform and screen enzyme proteins, but its random mutation characteristics often lead to excessively large mutation libraries, with disadvantages such as high cost, low efficiency and long cycle. Semi-rational design, which has gradually developed in recent years, uses bioinformatics methods to rationally select protein transformation targets and alternative amino acids through homologous protein sequence alignment and protein spatial structure comparison, while referring to known catalytic mechanisms and other information, so as to construct a more streamlined mutant library and transform proteins more targeted. Among them, site-directed saturation mutagenesis constructs a site-directed saturation mutation library by rationally selecting a certain amino acid site and mutating the amino acid at this site into 19 other amino acids. Site-directed saturation mutation libraries play an important role in quickly and efficiently exploring protein functions and active centers. Therefore, designing and constructing a library of efficient mutants based on the limited information on the structure and catalysis of bifunctional terpene synthases is of great significance for gaining a deeper understanding of the relationship between the structure and function of terpene synthases, expanding the library of terpene natural products, and adding more reserve forces of terpene compounds with potential pharmaceutical value. Summary of the invention

[0005] The purpose of the present invention is to provide a bifunctional terpene synthase and its mutants and catalytic products.

[0006] Based on homologous sequence alignment and protein model construction, the mutation sites of the bifunctional terpene synthase FoFS were designed to construct a streamlined and efficient mutation library. Through the modification of the catalytic function of terpene synthase and the acquisition of mutant products, we can gain a deeper understanding of the relationship between the structure and function of terpene synthase, expand the terpene natural product library, and add more reserve forces of terpene compounds with potential drug value.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The present invention first provides a bifunctional terpene synthase, the amino acid sequence of which is shown in SEQ ID NO. 1. In the present invention, the wild-type bifunctional terpene synthase is abbreviated as FoFS.

[0009] The bifunctional terpene synthase FoFS (wild-type bifunctional terpene synthase) is a bifunctional terpene synthase screened from Fusarium oxysporum (deposit number: CGMCC No. 21067).

[0010] The present invention also provides a nucleotide sequence of a bifunctional terpene synthase (which can also be referred to as the wild-type nucleotide, abbreviated as FoFS) as shown in SEQ ID NO.2. The nucleotide of the wild-type bifunctional terpene synthase was obtained by extracting RNA from Fusarium oxysporum (deposit number: CGMCC No.21067), obtaining cDNA by reverse transcription, and then sequencing.

[0011] The present invention also provides a bifunctional terpene synthase mutant, which is a protein composed of a new amino acid sequence formed by mutating the leucine at the 89th position of the amino acid sequence shown in SEQ ID NO.1 to glutamine, and is also abbreviated as FoFS-L89Q.

[0012] Specifically, the new amino acid sequence formed by mutating the leucine at the 89th position of the amino acid sequence shown in SEQ ID NO.1 to glutamine is shown in SEQ ID NO.3.

[0013] The present invention also provides an isolated nucleic acid, which encodes the bifunctional terpene synthase. The nucleotide sequence of the isolated nucleic acid is shown in SEQ ID NO.4, abbreviated as FoFS-L89Q.

[0014] The nucleotide sequence shown in SEQ ID NO.4 was obtained by mutating the bases at positions 265-267 of SEQ ID NO.2 from CTA to CAG by PCR.

[0015] The present invention also provides another bifunctional terpene synthase mutant, which is a protein composed of a new amino acid sequence formed by mutating the leucine at the 89th position of the amino acid sequence shown in SEQ ID NO.1 to asparagine, and is also abbreviated as FoFS-L89N.

[0016] Specifically, the new amino acid sequence formed by mutating the leucine at the 89th position of the amino acid sequence shown in SEQ ID NO.1 to asparagine is shown in SEQ ID NO.5.

[0017] The present invention also provides an isolated nucleic acid, which encodes the bifunctional terpene synthase. The nucleotide sequence of the isolated nucleic acid is shown in SEQ ID NO.6, abbreviated as FoFS-L89N.

[0018] The nucleotide sequence shown in SEQ ID NO.6 was obtained by mutating the bases at positions 265 - 267 of SEQ ID NO.2 from CTA to AAT through PCR.

[0019] The bifunctional terpene synthase and the bifunctional terpene synthase mutant provided by the present invention are sesterterpene synthases that simultaneously have an isopentenyl transferase domain and a terpene cyclase domain, and have the dual functions of catalyzing the head - to - tail connection and cyclization of the terpene synthesis precursors DMAPP and IPP.

[0020] The present invention also provides a recombinant expression vector, and the recombinant expression vector contains a nucleic acid encoding a bifunctional terpene synthase or a bifunctional terpene synthase mutant.

[0021] The present invention also provides a recombinant expression transformant, and the recombinant expression transformant contains the recombinant expression vector described above.

[0022] The present invention also provides the application of the bifunctional terpene synthase or the bifunctional terpene synthase mutant in the synthesis of compounds containing a sesterterpene skeleton.

[0023] The present invention also provides the catalytic products of the bifunctional terpene synthase, including,

[0024] The catalytic product 1 of the bifunctional terpene synthase is a compound containing a pentacyclic sesterterpene skeleton of 5 / 3 / 7 / 6 / 5, and its molecular formula is C 25 H 40 , and the structural formula is as shown in Formula 1, named: fusoxypene A.

[0025] The catalytic product 2 of the bifunctional terpene synthase is a compound containing a pentacyclic sesterterpene skeleton of 5 / 3 / 7 / 6 / 5, and its molecular formula is C 25 H 40 , and the structural formula is as shown in Formula 2, named: fusoxypene B.

[0026] The catalytic product 3 of the bifunctional terpene synthase is a compound containing a tricyclic sesterterpene skeleton of 5 / 12 / 5, and its molecular formula is C 25 H 40 , and the structural formula is as shown in Formula 3, named: fusoxypene C.

[0027] The catalytic product 4 of the bifunctional terpene synthase is a compound containing a pentacyclic sesterterpene skeleton of 5 / 4 / 7 / 6 / 5, and its molecular formula is C 25 H 40 , and the structural formula is as shown in Formula 4, named: (-)-astellatene.

[0028]

[0029] The present invention also provides the catalytic products of the bifunctional terpene synthase mutants, including,

[0030] The catalytic product 5 of the bifunctional terpene synthase mutant is a compound containing a 5 / 8 / 6 / 5 tetracyclic sesterterpene skeleton, and its molecular formula is C 25 H 40 , and the structural formula is as shown in Formula 5;

[0031] The catalytic product 6 of the bifunctional terpene synthase mutant is a compound containing a 5 / 3 / 7 / 6 / 5 pentacyclic sesterterpene skeleton, and its molecular formula is C 25 H 40 , and the structural formula is as shown in Formula 6;

[0032] The catalytic product 7 of the bifunctional terpene synthase mutant is a compound containing a 5 / 12 / 5 tricyclic sesterterpene skeleton, and its molecular formula is C 25 H 40 , and the structural formula is as shown in Formula 7;

[0033]

[0034] Due to the adoption of the above technical solutions, the present invention has the following advantages and beneficial effects:

[0035] The present invention provides a bifunctional terpene synthase and two bifunctional terpene synthase mutants. The two bifunctional terpene synthase mutants respectively contain an amino acid sequence in which the leucine at the 89th position of SEQ ID NO.1 is mutated to glutamine and an amino acid sequence in which the leucine at the 89th position of SEQ ID NO.1 is mutated to asparagine. Among them, the gene encoding the amino acid sequence represented by SEQ ID NO.1 is a bifunctional terpene synthase gene screened from Fusarium oxysporum.

[0036] The present invention also provides the extraction, detection and identification of the catalytic products of the mutant protein after the in vivo enzymatic catalytic reaction of the target bifunctional terpene synthase in Escherichia coli E.coli BL21(DE3).

[0037] By constructing a single-site saturation mutation library of the bifunctional terpene synthase and detecting the mutant products, the present invention has modified the catalytic function of the bifunctional terpene synthase and obtained terpene compounds with rich ring systems, which is of great significance for expanding the terpene natural product library and adding more terpene compounds with potential drug value to the reserve force.

[0038] The object of modification of the present invention, the bifunctional terpene synthase FoFS, simultaneously has a prenyltransferase domain and a terpene cyclase domain, and has the dual functions of catalyzing the head-to-tail connection and cyclization of the terpene synthesis precursors DMAPP and IPP. FoFS has sesquiterpene products with multiple different ring systems. By modifying FoFS, the mutant protein can catalyze sesquiterpene compounds with other complex ring systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Chemical structure diagrams and GC-MS chromatograms of the original products (1–4) and mutant products (compounds 5–10) of the bifunctional terpene synthase provided in the examples of the present invention

[0040] Figure 2 HR-EI-MS diagram of compound 5 provided in the examples of the present invention

[0041] Figure 3 For compound 5 provided in the examples of the present invention in Benzene-d6 1 1H NMR diagram

[0042] Figure 4 For compound 5 provided in the examples of the present invention in Benzene-d6 13 13C NMR diagram

[0043] Figure 5 HSQC diagram of compound 5 provided in the examples of the present invention in Benzene-d6

[0044] Figure 6 For compound 5 provided in the examples of the present invention in Benzene-d6 1 1H- 1 1H-1H COSY diagram

[0045] Figure 7 HMBC diagram of compound 5 provided in the examples of the present invention in Benzene-d6

[0046] Figure 8 NOESY diagram of compound 5 provided in the examples of the present invention in Benzene-d6

[0047] Figure 9 Relative configuration of compound 5 provided in the examples of the present invention and key two-dimensional NMR correlations.

[0048] Figure 10 HR-EI-MS diagram of compound 6 provided in the examples of the present invention

[0049] Figure 11 For compound 6 provided in the examples of the present invention in Benzene-d6 11H NMR spectrum

[0050] Figure 12 of Compound 6 provided in the examples of the present invention in Benzene-d6 13 13C NMR spectrum

[0051] Figure 13 HSQC spectrum of Compound 6 provided in the examples of the present invention in Benzene-d6

[0052] Figure 14 of Compound 6 provided in the examples of the present invention in Benzene-d6 1 H- 1 H COSY spectrum

[0053] Figure 15 HMBC spectrum of Compound 6 provided in the examples of the present invention in Benzene-d6

[0054] Figure 16 NOSEY spectrum of Compound 6 provided in the examples of the present invention in Benzene-d6

[0055] Figure 17 The relative configuration of Compound 6 provided in the examples of the present invention and key two-dimensional NMR correlations.

[0056] Figure 18 HR-EI-MS spectrum of Compound 7 provided in the examples of the present invention

[0057] Figure 19 of Compound 7 provided in the examples of the present invention in Benzene-d6 1 1H NMR spectrum

[0058] Figure 20 of Compound 7 provided in the examples of the present invention in Benzene-d6 13 13C NMR spectrum

[0059] Figure 21 HSQC spectrum of Compound 7 provided in the examples of the present invention in Benzene-d6

[0060] Figure 22 of Compound 7 provided in the examples of the present invention in Benzene-d6 1 H- 1 H COSY spectrum

[0061] Figure 23 HMBC spectrum of Compound 7 provided in the examples of the present invention in Benzene-d6

[0062] Figure 241H-1H NOESY spectrum of Compound 7 provided in the embodiments of the present invention in Benzene-d6

[0063] Figure 25 Relative configuration of Compound 7 provided in the embodiments of the present invention and key two-dimensional NMR correlations

[0064] Figure 26 HR-EI-MS spectrum of Compound 8 provided in the embodiments of the present invention

[0065] Figure 27 1H NMR spectrum of Compound 8 provided in the embodiments of the present invention in Benzene-d6 1 1H NMR spectrum

[0066] Figure 28 HR-EI-MS spectrum of Compound 9 provided in the embodiments of the present invention in methanol

[0067] Figure 29 1H NMR spectrum of Compound 9 provided in the embodiments of the present invention in Benzene-d6 1 1H NMR spectrum

[0068] Figure 30 HR-EI-MS spectrum of Compound 10 provided in the embodiments of the present invention

[0069] Figure 31 1H NMR spectrum of Compound 10 provided in the embodiments of the present invention in Benzene-d6 1 1H NMR spectrum

[0070] Figure 32 Key NOE correlation diagrams of Compounds 5-7 provided in the embodiments of the present invention

[0071] Figure 33 Schematic diagrams of the actually measured CD and calculated ECD of Compound 5 provided in the embodiments of the present invention

[0072] Figure 34 Schematic diagrams of the actually measured CD and calculated ECD of Compound 6 provided in the embodiments of the present invention

[0073] Figure 35 Schematic diagrams of the actually measured CD and calculated ECD of Compound 7 provided in the embodiments of the present invention

[0074] Figure 36 Schematic diagrams of the wild-type products 1-4 of a bifunctional terpene synthase and the new products 5-10 catalyzed after the 89th amino acid is mutated to glutamine and asparagine Detailed implementation manners

[0075] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0076] Example 1

[0077] 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 the present invention, the wild-type bifunctional terpene synthase is abbreviated as FoFS.

[0078] Among them, Fusarium oxysporum F. oxysporum 14005 is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, with the deposit number CGMCC No.21067, the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date being December 31, 2020. The deposit information about Fusarium oxysporum F. oxysporum 14005 has been disclosed in Patent CN113046332A.

[0079] The nucleotide sequence of the wild-type bifunctional terpene synthase (which can also be called wild-type nucleotide, abbreviated as FoFS) is shown in SEQ ID NO.2. The nucleotide of the wild-type bifunctional terpene synthase is obtained by extracting the RNA of Fusarium oxysporum (deposit number: CGMCC No.21067), obtaining cDNA by reverse transcription, and then sequencing.

[0080] This embodiment provides a bifunctional terpene synthase mutant, which is a protein composed of a new amino acid sequence formed by mutating the leucine at the 89th position of the amino acid sequence shown in SEQ ID NO.1 to glutamine, and is also simply referred to as FoFS-L89Q.

[0081] Specifically, the new amino acid sequence formed by mutating the leucine at the 89th position of the amino acid sequence shown in SEQ ID NO.1 to glutamine is shown in SEQ ID NO.3.

[0082] This embodiment also provides an isolated nucleic acid, and the nucleic acid encodes the bifunctional terpene synthase mutant. The nucleotide sequence of the isolated nucleic acid is shown in SEQ ID NO.4, abbreviated as FoFS-L89Q.

[0083] The nucleotide sequence shown in SEQ ID NO.4 was obtained by mutating the bases at positions 265-267 of SEQ ID NO.2 from CTA to CAG by PCR.

[0084] This example also provides another bifunctional terpene synthase mutant, which is a protein composed of a new amino acid sequence formed by mutating the leucine at position 89 of the amino acid sequence shown in SEQ ID NO.1 to asparagine, and is also abbreviated as FoFS-L89N.

[0085] Specifically, the new amino acid sequence formed by mutating the leucine at position 89 of the amino acid sequence shown in SEQ ID NO.1 to asparagine is shown in SEQ ID NO.5.

[0086] This example also provides a separated nucleic acid, and the nucleic acid encodes the bifunctional terpene synthase mutant. The nucleotide sequence of the separated nucleic acid is shown in SEQ ID NO.6, and is abbreviated as FoFS-L89N.

[0087] The nucleotide sequence shown in SEQ ID NO.6 was obtained by mutating the bases at positions 265-267 of SEQ ID NO.2 from CTA to AAT by PCR.

[0088] The two different bifunctional terpene synthases provided in this example are sesterterpene synthases that simultaneously have an isopentenyl transferase domain and a terpene cyclase domain.

[0089] This example further provides a method for preparing a bifunctional terpene synthase, including the following steps:

[0090] Retrieve the nucleotide sequence and protein sequence of the FoFS coding gene from the bifunctional terpene synthase gene of Fusarium oxysporum F.oxysporum 14005, import them into BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and retrieve in the PDB (http: / / www.rcsb.org / ) database to obtain the crystal structure of the first bifunctional terpene synthase PaFS from fungal origin with a similarity of 45%. Using this as a template, apply the SWISS-MODEL (https: / / swissmodel.expasy.org / ) online server to construct a protein model for the TC domain of FoFS.

[0091] The sequences of FoFS and the bifunctional terpene synthase BsPS studied previously were aligned using Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ). It was found that FoFS has a "triad" site similar to BsPS that affects the catalytic product ring system: F61, W69, L89. Given that previous studies on BsPS have shown that the amino acid at position 89 plays an important role in catalysis, saturation mutagenesis was performed on the leucine at position 89 of FoFS.

[0092] The coding gene of the wild-type bifunctional terpene synthase was recombined with the linearized plasmid pET28a fragment, and the recombinant product was then transformed into Escherichia coli BL21(DE3). Subsequently, the mutation sites were designed in the primers for whole plasmid PCR. The PCR product was digested with DpnI to remove the plasmid template. 2 μL of the digested product was transformed into Escherichia coli BL21(DE3). After screening with a resistant plate, monoclonal colonies were picked for sequencing, and the correctly sequenced sequence was selected for subsequent induction expression in E. coli BL21(DE3), and the wild-type bifunctional terpene synthase could be isolated.

[0093] This example provides some sesquiterpenoid compounds catalyzed by the bifunctional terpene synthase, including:

[0094] The catalytic product 1 of the bifunctional terpene synthase is a compound with a pentacyclic sesquiterpene skeleton of 5 / 3 / 7 / 6 / 5, and its molecular formula is C 25 H 40 , and its structural formula is shown in Formula 1, named fusoxypene A. Product 2 is a compound with a pentacyclic sesquiterpene skeleton of 5 / 3 / 7 / 6 / 5, and its molecular formula is C 25 H 40 , and its structural formula is shown in Formula 2, named fusoxypene B. Product 3 is a compound with a tricyclic sesquiterpene skeleton of 5 / 12 / 5, and its molecular formula is C 25 H 40 , and its structural formula is shown in Formula 3, named fusoxypene C. Product 4 is a compound with a pentacyclic sesquiterpene skeleton of 5 / 4 / 7 / 6 / 5, and its molecular formula is C 25 H 40 , and its structural formula is shown in Formula 4, named (-)-astellatene.

[0095]

[0096] The catalytic product 5 of the bifunctional terpene synthase is a compound with a tetracyclic sesquiterpene skeleton of 5 / 8 / 6 / 5, and its molecular formula is C 25 H 40, with the structural formula shown in Formula 5; Compound 6 is a compound containing a pentacyclic sesterterpene skeleton of 5 / 3 / 7 / 6 / 5, and its molecular formula is C 25 H 40 , with the structural formula shown in Formula 6; Compound 7 is a compound containing a tricyclic sesterterpene skeleton of 5 / 12 / 5, and its molecular formula is C 25 H 40 , with the structural formula shown in Formula 7; Compound 8 is a compound containing a bicyclic sesterterpene skeleton of 5 / 15, and its molecular formula is C 25 H 42 O, with the structural formula shown in Formula 8.

[0097]

[0098] The catalytic products 9 and 10 of the bifunctional terpene synthase are compounds containing a bicyclic sesterterpene skeleton of 5 / 15, and their molecular formula is C 25 H 40 , with the structural formulas shown in Formulas 9 and 10.

[0099]

[0100] The bifunctional terpene synthase is heterologously expressed in Escherichia coli E. coli BL21(DE3)

[0101] A method for preparing a sesterterpene compound as a catalytic product of a bifunctional terpene synthase, comprising the following steps:

[0102] The host bacterium is Escherichia coli E. coli BL21(DE3): Using the bifunctional terpene synthase in the cells obtained by fermentation culture of the genetically engineered bacterium containing the bifunctional terpene synthase coding gene as a catalyst, directly extracting the terpene product from the cells and detecting it by GC-MS.

[0103] The wet cells are recombinant Escherichia coli E. coli BL21(DE3) containing the bifunctional terpene synthase coding gene. The fermentation culture method is to inoculate recombinant Escherichia coli E. coli BL21(DE3) containing the bifunctional terpene synthase coding gene into LB medium (Yeast Extract 0.5% (w / v), Tryptone 1% (w / v), sodium chloride 1% (w / v), agar powder 2% (w / v)) supplemented with 50 μg / mL kanamycin, shake culture at 37 °C and 220 rpm for 8–10 h, transfer to the self-inducing medium supplemented with 50 μg / mL kanamycin at an inoculation amount of 2%, shake culture at 37 °C and 220 rpm, and when OD 600 reaches 0.6–1.0, change the culture conditions to 17 °C and 220 rpm, shake culture for 60–72 h to obtain wet Escherichia coli fermentation cells.

[0104] Auto-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.

[0105] 20× inorganic salt buffer: Na2HPO4 1M, KH2PO4 1M, NH4Cl 1M, Na2SO4 0.1M.

[0106] 20× carbon source: anhydrous glucose 2% (w / v), glycerol 10% (w / v), α-lactose 20% (w / v).

[0107] 500× MgSO4: anhydrous MgSO4 1M.

[0108] The wet cells of Escherichia coli fermented were lysed and extracted with acetone by ultrasound (130W, 30min) for 30min. After centrifugation, the supernatant was taken, and the lysis and extraction were repeated 3 times. The acetone from the 3 extractions was combined and extracted 3 times with an equal volume of ethyl acetate to obtain the crude extract of the fermented cells.

[0109] The crude extract of the fermented cells was purified and prepared by silica gel column chromatography and semi-preparative liquid chromatography (HPLC), including the following steps:

[0110] 0.23 g of the crude extract extracted from 200 g of E. coli BL21(DE3) / pET28a-FoFS-L89Q cells fermented was preliminarily separated by silica gel column chromatography. n-Hexane was used as the elution mobile phase for silica gel column chromatography. By monitoring with TLC, the fraction containing compounds 5 and 6 was obtained. Then, dichloromethane was used as the elution mobile phase for silica gel column chromatography. By monitoring with TLC, the fraction containing compound 8 was obtained.

[0111] Another 0.97 g of the crude extract extracted from 313 g of E. coli BL21(DE3) / pET28a-FoFS-L89Q cells fermented was preliminarily separated by silica gel column chromatography. n-Hexane was used as the elution mobile phase for silica gel column chromatography. By monitoring with TLC, the fraction containing compound 7 was obtained.

[0112] The fractions eluted from the silica gel column and containing the target compounds 5, 6, 7, and 8 were further separated and purified by a liquid chromatography column. For the fraction containing compound 5 and compound 6, it was first prepared by isocratic elution using a liquid chromatography column ACE Excel 5 C18-AR (Φ4.6×250 mm), 100% acetonitrile, and a flow rate of 1 mL / min. The retention time of compound 6 was 29 min, and the retention time of compound 5 was 27 min. Then, the fraction containing compound 5 was further purified by isocratic elution using a chromatography column COSMOSIL Cholester (Φ4.6×250 mm), 100% methanol, and a flow rate of 1 mL / min. The retention time of compound 5 was 51 min. For the fraction containing compound 7, it was first eluted isocratically using a semi-preparative liquid chromatography column Phenomenex C18 (Φ10×250 mm) with a volume ratio of 98:2 methanol and water and a flow rate of 4 mL / min. The retention time of compound 7 was 37 min. Then, it was purified by isocratic elution using a chromatography column ACE C18-PFP (Φ4.6×250 mm), 100% acetonitrile, and a flow rate of 1 mL / min. The retention time of compound 7 was 19 min. For the fraction containing compound 8, it was eluted isocratically using a semi-preparative liquid chromatography column ACE Excel 5 C18-AR (Φ10×250 mm) with a volume ratio of 95:5 acetonitrile and water and a flow rate of 4 mL / min. The retention time of compound 8 was 11.9 min.

[0113] 0.25 g of the crude extract extracted from 64 g of fermented E. coli BL21(DE3) / pET28a-FoFS-L89N cells was purified and prepared by silica gel column chromatography and semi-preparative liquid chromatography (HPLC), including the following steps:

[0114] 0.25 g of the crude extract of fermented E. coli BL21(DE3) / pET28a-FoFS-L89N cells was preliminarily separated by silica gel column chromatography. n-Hexane was used as the elution mobile phase for silica gel column chromatography, and by TLC monitoring, the fraction containing compounds 9 and 10 was obtained.

[0115] The fractions eluted from the silica gel column and containing the target compounds 9 and 10 were further separated and purified by a liquid chromatography column. For the fraction containing compound 9 and compound 10, it was first prepared by isocratic elution using a semi-preparative liquid chromatography column ACE Excel 5 C18-AR (Φ10×250 mm), 100% acetonitrile, and a flow rate of 4 mL / min. The retention times of both compound 9 and compound 10 were 17 min. Then, it was further purified by isocratic elution using a chromatography column COSMOSIL Cholester (Φ4.6×250 mm), 100% methanol, and a flow rate of 1 mL / min. The retention time of compound 9 was 17 min, and the retention time of compound 10 was 18 min.

[0116] The prepared compounds 5–10 were detected using a gas chromatography-mass spectrometry (GC-MS) instrument. The detection conditions were as follows: injection was carried out in high-pressure split mode, the inlet pressure was 110 kPa, the split ratio was 50, and the column flow rate was 1.77 mL / min. The initial temperature was 60 °C, which was then increased to 280 °C at a rate of 25 °C / min, and then further increased to 310 °C at a rate of 10 °C / min. The retention time of compound 5 was 9.750 min, m / z 340; the retention time of compound 6 was 10.117 min, m / z 340; the retention time of compound 7 was 10.250 min, m / z 340; the retention time of compound 8 was 11.342 min, m / z 358; the retention time of compound 9 was 10.517 min, m / z 340; the retention time of compound 10 was 10.583 min, m / z 340.

[0117] The catalytic products of the bifunctional terpene synthase FoFS mutants and the GC-MS chromatograms are as Figure 1 shown. Compared with the wild type, compound 8 was obtained in the mutant products of FoFS-L89A / L89C / L89Q / L89N / L89S / L89T / L89G (FoFS-L89A means that the L amino acid at position 89 of the amino acid sequence shown in SEQ ID NO.1 of FoFS was replaced with an A amino acid, and the same explanation applies to the others). Compound 8 is a compound containing a 5-15 bicyclic bisabolane skeleton, and its molecular formula is C 25 H 42 O. In addition, compounds 5, 6, 7, and 9 were also obtained in the mutant product of FoFS-L89Q. Compound 5 contains a 5 / 8 / 6 / 5 tetracyclic bisabolane skeleton, compound 6 contains a 5 / 3 / 7 / 6 / 5 pentacyclic bisabolane skeleton, compound 7 contains a 5 / 12 / 5 tricyclic bisabolane skeleton, and compound 9 contains a 5 / 15 bicyclic bisabolane skeleton. Their molecular formulas are all C 25 H 40 . Compounds 9 and 10 containing a 5 / 15 bicyclic bisabolane skeleton were also obtained in the mutant L89N, and their molecular formulas are both C 25 H 40 . Among them, compounds 5, 6, and 7 are new compounds not reported in the literature, and compounds 8, 9, and 10 are known compounds.

[0118] Compound 5 is a white powder, 14.1 (c 0.01, MeOH), and its quasi-molecular ion peak measured by HR-EI-MS was m / z 340.3134 [M + (the calculated value was 340.3130, Figure 1 ), the degree of unsaturation was 6, and the molecular formula was C25 H 40 ( Figure 2 )。For compound 5, 1 H NMR, 13 C NMR, and HSQC NMR spectral data analysis showed that this compound had 4 olefinic quaternary carbons (δ C 141.3; 136.0; 128.1; 134.1), 1 sp 3 quaternary carbon (δ C 42.9), 6 methyl groups (δ C / H 40.8 / 2.94; 39.0 / 1.50; 45.1 / 2.46; 44.4 / 1.88; 47.1 / 1.72; 30.7 / 1.60), 8 methylene groups (δ C / H 44.8 / 1.95&1.24; 29.6 / 2.36&1.24; 24.4 / 2.38&1.90; 32.1 / 2.00&1.33; 35.3 / 2.57&2.21; 29.0 / 2.41&2.38; 42.3 / 1.49&1.10; 30.1 / 1.78&1.49), 2 singlet methyl groups (δ C / H 16.9 / 1.81; 20.4 / 0.82) and 4 doublet methyl groups (δ C / H 18.2 / 1.06; 20.0 / 1.08; 24.5 / 1.02; 22.0 / 0.91)( Figure 3 –5, Table 1). The planar structure of 5 could be determined by 1D and 2D NMR experiments ( Figure 6 –7, Table 1). NOE effects existed between H-25 and H-17, H-23 and H-17, H-24 and H-13, H-13 and H-23, H-3 and H-14, H-2 and H-23, H-2 and H-20, H-20 and H-5, H-2 and H-5, and H-21 and H-5 in the NOESY spectrum, indicating that these protons had the same orientation, and thus the relative configuration of compound 5 could be determined ( Figure 8 , Figure 32 ). According to the ECD quantum chemical calculation based on TD-DFT, the absolute configuration of 5 was determined to be 2S,3R,7R,14R,15S,18S-5( Figure 9 ).

[0119] Compound 6 was a white powder, 20(c 0.015,MeOH), and its quasi-molecular ion peak measured by HR-EI-MS was m / z 340.3125 [M + (the calculated value was 340.3130), the degree of unsaturation was 6, and the molecular formula was C 25 H 40 ( Figure 10)。For compound 6 1 HNMR, 13 C NMR, and HSQC NMR spectral data analysis showed that this compound has one quaternary carbon of alkene (δ C 147.9), three sp 3 quaternary carbons (δ C 20.3; 41.5; 35.8), eight methyl groups (δ C / H 44.1 / 2.10; 45.0 / 1.20; 44.7 / 2.30; 38.5 / 1.46; 126.1 / 6.08; 48.2 / 2.21; 44.9 / 1.90; 29.4 / 1.72), seven methylene groups (δ C / H 47.6 / 1.94&1.13; 37.2 / 1.70&1.51; 36.5 / 2.00&1.91; 36.4 / 1.59&1.20; 25.5 / 2.01&1.78; 40.4 / 1.62&1.13; 28.0 / 1.76&1.64), two singlet methyl groups (δ C / H 17.5 / 1.35; 20.3 / 0.81) and four doublet methyl groups (δ C / H 21.4 / 0.98; 18.0 / 1.18; 24.6 / 1.00; 20.5 / 0.85) ( Figure 11 –13, Table 2). The planar structure of 6 can be determined by 1D and 2D NMR experiments ( Figure 14 –15, Table 2). NOE effects exist between H-25 and H-17, H-23 and H-17, H-23 and H-2, H-2 and H-22, H-22 and H-21, H-14 and H-10, H-10 and H-5, H-5 and H-7 in the NOESY spectrum, indicating the same orientation between these protons. Thus, the relative configuration of compound 6 can be determined ( Figure 16 , Figure 32 ). According to the ECD quantum chemical calculation based on TD-DFT, the absolute configuration of 6 is determined to be 2S,3R,6R,7R,10S,11S,14S,15S,18S-6 ( Figure 17 ).

[0120] Compound 7 is a white powder, 9.6 (c 0.04, MeOH). The quasi-molecular ion peak measured by HR-EI-MS is m / z 340.3128 [M + (the calculated value is 340.3130), the degree of unsaturation is 6, and the molecular formula is C 25 H 40 ( Figure 18 ). For compound 7 1 HNMR, 13Analysis of the data of the \(^{13}\)C NMR and HSQC NMR spectra showed that the compound had 4 olefinic quaternary carbons (δ C 136.2; 141.6; 140.9; 130.9), 1 sp 3 quaternary carbon (δ C 23.9), 6 methyl groups (δ C / H 124.8 / 5.47; 43.2 / 2.69; 131.6 / 5.50; 47.2 / 1.93; 53.5 / 1.53; 28.5 / 1.52), 8 methylene groups (δ C / H 43.5 / 2.08; 39.3 / 2.22&2.10; 27.2 / 2.31&2.16; 32.4 / 2.07&1.39; 35.0 / 2.52&2.36; 24.7 / 2.20&1.84; 45.1 / 1.45&1.36; 28.2 / 1.56&1.16), 3 singlet methyl groups (δ C / H 17.1 / 1.64; 15.7 / 1.72; 23.9 / 1.02) and 3 doublet methyl groups (δ C / H 20.0 / 1.09; 22.7 / 0.92; 22.3 / 0.91) ( Figure 19 –21, Table 3). The planar structure of 7 could be determined by 1D and 2D NMR experiments ( Figure 22 –23, Table 3). NOE effects were observed between H-25 and H-17, H-23 and H-17, H-24 and H-13, H-13 and H-23, H-23 and H-4, H-4 and H-21 in the NOESY spectrum, indicating that these protons had the same orientation, and thus the relative configuration of compound 7 could be determined ( Figure 24 , Figure 32 ). The three double bonds Δ 2,3 , Δ 6,7 , Δ 11,12 were all of E-configuration. Based on TD-DFT-based ECD quantum chemical calculations, the absolute configuration of 7 was determined to be 7R,14R,15S,18S-7 ( Figure 25 ).

[0121] Compound 8 was a white powder. By comparing the MS and NMR data of compound 8 with the data reported in the literature, its structure was determined. The quasi-molecular ion peak of HR-EI-MS was measured to be m / z 358.3233 [M + (calculated value: 358.3236), the degree of unsaturation was 5, and the molecular formula was C 25 H 40 ( Figure 26 , Figure 27 ).

[0122] Compound 9 is a white powder. By comparing the MS and NMR data of Compound 9 with the reported data in the literature, its structure was determined. The quasi-molecular ion peak of Compound 9 measured by HR-EI-MS was m / z 340.3128 [M + (the calculated value was 340.3130), the degree of unsaturation was 6, and the molecular formula was C 25 H 40 ( Figure 28 , Figure 29 ).

[0123] Compound 10 is a white powder. By comparing the MS and NMR data of Compound 10 with the reported data in the literature, its structure was determined. The quasi-molecular ion peak of Compound 10 measured by HR-EI-MS was m / z 340.3133 [M + (the calculated value was 340.3130), the degree of unsaturation was 6, and the molecular formula was C 25 H 40 ( Figure 30 , Figure 31 ).

[0124] The key NOE correlation diagrams of Compounds 5–7 provided in the embodiments of the present invention are as shown in Figure 32 ; the schematic diagrams of the actually measured CD and calculated ECD of Compounds 5, 6, and 7 provided in the embodiments of the present invention are as shown in Figure 33 , 34 , and 35.

[0125] Schematic diagrams of the wild-type products 1–4 of the bifunctional terpene synthase and the new products 5–10 catalyzed after the 89th amino acid is mutated to glutamine and asparagine are as shown in Figure 36 .

[0126] Table 1 1D and 2D NMR data table of Compound 5

[0127]

[0128]

[0129] Table 2 1D and 2D NMR data table of Compound 6

[0130]

[0131]

[0132] Table 3 1D and 2D NMR data table of Compound 7

[0133]

[0134] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent, without departing from the scope of the technical solution of the present invention, may make some changes or modifications using the technical content prompted above to form equivalent embodiments of equivalent changes. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the present invention's solution.

Claims

1. A bifunctional terpene synthase, characterized in that, The amino acid sequence is as shown in SEQ ID NO.

1.

2. A nucleotide of a bifunctional terpene synthase, characterized in that, The nucleotide sequence is as shown in SEQ ID NO.

2.

3. A bifunctional terpene synthase mutant, characterized in that, It is a protein composed of a new amino acid sequence formed by mutating the leucine at the 89th position of the amino acid sequence shown in SEQ ID NO.1 to glutamine, and the amino acid sequence is as shown in SEQ ID NO.

3.

4. A bifunctional terpene synthase mutant, characterized in that, It is a protein composed of a new amino acid sequence formed by mutating the leucine at the 89th position of the amino acid sequence shown in SEQ ID NO.1 to asparagine, and the amino acid sequence is as shown in SEQ ID NO.

5.

5. An isolated nucleic acid, characterized in that, The nucleic acid encodes the bifunctional terpene synthase mutant according to claim 3 or 4; the nucleotide sequence of the isolated nucleic acid is as shown in SEQ ID NO.4, or the nucleotide sequence of the isolated nucleic acid is as shown in SEQ ID NO.

6.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains a nucleic acid encoding the bifunctional terpene synthase according to claim 1 or the bifunctional terpene synthase mutant according to claim 3 or 4.

7. A recombinant expression transformant, characterized in that, The recombinant expression transformant contains the recombinant expression vector according to claim 6.

8. Use of the bifunctional terpene synthase according to claim 1 or the bifunctional terpene synthase mutant according to claim 3 or 4 in the synthesis of a compound containing a sesterterpene skeleton.

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