Terpene compound epoxidase and use thereof

Abstract

Provided are a terpene compound epoxidase and the use thereof. Provided is a terpene compound epoxidase. The terpene compound epoxidase has an amino acid sequence as shown in any one of SEQ ID NO. 1-SEQ ID NO. 8, shows epoxidase enzymatic activity, and is capable of performing epoxidation catalysis on taxadiene or a hydroxylation product of taxadiene or an acetylation product of taxadiene so as to generate a corresponding epoxidized terpene compound. The terpene compound epoxidase can be used to improve the taxane compound biocatalysis pathway so as to improve the biocatalysis efficiency of taxane compounds (for example, paclitaxel).

Description

Terpene compound epoxidase and application TECHNICAL FIELD

[0001] The present application relates to the technical field of biosynthesis, in particular to a terpene compound epoxidase and application. BACKGROUND

[0002] Taxanes are antitumor active ingredients isolated from plants, and taxane drugs mainly include paclitaxel, docetaxel, cabazitaxel, and derivatives with a taxane skeleton.

[0003] Paclitaxel is a diterpenoid alkaloid, which is widely used in the clinical treatment of various cancers. Paclitaxel has a complex 6-8-6 tricyclic carbon skeleton with 9 stereogenic centers, 1 obvious oxetane ring group and 1 phenyl isoserine chain. It can pass through the nanometer hole of the microtubule wall, interact with the tubulin on the surface of the microtubule cavity, and destroy the microtubule dynamics, thereby producing cytotoxicity to cancer cells.

[0004] In the background, taxadiene and baccatin III are key precursors in the paclitaxel biosynthetic pathway. Taxadiene is a class of diterpenoid compounds, which is generated by polymerization, cyclization and other enzymatic reactions from 4 molecules of isopentenyl pyrophosphate (IPP). Studies have found that taxadiene synthase derived from Taxus brevifolia can catalyze GGPP (geranylgeranyl pyrophosphate, geranylgeranyl pyrophosphate) to synthesize taxadiene. Taxadiene undergoes a series of oxidation, acylation and epoxidation to form the key intermediate baccatin III, and baccatin III finally combines with the C13 side chain to form paclitaxel.

[0005] The epoxidation of taxadiene is one of the key steps in the paclitaxel biosynthetic pathway. So far, there has been no report in the background art of a direct epoxidation catalytic epoxidase for taxadiene, which is not conducive to improving the biocatalytic efficiency of taxane compounds (e.g., paclitaxel). SUMMARY

[0006] In view of the above problems, the embodiments of the present application provide a terpene compound epoxidase and application to solve the technical problem of low catalytic efficiency of taxadiene epoxidation.

[0007] In a first aspect, the embodiments of the present application provide a terpene compound epoxidase, which has an amino acid sequence as shown in any one of SEQ ID NO. 1-SEQ ID NO. 8.

[0008] In a second aspect, the embodiments of the present application provide a biological material, which is any one of A1) to A3):

[0009] A1) a nucleotide sequence encoding a terpene compound epoxidase as described above;

[0010] A2) a recombinant vector comprising the nucleotide sequence of A1);

[0011] A3) a bacterial cell line or a fungal cell line comprising the recombinant vector of A2).

[0012] In a third aspect, the embodiments of the present application provide a biological catalytic method for producing an epoxy terpene compound, comprising:

[0013] contacting a terpene compound with a polypeptide having epoxidase activity to form the epoxy terpene compound;

[0014] separating the epoxy terpene compound;

[0015] wherein the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO. 1-8.

[0016] Optionally, the epoxy terpene compound is as shown in general formula I:

[0017] wherein R1 represents acetoxy or hydroxyl or H, and R2, R3, R4, R5, R6 and R7 each represent hydroxyl or H.

[0018] Optionally, the epoxy terpene compound is as shown in general formula II:

[0019] Optionally, the epoxy terpene compound is as shown in general formula III:

[0020] Optionally, contacting a terpene compound with a polypeptide having epoxidase activity to form the epoxy terpene compound comprises:

[0021] fermenting the host cell transformed with the first recombinant vector to obtain a first fermentation product; wherein the first recombinant vector comprises a nucleotide sequence encoding a terpene compound epoxidase as shown in any one of SEQ ID NO. 9-16.

[0022] Optionally, the host cell is further transformed with a second recombinant vector comprising a nucleotide sequence encoding a taxadiene synthase as shown in SEQ ID NO. 18.

[0023] In a fourth aspect, the embodiments of the present application provide an application of the terpene compound epoxidase described above in the biosynthesis of bacatin III or its precursor.

[0024] In a fifth aspect, the present application provides an application of the terpene compound epoxidase in paclitaxel biosynthesis.

[0025] The terpene compound epoxidase provided by the embodiments of the present application has an amino acid sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 8, shows enzymatic activity of an epoxidase, and can catalyze epoxidation of taxadiene or a hydroxylated product of taxadiene or an acetylated product of taxadiene to generate a corresponding epoxidized terpene compound. The terpene compound epoxidase can be used to improve a biological catalytic pathway of taxane compounds, so as to improve the biological catalytic efficiency of the taxane compounds (e.g., paclitaxel).

[0026] These aspects or other aspects of the present application will be more apparent in the following description of the embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0027] FIG. 1a shows a structural schematic diagram of a first recombinant vector in the embodiments of the present application.

[0028] FIG. 1b shows a structural schematic diagram of a second recombinant vector in the embodiments of the present application.

[0029] FIG. 2 shows fermentation results of the terpene compound epoxidase provided by the embodiments of the present application; wherein FIG. 2a is a gas chromatogram of tax-4(5), 11(12)-diene in a fermentation product; FIG. 2b is a mass spectrum of tax-4(5), 11(12)-diene in the fermentation product; FIG. 2c is a gas chromatogram of a compound shown in general formula II in the fermentation products of Example 1 to Example 8 and Comparative Example 1; and FIG. 2d is a mass spectrum of the compound shown in general formula II in the fermentation product.

[0030] FIG. 3 shows a comparison diagram of catalytic activities of the terpene compound epoxidases provided by the embodiments of the present application and comparative examples. DETAILED DESCRIPTION

[0031] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the drawings, in which the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as a limitation of the present application.

[0032] In order for those skilled in the art to better understand the scheme of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0033] It should be noted that, in the embodiments of the present application, the relationship terms such as first and second are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations.

[0034] In addition, the terms "comprises", "comprising", or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without more limitations, an element defined by the statement "comprising a" does not exclude the existence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0035] In the description of the embodiments of the present application, the words "example" or "for example" are used to represent an example, illustration, or description. Any embodiment or design scheme described as "example" or "for example" in the embodiments of the present application is not interpreted as more preferred or having more advantages than another embodiment or design scheme. The words "example" or "for example" are intended to present a relative concept in a clear manner.

[0036] In addition, "multiple" in the embodiments of the present application refers to two or more, and therefore "multiple" in the embodiments of the present application can also be understood as "at least two". "At least one" can be understood as one or more, for example, as one, two or more. For example, including at least one means including one, two or more, and does not limit which ones are included, for example, including at least one of A, B and C, and the included can be A, B, C, A and B, A and C, B and C, or A and B and C.

[0037] It should be noted that, in the embodiments of the present application, the relationship between the associated objects described by "and / or" represents that there can be three relationships, for example, A and / or B can represent the following three cases: A exists alone, A and B exist together, and B exists alone. In addition, the character " / ", if not specially stated, generally represents that the associated objects before and after are in a "or" relationship.

[0038] As used herein, the term "terpene" denotes acyclic and cyclic chemical hydrocarbon radical residues derived from the C5 building block isoprene, which can contain one or more such building blocks.

[0039] As used herein, the term "diterpene" or "diterpenyl" refers to a terpene compound or terpenyl residue comprising in its structure two isoprene units.

[0040] As used herein, the term "epoxidase (ODD)" or "epoxidizing enzyme" denotes an enzyme catalyzing the biosynthesis of an epoxide by converting a carbon bond into an epoxide group, in particular an enzyme catalyzing the biosynthesis of an epoxide by converting a carbon double bond into an epoxide group.

[0041] As used herein, the term "epoxidase (ODD)" or "epoxidizing enzyme" denotes an enzyme catalyzing the biosynthesis of an epoxide by converting a carbon bond into an epoxide group, in particular an enzyme catalyzing the biosynthesis of an epoxide by converting a carbon double bond into an epoxide group.

[0042] It is obvious that the polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO. in which some sequences are deleted, modified, substituted, conservatively substituted or added, can also be used in the present application, as long as it can exhibit the same or corresponding activity as the polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO.. For example, it is not excluded that a sequence which does not change the function of the protein, a naturally occurring mutation, a silent mutation thereof or a conservative substitution is added before or after the "polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO."; and, the polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO. when it is added with the above-mentioned sequence which does not change the function of the protein, naturally occurring mutation, silent mutation thereof or conservative substitution also falls within the scope of the present application, as long as it exhibits the same or corresponding activity as the amino acid sequence as shown in SEQ ID NO. after being added with the above-mentioned sequence.

[0043] As used herein, the term "mevalonate pathway" (also known as the "isoprene pathway" or "HMG-CoA reductase pathway") is an essential metabolic pathway in eukaryotes, archaea, and some bacteria. The mevalonate pathway begins with acetyl-CoA and produces two five-carbon building blocks, called isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Key enzymes in the mevalonate pathway are acetoacetyl-CoA thiolase (atoB), HMG-CoA synthase (mvaS), HMG-CoA reductase (HMGCR), mevalonate kinase (MvaK1), phosphomevalonate kinase (MvaK2), mevalonate diphosphate decarboxylase (MvaD), and isopentenyl pyrophosphate isomerase (IDI). Combining the mevalonate pathway with enzymatic activities to produce the terpene precursor GPP, FPP, or GGPP, particularly FPP synthase (ERG20), allows a recombinant cell to produce a terpene.

[0044] As used herein, the term "host cell" refers to a cell (or organism) that has been altered to harbor at least one nucleic acid molecule, e.g., a recombinant gene encoding a desired protein or nucleic acid sequence. The host cell can be, for example, a bacterial cell, a fungal cell, or a plant cell or plant. The host cell can contain a recombinant gene or genes, e.g., organized as an operon, that have been integrated into the nuclear or organelle genome of the host cell. Alternatively, the host can contain the recombinant gene extrachromosomally.

[0045] An embodiment of the present application provides a terpene compound epoxidase, which has an amino acid sequence as shown in any one of SEQ ID NO. 1 to SEQ ID NO. 8.

[0046] In the present application, the nucleotide sequence with epoxidation catalytic activity is mined and analyzed by annotating the assembly of the Taxus genome, and the terpene compound epoxidase of the present application is obtained.

[0047] The terpene compound epoxidase can convert the carbon bond of a terpene compound into an epoxy group to catalyze the synthesis of an epoxidized terpene compound.

[0048] The terpene compound epoxidase can convert the carbon double bond of a terpene compound into an epoxy group to catalyze the synthesis of an epoxidized terpene compound.

[0049] The terpene compound epoxidase exhibits taxane 4β,20 epoxidation activity.

[0050] As an embodiment, the terpene compound epoxidase can catalyze the reaction as shown in formula (1):

[0051] The terpene compound as the catalytic substrate is tax-4(5), 11(12)-diene, and the terpene compound epoxidase exhibits 4β, 20-epoxidation activity in a tricyclic diterpene skeleton.

[0052] As an embodiment, the terpene compound epoxidase can catalyze a reaction as shown in formula (2):

[0053] The terpene compound as the catalytic substrate is tax-4(20), 11(12)-diene, and the terpene compound epoxidase exhibits 4β, 20-epoxidation activity in a diterpene skeleton.

[0054] As an embodiment, the terpene compound epoxidase can catalyze a reaction as shown in formula (3):

[0055] R1 represents acetoxy or hydroxyl or H, and R2, R3, R4, R5, R6 and R7 each represent hydroxyl or H.

[0056] As an embodiment, the terpene compound epoxidase can catalyze a reaction as shown in formula (4):

[0057] R1 represents acetoxy or hydroxyl or H, and R2, R3, R4, R5, R6 and R7 each represent hydroxyl or H.

[0058] The present application also provides a polynucleotide encoding the terpene compound epoxidase, which comprises a nucleotide sequence corresponding to the amino acid sequence of the terpene compound epoxidase.

[0059] The polynucleotide is a DNA chain or an RNA chain formed by polymerization of a plurality of nucleotides.

[0060] The polynucleotide can be chemically modified as long as it can encode the terpene compound epoxidase.

[0061] As an embodiment, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO. 9-SEQ ID NO. 16.

[0062] The present application also provides a recombinant vector comprising the polynucleotide encoding the terpene compound epoxidase.

[0063] The recombinant vector refers to a DNA preparation containing a nucleic acid sequence of a polynucleotide encoding a terpene compound epoxidase, which can further contain a control sequence. In the recombinant vector, the nucleic acid sequence of the polynucleotide encoding the terpene compound epoxidase is operably linked to a suitable control sequence, so that the terpene compound epoxidase can be expressed in a suitable host. Specifically, the control sequence can include, but is not limited to, a promoter capable of initiating transcription, any operator sequence for regulating transcription, a suitable mRNA ribosome binding site, and a sequence for controlling the termination of transcription and translation. After being transformed into a suitable host cell, the recombinant vector can replicate or function independently of the host genome, or can be integrated into the genome itself for replication or function.

[0064] The type of the recombinant vector is not particularly limited as long as it can replicate in a host cell, and any vector known in the art can be used. Illustratively, the commonly used vectors in the art can include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as bacteriophage vectors or cosmid vectors, and pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0065] The present application also provides a host cell comprising the above-mentioned recombinant vector.

[0066] The above-mentioned recombinant vector is transformed into the host cell, so that the terpene compound epoxidase can be synthesized in the host cell of the present application. The recombinant vector is introduced into the host cell, and the polynucleotide encoding the terpene compound epoxidase in the recombinant vector can be expressed in the host cell, so that the host cell can synthesize the above-mentioned terpene compound epoxidase. The polynucleotide can be inserted into the chromosome of the host cell or located outside the chromosome of the host cell or inserted into the chromosome of the host cell and located outside the chromosome at the same time, and the polynucleotide can be DNA or RNA as long as it can be expressed in the host cell. Illustratively, the recombinant vector can be an expression cassette comprising a promoter, a transcription termination element, a ribosome domain, and a translation termination element operably linked to the polynucleotide.

[0067] As an embodiment, the host cell can be Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, or Brevibacterium; exemplarily, the host cell can be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or Aspergillus oryzae.

[0068] As an embodiment, the host cell can be a yeast, such as S. cerevisae, S. pombe, or K. lactis.

[0069] The embodiment of the present application further provides a biological catalysis method for producing an epoxy terpene compound, comprising the following steps:

[0070] a) contacting a terpene compound with a polypeptide having an epoxy enzyme activity to form the epoxy terpene compound;

[0071] b) separating the epoxy terpene compound;

[0072] wherein the polypeptide has an amino acid sequence as shown in any one of SEQ ID NO. 1-8.

[0073] As an embodiment, the epoxy terpene compound is as shown in general formula II:

[0074] wherein the terpene compound as a substrate can be taxa-4(5), 11(12)-dien or taxa-4(20), 11(12)-dien.

[0075] As an embodiment, the epoxy terpene compound is as shown in general formula I:

[0076] wherein R1 represents acetoxy or hydroxyl or H, and R2, R3, R4, R5, R6, and R7 respectively represent hydroxyl or H.

[0077] As an embodiment, the epoxy terpene compound is as shown in general formula III:

[0078] The terpene compound as the substrate can be 5-hydroxy-tax-4(5), 11(12)-dien or 5-hydroxy-tax-4(20), 11(12)-dien.

[0079] As an embodiment, in step a, the host cell is fermented to obtain the first fermentation product; wherein the host cell has a terpene compound epoxidase gene, and the terpene compound epoxidase gene has a nucleotide sequence encoding a terpene compound epoxidase as shown in any one of SEQ ID NO. 9 to SEQ ID NO. 16.

[0080] In some embodiments, the host cell further has a taxadiene synthase gene, and the taxadiene synthase gene has a nucleotide sequence encoding a taxadiene synthase as shown in SEQ ID NO. 18.

[0081] In some embodiments, the terpene compound epoxidase gene can be introduced into the host cell in the form of a recombinant vector, and thus, in step a, the host cell transformed with the first recombinant vector is fermented to obtain the first fermentation product; wherein the first recombinant vector comprises a nucleotide sequence encoding a terpene compound epoxidase as shown in any one of SEQ ID NO. 9 to SEQ ID NO. 16.

[0082] In some embodiments, the taxadiene synthase gene can be introduced into the host cell in the form of a recombinant vector, and thus, in step a, the host cell is further transformed with a second recombinant vector, and the second recombinant vector comprises a nucleotide sequence encoding a taxadiene synthase as shown in SEQ ID NO. 18.

[0083] Those skilled in the art should understand that the terpene compound epoxidase gene and the taxadiene synthase gene are constructed in the first recombinant vector and the second recombinant vector respectively, which is only one of the embodiments, and the terpene compound epoxidase gene and the taxadiene synthase gene can also be constructed in the same recombinant vector, or introduced into the host cell by other any feasible ways.

[0084] The host cell can be, for example, Saccharomyces cerevisiae or Escherichia coli, and the host cell itself has a mevalonate metabolic pathway, and GGPP (geranylgeranyl pyrophosphate) is a downstream metabolite of the mevalonate pathway, and the host cell can naturally produce GGPP.

[0085] By transferring a taxadiene synthase gene (TS) into a host cell, the taxadiene synthase synthesized by the host cell can catalyze GGPP to synthesize taxadiene, and the taxadiene synthesized in the above manner includes two isomers, i.e., taxa-4(5), 11(12)-diene and taxa-4(20), 11(12)-diene.

[0086] By transferring a terpene compound epoxidase gene into a host cell, the terpene compound epoxidase synthesized by the host cell can catalyze taxa-4(5), 11(12)-diene and taxa-4(20), 11(12)-diene to synthesize the compound shown in Formula II, respectively.

[0087] Exemplarily, the first recombinant vector has a result as shown in FIG. 1a, and the first recombinant vector includes, in sequence, a nucleotide sequence as shown in SEQ ID NO. 19, a nucleotide sequence encoding a terpene compound epoxidase, and a nucleotide sequence as shown in SEQ ID NO. 20. The nucleotide sequence encoding the terpene compound epoxidase is any one of SEQ ID NO. 9 to SEQ ID NO. 16.

[0088] Exemplarily, the second recombinant vector has a result as shown in FIG. 1b, and the first recombinant vector has a nucleotide sequence as shown in SEQ ID NO. 21. The nucleotide sequence as shown in SEQ ID NO. 21 has, at positions 5689 to 8277, a nucleotide sequence encoding a taxadiene synthase (TS gene).

[0089] The embodiments of the present application also provide a biological enzyme composition, including a taxadiene synthase (taxa diene synthase, TS) and the above-mentioned terpene compound epoxidase.

[0090] The terpene compound epoxidase has an amino acid sequence as any one of SEQ ID NO. 1 to SEQ ID NO. 8.

[0091] The taxadiene synthase has an amino acid sequence as shown in SEQ ID NO. 17.

[0092] The taxadiene synthase is used to cyclize GGPP to generate taxadiene, and forms a tricyclic diterpene skeleton structure.

[0093] In some embodiments, the biological enzyme composition can further include a hydroxylase.

[0094] The overexpression of the hydroxylase gene can up-regulate the expression of several known key enzymes in the paclitaxel biosynthesis pathway, and the content of each taxane in the cell is generally increased, which indicates that the hydroxylase is likely to be involved in the paclitaxel biosynthesis pathway. The key enzyme genes in the paclitaxel biosynthesis pathway include, for example, a taxadiene synthase gene (TS), a taxane 2α-hydroxylase gene (T2αH), a taxane 5α-hydroxylase gene (T5αH), a taxane 7β-hydroxylase gene (T7βH), a taxane 10β-hydroxylase gene (T10βH), and a taxane 13α-hydroxylase gene (T13αH).

[0095] In the present embodiment, the addition of the hydroxylase is beneficial to improving the synthesis efficiency of the taxane.

[0096] The present application also provides a recombinant vector comprising a nucleic acid molecule composition corresponding to the above-mentioned biological enzyme composition.

[0097] The recombinant vector can comprise a nucleotide sequence encoding a terpene compound epoxidase as shown in any one of SEQ ID NO. 9-SEQ ID NO. 16 and a nucleotide sequence encoding a taxadiene synthase as shown in SEQ ID NO. 18.

[0098] As an embodiment, the recombinant vector can be a YCPlac22 vector comprising a YCPlac22 vector fragment with a bidirectional terminator CYC1 and TDH1, a bidirectional strong promoter PGK1 and THD3 fragment, and a nucleotide sequence encoding a terpene compound epoxidase as shown in any one of SEQ ID NO. 9-SEQ ID NO. 16. In some embodiments, the recombinant vector further comprises a nucleotide sequence encoding a taxadiene synthase (as shown in SEQ ID NO. 18).

[0099] Examples and Comparative Examples

[0100] The genes encoding the terpene compound epoxidases in Examples 1-8 can be referred to as epoxidase genes. In Examples 1-8, the corresponding epoxidase genes were constructed in YCPlac22 plasmids to obtain a first recombinant vector as shown in FIG. 1a. The nucleotide sequence encoding a taxadiene synthase is a TS gene, which was constructed in a second recombinant vector as shown in FIG. 1b. The host cells in Examples 1-8 and Comparative Example 1 were transformed with the first recombinant vector and the second recombinant vector, respectively. The first recombinant vector in Comparative Example 1 does not comprise an epoxidase gene. The parameters of the first recombinant vector in Examples 1-8 and Comparative Example 1 are shown in Table 1.

[0101] The first and second recombinant vectors were transformed into S. cerevisiae respectively, and the S. cerevisiae transformed with the two recombinant vectors was inoculated into a test tube containing 3 mL of corresponding deficient medium, and cultured at 30°C and 800 rpm for 48 hours, and then the obtained seed culture was transferred into 40 mL of fresh medium at a ratio of 1:50, and 5 mL of n-dodecane and 5 mL of glucose (40%) were added after 10 hours for two-phase fermentation.

[0102] Fermentation was carried out at 30°C and 220 rpm for 3 days. Then the mixture was centrifuged at 3600 rpm for 10 min. The supernatant was taken for GC-MS detection, and the relative activity of the terpene compound epoxidase was calculated according to the content of the compound represented by general formula II in the fermentation product.

[0103] Table 1 Parameters of Examples 1-8 and Comparative Example 1

[0104] Figure 2a is a gas chromatogram of taxa-4(5), 11(12)-dien in the fermentation product, and the corresponding peak value of taxa-4(5), 11(12)-dien is 11.73; Figure 2b is a mass spectrum of taxa-4(5), 11(12)-dien in the fermentation product; Figure 2c is a gas chromatogram of the compound represented by general formula II in the fermentation product of Examples 1-8 and Comparative Example 1, and the corresponding peak value of the compound represented by general formula II is about 32.82; and Figure 2d is a mass spectrum of the compound represented by general formula II in the fermentation product. As shown in Figure 2c, the terpene compound epoxidases of Examples 1-8 respectively catalyze taxa-4(5), 11(12)-dien to synthesize the compound represented by general formula II, wherein the terpene compound epoxidases of Examples 1-8 respectively have the chromatographic peak of the compound represented by general formula II, the chromatographic peak of the compound represented by general formula II in the spectrum of Comparative Example 1 is not obvious, the terpene compound epoxidases of Examples 1-8 have 4β, 20 epoxidation activity in the tricyclic diterpene skeleton, and the chromatographic peak of the compound represented by general formula II in Example 1 has the highest peak height.

[0105] As shown in Figure 3, the relative activity of 4β, 20 epoxidation of the terpene compound epoxidases of Examples 1-8 and the epoxidase of Comparative Example 1 in the tricyclic diterpene skeleton, the terpene compound epoxidases of Examples 1-8 have 4β, 20 epoxidation activity in the tricyclic diterpene skeleton, and the 4β, 20 epoxidation activity of the epoxidase of Comparative Example 1 in the tricyclic diterpene skeleton is almost zero.

[0106] The above only describes the embodiments of the present application, and it should be noted that those skilled in the art can make improvements without departing from the inventive concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A terpene compound epoxidase, characterized in that, The terpene compound cyclooxygenase has an amino acid sequence as shown in any of SEQ ID NO.1 to SEQ ID NO.

8.

2. A biomaterial, characterized in that, The biomaterial is any one of A1) to A3): A1) The nucleotide sequence encoding the epoxidase of the terpene compound according to claim 1; A2) A recombinant vector containing the nucleotide sequence described in A1); A3) Bacterial or fungal cell lines containing the recombinant vector described in A2).

3. A biocatalytic method for producing epoxide terpene compounds, characterized in that, include: The terpene compound is contacted with a polypeptide having epoxygenase activity to form the epoxy terpene compound; The epoxy terpene compounds were separated; The polypeptide has an amino acid sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.

8.

4. The biocatalytic method for generating epoxy terpene compounds according to claim 3, characterized in that, The epoxy terpene compound is shown in general formula I: In this context, R1 represents acetoxy, hydroxyl, or H, while R2, R3, R4, R5, R6, and R7 represent hydroxyl or H, respectively.

5. The biocatalytic method for generating epoxy terpene compounds according to claim 4, characterized in that, The epoxy terpene compound is shown as general formula II:

6. The biocatalytic method for generating epoxide terpene compounds according to claim 4, characterized in that, The epoxy terpene compounds are shown in general formula III:

7. The biocatalytic method for generating epoxy terpene compounds according to claim 4, characterized in that, Contacting a terpene compound with a polypeptide having epoxygenase activity to form the epoxy terpene compound includes: The host cell transformed with the first recombinant vector is fermented to obtain a first fermentation product; wherein the first recombinant vector includes a nucleotide sequence encoding a terpene compound cyclooxygenase as shown in any one of SEQ ID NO.9 to SEQ ID NO.

16.

8. The biocatalytic method for generating epoxide terpene compounds according to claim 7, characterized in that, The host cell is also transformed with a second recombinant vector, which includes a nucleotide sequence encoding taxadiene synthase as shown in SEQ ID NO.

18.

9. The use of the terpene compound epoxidase as described in claim 1 in the biosynthesis of Baccatin III or its precursor.

10. The application of the terpene compound epoxidase as described in claim 1 in the biosynthesis of paclitaxel.

Images