CN116790572B

CN116790572B - Use of SoSTPS5 as a sesquiterpene synthase

Info

Publication number: CN116790572B
Application number: CN202310683458.0A
Authority: CN
Inventors: 虞慕瑶; 邰巴达拉胡; 郑汉; 黄璐琦; 拉喜那木吉拉; 刘琪
Original assignee: Institute of Materia Medica of CAMS; Inner Mongolia University for Nationlities
Current assignee: Institute of Materia Medica of CAMS; Inner Mongolia University for Nationlities
Priority date: 2023-06-09
Filing date: 2023-06-09
Publication date: 2024-04-09
Anticipated expiration: 2043-06-09
Also published as: CN116790572A

Abstract

The invention discloses an application of SoSTPS5 as sesquiterpene synthase. The SoSTPS5 disclosed by the invention is derived from Syringa oblata and is A1), A2) or A3) as follows: a1 A protein having an amino acid sequence of SEQ ID No. 9; a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID No.9 in the sequence table and has sesquiterpene synthase activity; a3 A protein derived from Syringa oblata and having 98% or more identity with the protein of A1) and sesquiterpene synthase activity. Experiments prove that the SoSTPS5 has sesquiterpene synthase activity, can catalyze farnesyl pyrophosphoric acid or salt thereof to generate (+) -germacrene D-4-ol and shyobinol, and has good application prospect.

Description

Use of SoSTPS5 as a sesquiterpene synthase

Technical Field

The invention relates to the field of biotechnology, and relates to the application of SoSTPS5 as sesquiterpene synthase.

Background

The number of terpenoids reported at present is more than 8 ten thousand, wherein sesquiterpenoids are the most complex class of structural diversity, more than 300 basic frameworks are found at present and widely distributed in tissues of plants, marine organisms, microorganisms, insects and the like, and the terpenoids exist in the form of esters, alcohols, ketones or glycosides. Sesquiterpenes (sesterpenoids) are a class of compounds consisting of 3 isoprene units and having 15 carbon atoms and derivatives thereof, most of which have strong biological activity and important biological functions. Sesquiterpenes are the main constituent of high boiling aromatic essential oils and are also important compounds that lead to the development of differences in fragrance notes in aromatic oils. Sesquiterpenes produce complex derivatives in several secondary metabolic processes involving multiple enzymes in the organism. The study of sesquiterpenes has been a very important area of research in natural product chemistry.

Sesquiterpenes active ingredient is an important component of plant volatile oil, and consists of 3 isoprene units such as artemisinin, alantolactone, etc. The biosynthesis of sesquiterpenes is mainly carried out by the mevalonate pathway (Mevalonate pathway, MVA) pathway, 2 molecules of prenyl diphosphate (Isopentenyl pyrophosphate, IPP) and 1 molecule of dimethylallyl pyrophosphate (Dimethylallyl pyrophosphate, DMAPP) are catalyzed by the action of farnesyl diphosphate synthase (Farnesyl diphosphate synthase, FPPS) to form farnesyl pyrophosphate (Farnesyl diphosphate, FPP), the sesquiterpenes basic skeleton is formed by the action of the sesquiterpene synthase (Sesquiterpene synthase), and complex and various sesquiterpenes active ingredients are formed by the action of CYP450 modification enzyme. The process involves 3 acetyl coas, 3 acetyl coas generating Acetoacetyl-CoA (atat) under the action of Acetoacetyl-CoA thiolase, another acetyl CoA generating 3-hydroxy-3-methylglutaryl CoA (HMGS) under the action of hydroxymethylglutaryl-CoA synthase (HMGS) and acetyl CoA acyltransferase, and then converting to MVA under the catalysis of hydroxymethylglutaryl-CoA reductase (HMGR). The MVA is phosphorylated under the action of Mevalonate kinase (Mevalonate kinase, MK) to form Mevalonate-5-phosphate (MVAP), and further decarboxylated under the action of MVA pyrophosphoric acid decarboxylase (Mevalonate pyrophosphate decarboxylase, MPD) to form IPP, which is converted to DMAPP by an isomerase. HMGR is the first and critical rate limiting enzyme of the MVA pathway.

Sesquiterpene synthases (sesquiterpene synthase) catalyze the formation of sesquiterpene intermediates from farnesylphosphoric acid (FPP). Sesquiterpene synthases are one of the terpene synthases, mainly present in the aerial tissues of angiosperms and gymnosperms. In 1992, the sesquiterpene synthase gene has been cloned in 40 plants, including crop plants, medicinal plants, microorganisms, and Arabidopsis thaliana, from the time of successful cloning of two sesquiterpene synthases from tobacco.

Disclosure of Invention

The technical problem to be solved by the invention is how to prepare sesquiterpene synthases.

To solve the above technical problems, the present invention provides a protein derived from Syringa oblata lindl, which is named SoSTPS5, soSTPS5 being A1), A2), A3) or A4) as follows:

a1 A protein having an amino acid sequence of SEQ ID No. 9;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID No.9 in the sequence table and has sesquiterpene synthase activity;

a3 A protein derived from Syringa oblata and having 98% or more identity to the protein of A1) and sesquiterpene synthase activity;

a4 A fusion protein obtained by ligating a tag to the N-terminal or/and C-terminal of A1), A2) or A3).

In order to facilitate purification of the protein of A1), a tag as shown in the following table may be attached to the amino-terminus or the carboxyl-terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.9 of the sequence Listing.

Table: tag sequence

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein in A2) has 75% or more identity with the amino acid sequence of the protein shown in SEQ ID No.9 and has the same function. The identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

The protein in A2) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

The gene encoding the protein in A2) above can be obtained by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID No.10, and/or performing missense mutation of one or more base pairs, and/or ligating the coding sequences of the tags shown in the above table at the 5 'and/or 3' ends thereof. Wherein the DNA molecule shown in SEQ ID No.10 encodes the protein shown in SEQ ID No. 9.

The present invention also provides a biological material related to SoSTPS5, which is any one of the following B1) to B12):

b1 A nucleic acid molecule encoding SoSTPS 5;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

b4 A recombinant vector comprising the expression cassette of B2);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1);

b6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism comprising the recombinant vector of B4);

b9 A transgenic plant cell line comprising the nucleic acid molecule of B1);

b10 A transgenic plant cell line comprising the expression cassette of B2);

b11 A transgenic plant cell line containing the recombinant vector of B3);

b12 A transgenic plant cell line containing the recombinant vector of B4).

In the above biological material, the nucleic acid molecule may be 1) or 2) or 3) or 4) as follows:

1) The coding sequence is a DNA molecule of SEQ ID No.10 in a sequence table;

2) A DNA molecule shown in SEQ ID No.10 of the sequence Listing;

3) A DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) or 2) and encoding SoSTPS 5;

4) A DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) or 3) and which encodes SoSTPS5.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the SoSTPS5 protein of the present invention can be easily mutated by a person skilled in the art using known methods such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the SoSTPS5 protein isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the SoSTPS5 protein and function as the SoSTPS5 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID No.9 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

In the above biological material, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5MNaPO ₄ Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: at 50 DEG C7％SDS、0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; the method can also be as follows: hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; the method can also be as follows: hybridization and washing of membranes were performed at 65℃in 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above biological material, the expression cassette (SoSTPS 5 gene expression cassette) described in B2) containing a nucleic acid molecule encoding a SoSTPS5 protein refers to DNA capable of expressing a SoSTPS5 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of a SoSTPS5 gene but also a terminator for terminating transcription of a SoSTPS5 gene. Further, the expression cassette may also include an enhancer sequence.

Recombinant vectors containing the SoSTPS5 gene expression cassette can be constructed using existing expression vectors.

In the above biological material, the vector may be a plasmid, cosmid, phage or viral vector. The plasmid may specifically be a pMAL-c2X vector.

B3 Specifically, the recombinant vector can be pMAL-c2X-SoSTPS5, and pMAL-c2X-SoSTPS5 is a recombinant vector obtained by inserting the SoSTPS5 gene shown in SEQ ID No.10 into the pMAL-c2X vector by utilizing BamHI. pMAL-c2X-SoSTPS5 can express a fusion protein formed by SoSTPS5 and MBP shown in SEQ ID No.9 in a sequence table.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacterium may be E.coli, such as E.coli Rosetta (DE 3).

In the above biological material, the transgenic plant cell line does not include propagation material.

The use of SoSTPS5 as a sesquiterpene synthase is also within the scope of the present invention.

The use of SoSTPS5 or the biological material in the preparation of sesquiterpene synthases also falls within the scope of the present invention.

The invention also provides a method for preparing a protein with sesquiterpene synthase activity, which comprises the step of expressing a gene encoding SoSTPS5 in a biological cell to obtain the protein with sesquiterpene synthase activity;

the biological cells are microbial cells, plant cells or non-human animal cells.

In the above method, the expressing the SoSTPS5 encoding gene in the biological cell may comprise a step of introducing the SoSTPS5 encoding gene into the biological cell.

In the above method, the coding gene may be the nucleic acid molecule of B1).

The gene encoding SoSTPS5 may be introduced into the biological cell by a recombinant vector containing the gene encoding SoSTPS5.

In the above method, the microorganism may be yeast, bacteria, algae or fungi.

Further, the bacterium may be E.coli.

The E.coli can be E.coli Rosetta (DE 3) and the recombinant vector can be pMAL-c2X-SoSTPS5.

The expression of the encoding gene of SoSTPS5 in biological cells comprises the steps of introducing the recombinant vector pMAL-c2X-SoSTPS5 into the escherichia coli Rosetta (DE 3) to obtain recombinant escherichia coli, and expressing the encoding gene of SoSTPS5 in the recombinant escherichia coli to obtain the protein with sesquiterpene synthase activity.

The invention also provides a preparation method of the sesquiterpene synthase, which comprises purifying the protein with the sesquiterpene synthase activity obtained by the preparation method of the protein with the sesquiterpene synthase activity to obtain the sesquiterpene synthase.

The invention also provides a method for carrying out catalytic reaction by taking farnesyl pyrophosphate as a substrate, which comprises the following steps: the method takes farnesyl pyrophosphoric acid or salt thereof as a substrate to carry out catalytic reaction by SoSTPS5, thus realizing the purpose catalytic reaction.

The invention also provides a method for preparing (+) -germacrene D-4-ol and/or shyobinol, the method comprising: the reaction product containing (+) -germacrene D-4-ol and/or shyobinol is obtained by using farnesyl pyrophosphoric acid or salt thereof as a substrate and using SoSTPS5 to perform catalytic reaction.

The reaction system of the catalytic reaction can be as follows: soSTPS4, ammonium farnesyl pyrophosphate (FPP, substrate), magnesium chloride, glycerol, DTT (dithiothreitol) were mixed with 20. Mu.L of 0.5M HEPES (pH 7.5) buffer, and the volumes were adjusted to 20. Mu.L with water, wherein the concentrations of FPP, magnesium chloride, glycerol, DTT in the reaction system were 50. Mu.M, 10mM, 5% (volume percent), 1M, respectively.

The catalytic reaction may be carried out at 30 ℃ and the reaction time may be 12 hours (overnight).

The use of SoSTPS5 or the biological material for the preparation of (+) -germacrene D-4-ol and/or shyobinol also falls within the scope of the invention.

The use of SoSTPS5 in catalyzing the formation of (+) -germacrene D-4-ol and/or shyobinol from farnesyl pyrophosphate or a salt thereof is also within the scope of the present invention.

The application of SoSTPS5 in catalytic reaction with farnesyl pyrophosphoric acid or salt thereof as substrate also belongs to the protection scope of the invention.

Experiments prove that the SoSTPS5 has sesquiterpene synthase activity, can catalyze FPP to generate (+) -germacrene D-4-ol and shyobinol, and has good application prospect.

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

Drawings

FIG. 1 shows the detection results of the purified proteins. Lanes on the left are in turn from left to right: protein molecular weight standard, MBP (i.e. protein obtained by purifying the expression of the protein of the control recombinant strain Rosetta-pMAL-c 2X), MBP-SoSTPS3, MBP, MBP-SoSTPS2, MBP, MBP-SoSTPS1; the right lanes are, from left to right, protein molecular weight standards, MBP, MBP-SoSTPS4, MBP, MBP-SoSTPS5. Each arrow represents a respective fusion protein of interest. FIG. 2 is a GC-MS diagram of the MBP-SoSTPS1 product. FIG. 3 is a mass spectrum of MBP-SoSTPS1 product. FIG. 4 is a GC-MS diagram of the MBP-SoSTPS2 product. FIG. 5 is a mass spectrum of MBP-SoSTPS2 product. FIG. 6 is a GC-MS diagram of the MBP-SoSTPS3 product; copu2 represents the catalytic product of Copu2, and the product of SoSTPS3 is different from Copu2. FIG. 7 is a mass spectrum of MBP-SoSTPS3 product. FIG. 8 is a GC-MS diagram of the MBP-SoSTPS4 product. FIG. 9 is a mass spectrum of MBP-SoSTPS4 product. FIG. 10 is a GC-MS diagram of the MBP-SoSTPS5 product. FIG. 11 is a mass spectrum at 19.26min of MBP-SoSTPS5 product retention time. FIG. 12 is a mass spectrum at a retention time of 20.96min for MBP-SoSTPS5 product.

Detailed Description

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

Farnesyl ammonium pyrophosphate salt (Farnesylpyrophosphate ammonium salt, FPP): sigma-Aldrich product.

Examples 1, soSTPS1-5 each have sesquiterpene synthase Activity

This example provides 5 proteins with sesquiterpene synthase activity derived from Syringa oblata Lindl, designated SoSTPS1-5. The protein sequences of SoSTPS1-5 are SEQ ID No.1, 3, 5, 7 and 9 in the sequence table, and the coding genes are shown as SEQ ID No.2, 4, 6, 8 and 10 in the sequence table.

1. Construction of recombinant vectors

Extracting total RNA of heartwood of syringa amurensis and reversely transcribing the total RNA into cDNA; the obtained cDNA was used as a template, and the primers for each gene in Table 1 were used to amplify the entire gene length, thereby obtaining PCR products.

TABLE 1 full length PCR primer sequences

The PCR products were ligated with cloning vectors (i.e., vector pEASY-Blunt Zero Cloning Kit) (the kit was a product of Beijing full gold Biotechnology Co., ltd., catalog number CB 501) to obtain cloning vectors 1-5, respectively. And respectively taking cloning vectors 1-5 as templates, and carrying out PCR amplification by using seamless cloning primers with vector sequences and enzyme cutting sites to obtain five PCR products. Single enzyme digestion is carried out on the pMAL-c2X vector by using restriction enzyme BamHI to obtain a linearization vector; the obtained linearized vector was ligated with five PCR products, and the recombinant vectors with correct sequences were designated as pMAL-c2X-SoSTPS1, pMAL-c2X-SoSTPS2, pMAL-c2X-SoSTPS3, pMAL-c2X-SoSTPS4, pMAL-c2X-SoSTPS5, respectively.

The vector characteristics of pMAL-c2X-SoSTPS1, pMAL-c2X-SoSTPS2, pMAL-c2X-SoSTPS3, pMAL-c2X-SoSTPS4, pMAL-c2X-SoSTPS5 are described as follows: pMAL-c2X-SoSTPS1, pMAL-c2X-SoSTPS2, pMAL-c2X-SoSTPS3, pMAL-c2X-SoSTPS4, pMAL-c2X-SoSTPS5 are recombinant vectors obtained by inserting the SoSTPS1-5 gene shown in SEQ ID No.2, 4, 6, 8, 10 into the pMAL-c2X vector by BamHI, respectively, and can express fusion proteins (MBP-SoSTPS 1, MBP-SoSTPS2, MBP-SoSTPS3, MBP-SoSTPS4, MBP-SoSTPS 5) formed by the SoSTPS1-5 shown in SEQ ID No.1, 3, 5, 7, 9 in the sequence table respectively, and the expression of the proteins is driven by 35 s.

The pMAL-c2X vector sequence is as follows:

2. expression and purification of proteins

The pMAL-c2X-SoSTPS1, pMAL-c2X-SoSTPS2, pMAL-c2X-SoSTPS3, pMAL-c2X-SoSTPS4, pMAL-c2X-SoSTPS5 obtained in the step one were respectively introduced into E.coli Rosetta (DE 3) (Beijing all-round gold Biotechnology Co., ltd.) to obtain recombinant bacteria Rosetta-pMAL-c2X-SoSTPS1, rosetta-pMAL-c2X-SoSTPS2, rosetta-pMAL-c2X-SoSTPS3, rosetta-pMAL-c2X-SoSTPS4, rosetta-pMAL-c2X-SoSTPS5, and the pMAL-c2X vector was introduced into E.coli Rosetta (DE 3) to obtain control recombinant bacteria Rosetta-pMAL-c2X. The recombinant bacteria are respectively expressed and purified, and the specific steps are as follows:

using 30mL of LB medium containing ampicillin, the recombinant bacteria were grown to turbidity, and then the resulting bacterial liquid was prepared in a ratio of 1:100 proportion in 100mL of LB medium containing ampicillin was cultured to OD by expansion ₆₀₀ The value was between 0.6 and 0.8, and IPTG was then added to a concentration of 0.4mM,25℃and 200rpm in the culture system, and the culture was continued for 8 hours.

After the culture is finished, taking out bacterial liquid, placing the bacterial liquid into a 50mL round bottom centrifuge tube, centrifuging at 4 ℃ and 4000rpm for 10min, discarding supernatant, and collecting bacterial cells; then adding 5mL of PBS buffer solution into the thalli to wash and precipitate, centrifuging at 4 ℃ and 4000rpm for 10min, discarding the supernatant, and collecting the thalli; the precipitate was weighed and Column Buffer (1 MHris-HCl (pH 7.4) 20ml,NaCl 11.7g,0.5M EDTA (pH 8.0) 2mL, dd H was added to the cells at a rate of 5 mL/g cells ₂ O is added to 1L), DTT is added to the concentration of 1mM in the bacterial suspension, the bacterial suspension is crushed by ultrasonic on ice, the ultrasonic is carried out for 5s at 30Hz, the suspension is suspended for 5s, the total time is 10min, and the temperature is 120 ℃ after the bacterial suspension is semitransparentCentrifuging at 00rpm for 15min, and collecting supernatant to obtain crude protein solution.

Taking a crude protein solution in a 15mL centrifuge tube, adding Amylose Resin (BioLabs product, product number E8021S) and Columb Buffer equal volume mixed solution into the centrifuge tube, mixing the mixture for 2h at 4 ℃ in a rotating way, taking out the mixture, centrifuging the mixture at 500rpm for 3min at 4 ℃, and discarding supernatant; adding 1mL Column Buffer into the precipitate, centrifuging at 4deg.C and 500rpm for 1min, discarding supernatant, repeatedly washing impurity protein with Column Buffer for 4-5 times, discarding supernatant; 200. Mu.L of an absorption Buffer (maltose 0.316g,Column Buffer is added to 100 mL) was added to the precipitate, the mixture was spun and mixed at 4℃for 1 hour, and then centrifuged at 500rpm for 3 minutes at 4℃to obtain a supernatant as a purified protein solution, namely purified MBP-SoSTPS1, MBP-SoSTPS2, MBP-SoSTPS3, MBP-SoSTPS4, MBP-SoSTPS5 and MBP (obtained from control recombinant bacteria Rosetta-pMAL-c 2X), respectively. The marked product is preserved at the temperature of minus 80 ℃ for standby.

The results of SDS-PAGE of each purified protein are shown in FIG. 1. The results showed that the fusion proteins of interest were obtained relatively pure.

3. Detection of sesquiterpene synthase Activity

Detecting the sesquiterpene synthase activity of the fusion proteins MBP-SoSTPS1, MBP-SoSTPS2, MBP-SoSTPS3, MBP-SoSTPS4 and MBP-SoSTPS5 obtained in the step II, and taking the MBP obtained in the step II as a control:

the enzymatic reaction system was 200. Mu.L in total, wherein 100. Mu.L of the purified protein solution of the second step was added with an ammonium farnesyl pyrophosphate (FPP, substrate), magnesium chloride, glycerol, DTT (dithiothreitol) and 0.5M HEPES (pH 7.5) buffer, wherein the concentrations of FPP, magnesium chloride, glycerol, DTT in the reaction system were 50. Mu.M, 10mM, 5% (by volume), and the addition amount of 1M,0.5M HEPES (pH 7.5) buffer was 20. Mu.L, respectively, with the balance being water.

After the above system was mixed uniformly, 300. Mu.L of n-hexane was added to cover the mixture to prevent the product from losing due to volatilization, and the mixture was reacted overnight (12 hours) in a water bath at 30℃to complete the enzymatic reaction.

After the reaction was completed, the enzymatic reaction product was extracted with 600. Mu.L of n-hexane, the upper organic phase was extracted three times repeatedly, and the organic phases were combined. Blow-dried by nitrogen blowing instrument, dissolved in 200. Mu.L of n-hexane, filtered through 0.22 μm polytetrafluoroethylene organic filter membrane, and subjected to GC-MS component measurement. GC-MS detection conditions: the sample volume was 1. Mu.L, and in the no-split mode, the initial temperature was 50deg.C for 2min, and the temperature was raised to 280℃at a rate of 8deg.C/min for 10min. The injector temperature and the ion trap heating temperature are 250 ℃, the ion source is EI, the electron energy is 70eV, and the scanning range is 20-650m/z.

MBP, MBP-SoSTPS1, MBP-SoSTPS2, MBP-SoSTPS3, MBP-SoSTPS4, MBP-SoSTPS5 were detected by GC-MS to catalyze the sesquiterpene products after FPP. The results showed that the control MBP appeared to be unimodal at 17.87min and that the catalytic product of MBP-SoSTPS1 had a new single product peak at 18.15min, which was consistent with the retention time and mass spectrum peak of the product of the Salvia Miltiorrhiza (Salvia miltiorrhiza Bunge) SmSTPS3 catalytic FPP, buddha, sci (2017), identification of a Novel (-) -5-Epieremophilene Synthase from Salvia miltiorrhiza via Transcriptome Mining, DOI: 10.3389/fpls.2017.00627), thus confirming that MBP-SoSTPS1 can catalyze FPP to produce Buddha, (-) -5-epi remophilene, FIGS. 2 and 3.

The catalytic products of MBP-SoSTPS2 had a new single product peak at 18.15min retention time, consistent with the retention time and mass spectrum peaks of the catalytic products of flammulin of SoSTPS1 and SmSTPS3. From this, it was determined that MBP-SoSTPS2 can catalyze FPP to produce friedel (-) -5-epi-remophilene, FIGS. 4 and 5.

The catalytic product of MBP-SoSTPS3 had a new single product peak at 17.71min of retention time, which was searched to find that it should be the Copaane type sesquiterpene beta-Copaene, thus determining that MBP-SoSTPS3 can catalyze FPP to produce the Copaane type sesquiterpene beta-Copaene, FIGS. 6 and 7.

The catalytic product of MBP-SoSTPS4 had new product peaks at retention times of 18.04min and 20.18min, which were searched for alpha-luteole (alpha-muurolene) and alpha-Bi Cheng solanol (alpha-candiol). The product retention time and mass spectrum peak of SoSTPS4 at retention time 18.04min was consistent with that of the product alpha-luteolin (alpha-luteolin) catalyzed by PpolyTPS4 of F.multiflorum (Physarum polycephalum) (X Chen et al, beilstein J Org Chem (2019), emission and biosynthesis of volatile terpenoids from the plasmodial slime mold Physarum polycephalum, DOI: 10.3762/bjoc.15.281), thereby determining that the product catalyzed by MBP-SoSTPS4 at retention time 18.04min was alpha-luteolin (alpha-luteolin); the product at 20.18min of retention time of SoSTPS4 was consistent with the retention time and mass spectrum peak of standard α -candol (CAS: 481-34-5), thereby determining that the catalytic product at 20.18min of retention time of MBP-SoSTPS4 was α -Bi Cheng solanol (α -candol), FIGS. 8 and 9. It is illustrated that MBP-SoSTPS4 can catalyze FPP to produce alpha-luteolin (alpha-muurolene) and alpha-Bi Cheng solanol (alpha-candiol).

The catalytic product of MBP-SoSTPS5 had new product peaks at retention times of 19.26min and 20.96min, which were searched and found to be (+) -germacreneD-4-ol and shyobinol. The product retention time and mass spectrum peak of MBP-SoSTPS5 at 19.26min was consistent with that of the termite umbrella fungus (Terminalia) STC15 catalyzed FPP, (+) -germacroend-4-ol (I Burkhardt et al org Biomol Chem (2019), mechanistic Characterization of Three Sesquiterpene Synthases from the Termite-Associated Fungus Termitomyces, DOI:10.1039/c8ob02744 g), thus determining that the product of MBP-SoSTPS5 at 19.26min was (+) -germacroend-4-ol, FIGS. 10 and 11. The mass spectrum of the product of MBP-SoSTPS5 at 20.38min of retention time is shown in FIG. 12. It is illustrated that MBP-SoSTPS5 can catalyze FPP to produce (+) -germacreneD-4-ol and shyobinol.

In the above, the steps of the Salvia Miltiorrhiza (Salvia miltiorrhiza Bunge) SmSTPS3 catalyzing FPP reaction, the Rhizopus polycephalum (Physarum polycephalum) PpolyTPS4 catalyzing FPP reaction, and the Terminalia fungus (Terminalia) STC15 catalyzing FPP reaction are as follows:

1. construction of recombinant vectors

Extracting total RNA of root of red sage root (Salvia miltiorrhiza Bunge) and reverse transcribing into cDNA; and (3) using the obtained cDNA as a template, and respectively amplifying the full length of the SmSTPS3 gene by using a SmSTPS3 primer to obtain a PCR product. The PCR products were ligated with cloning vector (the vector was pEASY-Blunt Zero Cloning Kit) to obtain cloning vector SmSTPS3. And (3) taking a cloning vector SmSTPS3 as a template, and carrying out PCR amplification by using a seamless cloning primer with a vector sequence and an enzyme cutting connection site to obtain a PCR product. The obtained PCR product was ligated with a linearized vector obtained by BamHI cleavage of the pMAL-c2X vector, and the resulting recombinant vector having the correct sequence was designated as pMAL-c2X-SmSTPS3 (the vector was obtained by synthesizing the SmSTPS3 gene containing BamHI cleavage site, and sequencing the resulting vector after cleavage by ligation with the pMAL-c2X vector). pMAL-c2X-SmSTPS3 contains the SmSTPS3 gene shown in SEQ ID No.12, and can express the fusion protein formed by SmSTPS3 shown in SEQ ID No.11 and MBP (marked as MBP-SmSTPS 3).

pMAL-c2X-PpolyTPS4 was constructed as described above using the template cDNA of F.polycephalum (Physarum polycephalum). pMAL-c2X-PpolyTPS4 can also be obtained by synthesizing a PpolyTPS4 gene containing a BamHI cleavage site, and connecting the gene with a pMAL-c2X vector for sequencing after cleavage. pMAL-c2X-PpolyTPS4 contains the PpolyTPS4 gene shown in SEQ ID No.14, and can express fusion protein (marked as MBP-PpolyTPS 4) formed by PpolyTPS4 shown in SEQ ID No.13 and MBP.

pMAL-c2X-STC15 was constructed as described above using a template cDNA of the fungus Umbelliferae. pMAL-c2X-STC15 can also be obtained by synthesizing STC15 gene containing BamHI cleavage site, and sequencing by ligation with pMAL-c2X vector after cleavage. pMAL-c2X-STC15 contains the STC15 gene shown in SEQ ID No.16, and can express a fusion protein (called MBP-STC 15) formed by the STC15 shown in SEQ ID No.15 and MBP.

2. Expression and purification of proteins

The recombinant vectors pMAL-c2X-SmSTPS3, pMAL-c2X-PpolyTPS4 and pMAL-c2X-STC15 are respectively introduced into escherichia coli Rosetta (DE 3) to respectively obtain recombinant bacteria Rosetta-pMAL-c2X-SmSTPS3, rosetta-pMAL-c2X-PpolyTPS4 and Rosetta-pMAL-c2X-STC15. According to the method of the second step, the recombinant bacteria are respectively replaced by the recombinant bacteria to express and purify the protein, and the purified MBP-SmSTPS3, MBP-PpolyTPS4 and MBP-STC15 are respectively obtained.

3. Catalytic reaction with FPP as substrate

According to the method of the third step, the fusion protein is respectively replaced by the purified MBP-SmSTPS3, MBP-PpolyTPS4 and MBP-STC15 obtained in the step 2, other steps are unchanged, the catalytic reaction with FPP as a substrate is carried out, and the GC-MS detection is carried out on the obtained reaction product.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims

1. Protein, which is A1) or A2) as follows:

a1 A protein having an amino acid sequence of SEQ ID No. 9;

a2 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1).

2. A biological material related to the protein of claim 1, which is any one of the following B1) to B6):

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1);

b5 A recombinant microorganism comprising the expression cassette of B2);

b6 A recombinant microorganism containing the recombinant vector of B3).

3. The biomaterial according to claim 2, characterized in that: the nucleic acid molecule is 1) or 2) as follows:

1) The coding sequence is a DNA molecule of SEQ ID No.10 in a sequence table;

2) A DNA molecule shown in SEQ ID No.10 of the sequence Listing.

4. Use of the protein of claim 1 as a sesquiterpene synthase.

5. Use of the biomaterial according to claim 2 or 3 for the preparation of sesquiterpene synthases.

6. A method for producing a protein having sesquiterpene synthase activity, comprising the step of expressing a gene encoding the protein of claim 1 in a biological cell to obtain a protein having sesquiterpene synthase activity;

7. The method according to claim 6, wherein: the step of expressing the gene encoding the protein in a biological cell includes the step of introducing the gene encoding the protein into the biological cell.

8. A process for producing a sesquiterpene synthase, comprising purifying a protein having a sesquiterpene synthase activity obtained by the process according to claim 6 or 7 to obtain a sesquiterpene synthase.

9. A method for performing a catalytic reaction using farnesyl pyrophosphate as a substrate, comprising: the method comprises the step of carrying out catalytic reaction by using the protein of claim 1 with farnesyl pyrophosphoric acid or salt thereof as a substrate to realize the target catalytic reaction.

10. A method for preparing (+) -germacrene D-4-ol and/or shyobinol, comprising: a method for preparing a reagent containing (+) -germacrene D-4-ol and/or shyobinol, which comprises the step of carrying out a catalytic reaction on a protein of claim 1 using farnesyl pyrophosphate or a salt thereof as a substrate.

11. Use of a protein according to claim 1 or a biomaterial according to claim 2 or 3 for the preparation of (+) -germacrene D-4-ol and/or shyobinol.

12. Use of a protein as claimed in claim 1 for catalyzing the formation of (+) -germacrene D-4-ol and/or shyobinol from farnesyl pyrophosphate or a salt thereof.

13. Use of the protein according to claim 1 for catalytic reactions with farnesyl pyrophosphate or a salt thereof as a substrate.