WO2019148609A1

WO2019148609A1 - Terpene synthase and application thereof

Info

Publication number: WO2019148609A1
Application number: PCT/CN2018/079910
Authority: WO
Inventors: 叶紫玲
Original assignee: 深圳艾格鑫科技有限公司
Priority date: 2018-01-30
Filing date: 2018-03-21
Publication date: 2019-08-08
Also published as: CN110093329B; CN110093329A

Abstract

Provided are a terpene synthase and an application thereof. In particular, provided are uses of the terpene synthase and a plasmid or vector expressing the terpene synthase in catalyzing the synthesis of terpenes or in preparing catalysts for catalyzing the synthesis of terpenes. Also provided is a method for preparing terpene.

Description

Indole synthase and its application

Priority information

Priority is claimed on Japanese Patent Application No. 201810091964.X filed on Jan. 30, 2008, the disclosure of which is hereby incorporated by reference.

Technical field

The present invention relates to the field of bioengineering, and in particular to steroid synthase and its use.

Background technique

The sesquiterpene compound is synthesized from two molecules of isopentenyl pyrophosphate (IPP) and one molecule of dimethyl propylene pyrophosphate (DMAPP) catalyzed by farnesyl pyrophosphate synthase (farnesyl pyrophosphate synthase). Farnesyl pyrophosphate (FPP) is subsequently formed under the catalysis of sesquiterpene synthase. To date, at least 122 different sesquiterpene compound skeletons have been discovered (Klapschinski et al., 2016). Some of the skeleton compounds, such as the 5-7 binary ring and the 3-5-6 ternary ring sesquiterpene compound, have various biological activities such as bacteriostatic and antitumor. However, since the sesquiterpenoids only contain three isoprene units, the length of the carbon chain limits the diversity of the skeleton, which greatly increases the difficulty of excavating a new sesquiterpene skeleton.

Thus, the method of synthesizing sesquiterpenes needs to be improved.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a novel indole synthase which can be transferred into a recombinant cell containing a high-producing terpenoid precursor material to isolate a plurality of terpenoids, especially sesquiterpenes. Compounds, including C ₅ -C ₇ bicyclic sesquiterpenes and C ₃ -C ₅ -C ₆ ternary sesquiterpene novel skeleton compounds, enrich the skeleton composition of sesquiterpene compounds.

Thus, in accordance with a first aspect of the invention, the invention provides an isolated polypeptide. According to an embodiment of the invention, the isolated polypeptide has the amino acid sequence set forth in SEQ ID NO: 1. The isolated polypeptide has the activity of a terpene synthase and can catalyze the synthesis of terpenoids.

As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify the polypeptide using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed using amino acid sequences.

The active polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptide of the present invention may be a naturally purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plant) using recombinant techniques.

"萜" is an aliphatic or cyclic isoprene-based (C ₅ H ₈ )-based hydrocarbon. "萜" includes, but is not limited to, fusarium diene, trans-fusagramineol, fusagramineol, nerolidol, acorenol, beta - farnesene, bisabolol, Acoradiene, cis-α-bergamotene, trans-α-bergamotene, (+)-epi-β-santalene, β-red Myrrhene, and trans-gamma-red myrrhene.

The "oxime" and "terpenoids" of the present invention include derivatives of ruthenium and osmium, including functionalized one or more steps such as hydroxylation, isomerization, redox, dimethylation or acylation. Compound. As used herein, "sesquivalent" is a C15-based oxime and includes sesquiterpene and sesquiterpene derivatives of a compound that undergoes one or more functionalization steps.

The "indole synthase" of the present invention is any enzyme which catalyzes the synthesis of an anthraquinone, and the isolated polypeptide is the indole synthase, and the two may be used interchangeably herein.

According to a second aspect of the invention, the invention provides an isolated nucleic acid. According to an embodiment of the invention, the isolated nucleic acid is a sequence selected from the group consisting of:

(1) A nucleotide sequence encoding the above-described isolated polypeptide, wherein it is to be noted that the nucleotide sequence may be a nucleotide sequence encoding the polypeptide, or may include additional coding and/or non- The nucleotide sequence of the coding sequence.

(2) Having the nucleotide sequence shown by SEQ ID NO: 2.

The polypeptides and nucleic acids of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

Those skilled in the art will appreciate that once the nucleic acids associated with the present invention are obtained, recombinant sequences can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant nucleic acid sequence from the proliferated host cell by conventional methods.

Further, a method of amplifying DNA/RNA using a PCR technique is preferably used to obtain the gene of the present invention. The primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.

According to a third aspect of the invention, the invention provides a carrier. According to an embodiment of the invention, the vector contains the aforementioned isolated nucleic acid.

A "vector" of the invention may include any recombinant vector, including but not limited to viral vectors, bacteriophages, and plasmids. The skilled person is able to select a suitable vector based on the expression system. In some embodiments, the expression vector comprises a cDNA sequence encoding a polypeptide operably linked to a regulatory sequence, an mRNA ribosome binding site, and a suitable sequence that regulates initiation and termination of transcription and translation, the regulatory sequence For example, a transcriptional promoter, a terminator, an operator or an enhancer. When a regulatory sequence is functionally linked to a cDNA sequence of the invention, then the nucleotide sequence is "operably linked."

According to a fourth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell comprises the aforementioned polypeptide, the aforementioned isolated nucleic acid or the aforementioned vector.

According to an embodiment of the present invention, the kind of the host cell used for preparing the recombinant cell is not particularly remarkable as long as the nucleic acid can be expressed in the host cell, and may be a prokaryotic cell such as a bacterial cell or Escherichia coli; or Eukaryotic cells, such as yeast, fungi. According to an embodiment of the invention, the host cell may be yeast, Escherichia coli, Bacillus subtilis or fungi.

According to a fifth aspect of the present invention, the present invention provides the aforementioned isolated polypeptide, the aforementioned isolated nucleic acid, the aforementioned vector or the aforementioned recombinant cell for catalyzing the synthesis of a steroid or for preparing a reaction for catalyzing the synthesis of a steroid. The use of the catalyst. The inventors have found that the aforementioned isolated polypeptide, ie, a guanidine synthase, and a vector and recombinant cell expressing the guanidine synthase can separate a plurality of steroids in a recombinant cell containing a precursor substance which produces a high steroid type compound. In particular, sesquiterpene compounds, including C ₅ -C ₇ bicyclic sesquiterpenes and C ₃ -C ₅ -C ₆ ternary sesquiterpene new skeleton compounds, enrich the skeleton composition of sesquiterpene compounds.

According to an embodiment of the invention, the moss may be sesquiterpenes. According to a preferred embodiment of the invention, the moss is at least one selected from the group consisting of:

Among them, compounds No. 1-8 are fusarium diene, trans-fusagramineol, fusagramineol, trans-nerolidol, -)-α-caloricol, (E)-β-farnesene, (+)-α-bisabolol, (-)-acoradiene.

According to a sixth aspect of the invention, the invention provides a method of preparing a moss. According to an embodiment of the invention, the method is carried out in the presence of the aforementioned polypeptide, the aforementioned vector or the aforementioned recombinant cells. The inventors have found that the above-mentioned isolated polypeptide, ie, a guanidine synthase, a steroid-like enzyme-expressing vector and a recombinant cell, can be used as a substrate for high-producing steroid precursors, and a plurality of steroids can be isolated, especially Semiquinone compounds, including C ₅ -C ₇ binary ring sesquiterpenes and C ₃ -C ₅ -C ₆ ternary ring sesquiterpene new skeleton compounds, enrich the skeleton composition of sesquiterpene compounds.

The inventors have found that the indole synthase of the embodiments of the invention contains a terpenoid cyclized (TC) domain and does not contain an isopentenyl transferase (PT) domain, according to some embodiments of the invention, the bottom of the method The product contains at least one of geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), and the product is formed by the reaction of the aforementioned substrate containing 2 isoprene At least one of a monoterpene of an olefin unit, a sesquiterpene of three isoprene units, and a diterpene of four isoprene units cannot be synthesized as a corresponding sesquiterpene.

The steroid synthase according to an embodiment of the present invention has at least one of the following advantages:

(1) The hydrazine synthase of the examples of the present invention can specifically and efficiently synthesize a plurality of sesquiterpene compounds using a steroid precursor material as a substrate;

(2) The hydrazine synthase of the embodiment of the present invention can synthesize a variety of high economic value steroids, such as nerolidol, using GPP, FPP and GGPP as substrates, and the compound is widely used. Used in industries such as flavoring and perfume production, the compound enhances the skin's ability to absorb drugs that are permeable to skin through the skin; beta-farnesene, which is used as a new aviation fuel and synthetic vitamin E; bisabolol, which has biological effects such as antibacterial, anti-inflammatory, anti-microbial and therapeutic psoriasis.

(3) In the recombinant cells (such as yeast), a synthetic route for the synthesis of terpenoids, especially high-economic value steroids such as nerolidol, farnesene and bisabolol, has been constructed, thereby realizing GPP, FPP and GGPP is a substrate, which is fermented by yeast to form hydrazines, especially high-value steroids, and the production cost is significantly reduced.

The additional aspects and advantages of the invention will be set forth in part in the description which follows.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

1 is a schematic view showing the results of in vitro reaction product analysis and detection of J1-018-A according to an embodiment of the present invention;

2 is a schematic view showing the results of in vitro reaction and in vivo fermentation product analysis of J1-018A according to an embodiment of the present invention;

3 is a schematic view showing the results of analysis and analysis of fermentation products of J1-018-A according to an embodiment of the present invention;

Figure 4 shows a schematic diagram of the results of purifying Compound 2 according to one embodiment of the present invention;

Figure 5 shows a schematic diagram of the results of purification of Compound 3 according to one embodiment of the present invention;

Figure 6 shows a schematic diagram of the results of purifying compound 4 according to one embodiment of the present invention;

Figure 7 shows a schematic diagram of the results of purifying compound 5 according to one embodiment of the present invention;

Figure 8 shows a schematic diagram of the results of purifying compound 7 according to one embodiment of the present invention;

[Correct according to Rule 91 18.07.2018]
9a-9f are schematic diagrams showing the results of nuclear magnetic resonance of purified compound 1 according to an embodiment of the present invention, wherein Fig. 9a is a ¹ H NMR spectrum, Fig. 9b is a ¹³ C NMR spectrum, and Fig. 9c is an HMQC spectrum, and Fig. 9d is ¹ H- ¹ H COSY spectrum, HMBC spectrum of FIG. 9e, 9f FIG. 1 and the structure of the key ¹ H- ¹ H COSY, HMBC correlation signal schematic compound;

[Correct according to Rule 91 18.07.2018]
10a to 10g are graphs showing the results of nuclear magnetic resonance of purified compound 2 according to an embodiment of the present invention, wherein Fig. 10a is a ¹ H NMR spectrum, Fig. 10b is a ¹³ C NMR spectrum, and Fig. 10c is an HSQC spectrum, and Fig. 10d is ¹ H- ¹ H COSY spectrum, HMBC spectrum of FIG. 10e, FIG. 10f is a NOESY spectrum, the structure of FIG. 10g of compound 2 and key ^¹ H- ¹ H ^COSY, HMBC and NOESY correlation signal is schematic;

Figure 11 is a graph showing the results of nuclear magnetic resonance of purified Compound 3 according to an embodiment of the present invention, wherein Figure 11a is a ¹ H NMR spectrum, Figure 11b is a ¹³ C NMR spectrum, Figure 11c is an HSQC spectrum, and Figure 11 is a ¹ H- ¹ H COSY map, Figure 11e is HMBC map, Figure 11f is NOESY map, Figure 11g is the structure of compound 3 and key ¹ H- ¹ H COSY, HMBC and NOESY related signals;

Figure 12 is a graph showing the results of nuclear magnetic resonance of purified Compound 4 according to one embodiment of the present invention, wherein Figure 12a is a ¹ H NMR spectrum and Figure 12b is a ¹³ C NMR spectrum;

Figure 13 is a graph showing the results of nuclear magnetic resonance of purified Compound 5 according to one embodiment of the present invention, wherein Figure 13a is a ¹ H NMR spectrum and Figure 13b is a ¹³ C NMR spectrum;

Figure 14 is a graph showing the results of nuclear magnetic resonance of purified compound 6 according to one embodiment of the present invention, wherein Figure 14a is a ¹ H NMR spectrum and Figure 14b is a ¹³ C NMR spectrum;

Figure 15 is a graph showing the results of nuclear magnetic resonance of purified compound 7 according to one embodiment of the present invention, wherein Figure 15a is a ¹ H NMR spectrum and Figure 15b is a ¹³ C NMR spectrum;

Figure 16 is a graph showing the results of nuclear magnetic resonance of purified Compound 8 according to one embodiment of the present invention, wherein Figure 16a is a ¹ H NMR spectrum, Figure 16b is a ¹³ C NMR spectrum Figure 16c is an HSQC spectrum, and Figure 16d is ¹ H- ¹ H COSY map, Fig. 16e is the HMBC map, Fig. 16f is the structure of compound 8 and the key ¹ H- ¹ H COSY, HMBC related signals.

Detailed description of the invention

The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are intended to be illustrative of the invention and are not to be construed as limiting.

The invention is described below with reference to the specific embodiments, which are intended to be illustrative, and are not to be construed as limiting.

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not mentioned, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially, for example, can be purchased from Sigma.

Example 1

In the present embodiment, the cDNA sequence of the hydrazine synthase J1-018-A (J1-018-A for short) is amplified by PCR, and the plasmid and the sesquiterpene yeast platform are constructed using the cDNA sequence, wherein The strains and plasmids used in this example are shown in Table 1, and the primers used to construct the relevant plasmids are shown in Table 2.

The main characteristics of the strains and plasmids used in Table 1

Table 2 Primers used for plasmid construction

1. J1-018-A plasmid construction and protein purification

Using the cDNA of the reverse-transcribed F. graminearum J1-012 as a template, the cDNA sequence of J1-018-A was amplified by primer P1/P2 and ligated into plasmid pET28a(+) to obtain plasmid pGB152, as follows:

(1) Obtaining cDNA: The fungal RNA was extracted using the QIAGEN RNeasy Plant Mini Kit and operated according to the instructions. According to the measured RNA concentration, the same amount of total RNA was taken, and the kit was added with a primer mixture, 1 μL of dNTP, and the volume was adjusted to 13 μL with sterile water. The reaction conditions were: 65 ° C for 5 min, 4 ° C for 1 min, and ice bath for 2 min. After a brief centrifugation, add 4 μL of 5× first strand synthesis reaction buffer, 1 μL of 0.1 M DTT, 1 μL of RNase inhibitor and 1 μL of SuperScriptTM III RT reverse transcriptase (200 U/μL). The reaction procedure was 25 ° C for 5 min, 55 ° C. 60 min, 70 ° C for 15 min. After completion of the reaction, the product was purified using QIAGEN PCR Product Purification Kit to obtain cDNA.

(2) Digestion enzyme reaction: The extracted plasmid or the purified DNA fragment can be directly digested with a restriction enzyme, and the restriction endonuclease is used for restriction enzyme digestion by Fermentas. The enzyme digestion system is as follows:

After mixing, the reaction was carried out at 37 ° C for 0.5-1 h, and the result of the enzyme digestion was detected by electrophoresis.

The vector DNA and the gene DNA fragment can be directly used for ligation by restriction endonuclease digestion, and the enzyme is ligated using Fermentous T4 DNA ligase. The reaction system is as follows:

After mixing, the reaction was carried out at 22 ° C for 30 min or at 4 ° C overnight. The ligation product is used directly for transformation.

(3) Plasmid pGB152 was transformed into E. coli BL21 (DE3) for protein expression.

(4) The cells were lysed, and the protein was purified by Ni-NTA affinity chromatography to obtain a purified protein, which was J1-018-A.

2. Detection of J1-018-A substrate recognition ability

In order to fully understand the potential of J1-018-A to catalyze the synthesis of terpenoids with different chain length substrates, the following in vitro enzymatic reaction system was established, as follows:

(1) Add 10 μM of purified protein (J1-018-A) to 200 μL of a final concentration of 50 mM PB buffer (pH 7.6) buffer containing 10% glycerol, and then add 100 μM of the substrate (the substrate is GPP, respectively). FPP, GGPP, GFPP and IPP+DMAPP), and 2 mM Mg ²⁺ , respectively, to obtain GPP, FPP, GGPP, GFPP and IPP + DMAPP substrate solutions;

(2) The GPP, FPP, GGPP, GFPP and IPP+DMAPP substrate solutions were reacted overnight at 30 ° C to obtain a reaction solution corresponding to each substrate.

(3) Each reaction solution was extracted twice with an equal volume of n-hexane, and the organic phases were combined and the products obtained by enzymatic catalysis of each substrate were examined by GC-MS.

The results of GC-MS detection are shown in Fig. 1. Fig. 1 is a product analysis result of a reaction solution using GPP as a substrate, and Fig. b is a product analysis result of a reaction solution using FPP as a substrate, and Fig. c is a GGPP As a result of product analysis of the reaction solution of the substrate, Fig. d is the result of the reaction solution using GFPP as a substrate, and Fig. e is the result of the reaction solution using IPP + DMAPP as a substrate. The results showed that J1-018-A was able to synthesize the corresponding monoterpenes, sesquiterpenes and diterpenes using three substrates of GPP, FPP and GGPP, but could not synthesize sesquiterpenes using GFPP and IPP+DMAPP, showing J1 -018-A has a relatively broad substrate and reaction heterogeneity. At the same time, the results of in vitro reaction showed that J1-018-A could not carry out chain extension with IPP and DMAPP as substrates, indicating that J1-018-A contains only indole cyclized (TC) domain and does not contain isopentenyl transfer. Enzyme (PT) domain.

3. Construction of a high-yield sesquiterpene yeast platform

3.1 Construction of mutant S. cerevisiae YZL141

(1) Using the P46/P47 as a primer to amplify the left homology arm of GaL1710 with CENPK2-1D as a template;

The Trp tag was amplified with p48424 as a template using P48/P49 as a primer;

The ACT1 terminator tag was amplified with P50/P51 as primer and S288C as template.

The tHMG1 tag was amplified with P52/P53 as primer and S288C as template.

Amplification of PGAL10-PGAL1 tag with CE54KD as a primer using P54/P55 as a primer;

The PPS1 terminator was amplified with PNP/P57 as primer and CENPK2-1D as template.

The homologous arm of Gal1710 was amplified with P58/P59 as primer and CENPK2-1D as template.

The plasmid backbone pRS426 was amplified with p60426 as a primer using P60/P61 as a primer;

(2) Using P46/P51 as a primer, the left homologous arm, the Trp tag and the ACT1 terminator of Gal1710 were ligated by OE-PCR;

(3) Using P52/P57 as a primer, the tHMG1, PGAL10-PGAL1, and CPS1 terminator were ligated by OE-PCR.

(4) The two fragments obtained by OE-PCR, the homologous arm of the right side of Gal1710 and the plasmid backbone pRS426 were ligated by the yeast assembly method to obtain plasmid pZY141. The yeast assembly method is based on the efficient homologous recombination efficiency of S. cerevisiae, which can realize one-step assembly of multiple fragments, and the operation steps are as follows:

1 The overlapping sequence of 40-80 bp needs to be included between the two assembled fragments (the longer the homologous fragment is, the better the assembly is), and the assembled fragment and the vector fragment containing the homology arm are obtained by a PCR method.

After the 2 PCR product was purified, its concentration was determined by Nanodrop.

3 Take 300 ng of the fragments to be assembled and calculate the total volume.

4 Add 10% by volume of 3M NaAc, 2% by volume of 10 mg/mL glycogen and mix well, then add 2 volumes of pre-cooled absolute ethanol and mix well.

5 placed in a -80 ° C refrigerator for about 2h.

Centrifuge at 64 ° C for 20 min at 64 ° C and discard the supernatant.

7 Add 500 μL of 70% ethanol, 13 200 rpm, centrifuge at room temperature for 3 min, and discard the supernatant.

8 freeze-precipitate the precipitate, do not dry for a long time, add 10 μL of deionized water to re-precipitate.

9 Transform S. cerevisiae and coat the corresponding plates.

10 After growing out of the single bacteria, the medium with the corresponding screening conditions was inoculated, and the plasmid was extracted overnight and transformed into E. coli competent cells for amplification of the target plasmid. The plasmid was extracted and verified by enzyme digestion.

(5) The plasmid pZY141 overexpressing tHMG1 was linearized and integrated into the Gal1710 locus of S. cerevisiae 30000B by homologous recombination to obtain the mutant strain S. cerevisiae YZL141, thereby constructing a high-yielding terpenoid S. cerevisiae. The platform provides a solid foundation for the efficient synthesis of terpenoids in S. cerevisiae.

3.2 Construction of mutant S. cerevisiae T16

(1) using pGB152 as a template, and amplifying the primer P3/P4 to obtain a J1-018-A fragment;

The ERG20 fragment was obtained by PCR amplification using S. cerevisiae 30000B as a template and P7/P8 as a primer.

Using S. cerevisiae 30000B as a template, primers P5/P6 were amplified to obtain the promoter GAL1-GAL10;

Using pRS426 as a template, primers P9/P10 were amplified to obtain a plasmid backbone;

(2) The above four fragments were ligated by Gibson method to obtain plasmid pYeastJ1-018;

(3) The plasmid pYeast J1-018 was linearized with NotI and integrated into the His3 site of the S. cerevisiae YZ141 genome overexpressing tHMG1, and finally the mutations overexpressing tHMG1, ERG20 (FPPS) and J1-018-A were obtained. S. cerevisiae T16 for efficient synthesis of sesquiterpene compounds.

4. Fermentation and detection of J1-018-A mutant strain

The mutant strain S. cerevisiae T16 was picked and transferred to a PA bottle containing YPD medium, cultured overnight at 30 ° C, and transferred to YPD medium containing 1% galactose at a final concentration of 0.25 OD ₆₀₀ inoculum. Fermentation for 72 h. The product was detected by GC-MS and the cells and fermentation broth were collected. It was then extracted twice with an equal volume of n-hexane, and the methanol was reconstituted after distillation under reduced pressure (a small amount of DMSO was added to the solution before the addition of methanol) for product purification.

The results of GC-MS detection are shown in Fig. 2, wherein Fig. a is a schematic diagram of the product detection result of J1-018A in vitro reaction, and Fig. b is a schematic diagram of the detection result of the fermentation product of J1-018A in vivo, and the result shows that S. cerevisiae T16 mutant strain The product type of sesquiterpene is consistent with the results of in vitro reaction, and it is possible to synthesize eight sesquiterpene compounds. The product composition ratios of the in vitro reaction and the in vivo fermentation result are different, but the S. cerevisiae T16 mutant strain constructed in this example can efficiently synthesize the sesquiterpene product, thereby being able to isolate those products having a very low content. Thus, this example provides a necessary basis for verifying the biological function of the enzyme and excavating the product by using the metabolically engineered S. cerevisiae YZ141, a high-yield isoprene precursor material.

Example 2

In this example, the J1-018-A fermentation product obtained in the first embodiment was purified and identified as follows:

1. Purification of S. cerevisiae T16 mutant product

The purified mobile phase A was ultrapure water, the mobile phase B was acetonitrile, and the ultraviolet absorption wavelength was 210 nm. The J1-018-A fermentation product was purified by semi-preparative HPLC according to the conditions described in Table 1, and the target product was determined by GC-MS. On the peak time on HPLC, the fractions containing the sesquiterpene product F1-F7 were collected, wherein F1-F4 was the main sesquiterpene component, and the results are shown in Fig. 3.

Table 1 Purification conditions of compounds in S. cerevisiae T16 mutant strain

The analysis results show that the purity of the compound 1 in the component F4, the compound 6 in the component F6 and the compound 8 in the component F7 can be directly used for the NMR detection without further purification, and the structures of the compounds 1-8 are shown in Table 2. .

Table 2 8 sesquiterpene compounds in the J1-018-A fermentation product

2. Purification of compound 2

The target compound 2 was purified from the component F1 by semi-preparative high performance liquid chromatography with a mobile phase of 75% acetonitrile at a flow rate of 0.75 mL/min. The results are shown in FIG.

3. Purification of compound 3

The target compound 3 was purified from the component F2 by semi-preparative high performance liquid chromatography with a mobile phase of 70% acetonitrile at a flow rate of 2 mL/min. The results are shown in FIG.

4. Purification of compound 4

The target compound 4 was purified from the component F2 by semi-preparative high performance liquid chromatography with a mobile phase of 80% acetonitrile at a flow rate of 0.6 mL/min. The results are shown in FIG.

5. Purification of compound 5

The target compound 5 was purified from the component F3 by semi-preparative high performance liquid chromatography with a mobile phase of 92% methanol at a flow rate of 0.6 mL/min. The results are shown in Fig. 7.

6. Purification of compound 7

The target compound 7 was purified from the component F5 by semi-preparative high performance liquid chromatography with a mobile phase of 96% acetonitrile at a flow rate of 0.6 mL/min. The results are shown in FIG.

7. Summary of purification results

The results of GC-MS analysis showed that the fermentation product of the mutant S. cerevisiae T16 contained 8 sesquiterpene compounds. Subsequently, the above 8 compounds were separated and concentrated under reduced pressure and identified by NMR techniques.

(1) Compound 1:

^{The 1} H NMR data is shown in Table 1 and Figure 9a, showing the presence of two single-peak methyl signals for compound 1 (Me-12, Me-13) and two bimodal methyl signals (Me-14, Me- 15), and 1 enemethyl signal (H-4). ¹³ C NMR and HMQC spectral data show that compound 1 contains 15 carbon atoms, one of which has a sp ³ hybrid quaternary carbon atom (C-10), and three sp ² hybridized quaternary carbon atoms (C-3, C -6, C-7), 1 methine group, 1 aliphatic methine group, 5 aliphatic methylene groups, and 4 methyl groups (Fig. 9b, Fig. 9c). These data show that Compound 1 is a bicyclic structure. The coupling relationship of ¹ H- ¹ H COSY is: H-1/H-2, H-4/H-5, H-8/H-9 and H-14/H-11/H-15 (Fig. 9d) ). The HMBC map shows that the signals related to methyl hydrogen are Me-12 and C-2, C-3 and C-4; Me-13 and C-6, C-7 and C-8; Me-14 and C- 10, C-11 and C-15; M-15 with C-10, C-11 and C-14. In addition, the HMBC map suggests H-1 and C-3, C-6, C-10; H-5 and C-3, C-4, C-6, C-7, C-10; H-9 and C. There is a coupling relationship between -10 and C-11 (Fig. 9e). Therefore, the planar structure of Compound 1 is a 5-7 membered bicyclic sesquiterpene, as shown in Figure 9f.

Table 1. NMR data for Compound 1

Compound 2:

¹ H NMR data showed 1 singlet methyl (Me-12), 3 bimodal methyl signals (Me-13, Me-14 and Me-15) (Table 2 and Figure 10a). ¹³ C NMR spectroscopy and HSQC spectroscopy confirmed that compound 2 has 15 carbon atoms, including 3 sp ³ hybrid quaternary carbons (C-4, C-6, C-10), 3 aliphatic methine groups, 5 Aliphatic methylene and 4 methyl groups (Fig. 10b, Fig. 10c). These data show the tricyclic skeleton of Compound 2. The coupling relationship of ¹ H- ¹ H COSY suggests H-1/H-2, H-4/H-5, H-8/H-7/H13 and H-14/H-11/H-15 (below Figure, Figure 10d). In addition, the HMBC map can see that the related signals of methyl hydrogen are Me-12 and C-2; C-3 and C-4; Me-13 and C-6; C-7 and C-8; Me-14 and C-10, C-11 and C-15; M-15 and C-10, C-11 and C-14. In addition, the HMBC map can see that the related signals of methyl hydrogen are: H-11 and C-1, C-9, H-7 and C-4, C-5, H-5 and C-6. Between C-7, between H-8 and C-9, C-10 (Fig. 10e). Therefore, the planar structure of Compound 2 was identified as a 5-6-3 membered tricyclic sesquiterpene. The results of NOESY analysis showed that there is a coupling relationship between H-5/H-11, H7/Me-14, Me-12 and H-4/H-1α (Fig. 10f, Fig. 10g), and compound 2 can be further determined. The relative configuration is 3R*, 4S*, 6S*, 7R*, 10R*.

Table 2 NMR data of Compound 2

Compound 3:

^{The 1} H NMR data showed a singlet methyl group (Me-12) and three bimodal methyl signals (Me-13, Me-14, Me-15) (Table 3 and Figure 11a). ¹³ C NMR, DEPT135 and HSQC spectra confirmed that compound 2 has 15 carbon atoms, including 3 sp3 hybrid quaternary carbons (C-4, C-6, C-10), 3 aliphatic methine groups, 5 aliphatic methylene groups and 4 methyl groups (Fig. 11b, Fig. 11c). These data show the tricyclic skeleton of Compound 3. The coupling relationship of ¹ H- ¹ H COSY suggests H-1/H-2, H-4/H-5, H-8/H-7/H13 and H-14/H-11/H-15 (Fig. 11d). In addition, the HMBC map can be seen that the signals related to methyl hydrogen are Me-12 and C-2, C-3 and C-4; Me-13 and C-6, C-7 and C-8; Me-14 and C-10, C-11 and C-15; M-15 with C-10C-11 and C-14. HMBC spectra indicate between H-2 and C-10, between H-5 and C-6, between C-7 and C-10, between H-7 and C-10, and between H-8 and C-9. Correlation between C-10 (Fig. 11e). Therefore, the planar structure of Compound 3 was identified as a 5-6-3 membered tricyclic sesquiterpene. The results of NOESY analysis showed that there was a coupling relationship between H-5b/H-11/Me-12/Me-14, H-7/Me-14, Me-13 and H-4 (Fig. 11f, 11g). It can be further determined that the relative configuration of Compound 2 is 3S*, 4S*, 6S*, 7R*, 10R*.

Table 3. NMR data for compound 3

Compound 4:

According to the GC-MS retention time (11.11 min) and the fragment ions compared with the standard library, compound 4 is a linear trans-nerolidol, and the nuclear magnetic results show ¹ H NMR (400 MHz, chloroform-d) δ 5. 91 (dd, 17.3, 10.8, 1H), 5.21 (dd, 17.3, 1.3, 1H), 5.13 (t, 5.8, 1H), 5.10–5.05 (m, 1H), 5.06 (dd, 10.8, 1.3, 1H) , 2.11–2.00 (m, 4H), 1.99–1.95 (m, 2H), 1.67 (s, 3H), 1.59 (s, 6H), 1.58 (m, 2H), 1.27 (s, 3H) (Fig. 12a) . ¹³ C NMR (101 MHz, CDCl ₃ ) δ 145.02, 135.60, 131.46, 124.20, 124.17, 111.66, 73.52, 42.01, 39.69, 27.90, 26.62, 25.70, 22.71, 17.69, 16.01 (Fig. 12b). The theoretical m/z value for C ₁₅ H ₂₅ [M-OH] ⁺ is 205.1951; the actual value of m/z (HRMS, ESI) is 205.1939.

Compound 5:

It is an acorenol. ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.45-5.39 (m, 1H), 2.40-2.30 (m, 1H), 2.03-1.90 (m, 3H), 1.89-1.85 (m, 2H), 1.85-1.80 (m,1H),1.80-1.70 (m,1H),1.67-1.64 (m,3H),1.55-1.45 (m,2H),1.43-1.35 (m,1H),1.26-1.24 (m,1H) , 1.21 (s, 3H), 1.21 (s, 3H), 0.85 (d, 6.8, 3H) (Fig. 13a). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 135.24, 121.22, 73.79, 54.77, 45.01, 41.76, 31.53, 30.64, 30.19, 29.08, 27.92, 27.86, 26.11, 23.34, 14.94 (Fig. 13b). This result is consistent with the literature reported by the Zhangpu alcohol nuclear magnetic data (Brock et al., 2013, Braun et al., 2003).

Compound 6:

Compound 6 was (E)-β-farnesene ((E)-β-farnesene) with a retention time of 9.72 min on GC-MS. ^{_{1 H NMR (400MHz, CDCl 3}} ) δ6.38 (dd, 17.6,10.8,1H), 5.25 (dd, 17.6,1.1,1H), 5.19-5.08 (m, 2H), 5.08-4.98 (m, 3H) , 2.28–2.15 (m, 3H), 2.12–1.96 (m, 5H), 1.68 (d, 1.5, 3H), 1.60 (d, 1.5, 6H) (Fig. 14a). 13C NMR (101MHz, CDCl3) δ 146.09, 138.97, 135.37, 131.31, 124.32, 123.99, 115.73, 113.04, 39.69, 31.37, 26.69, 26.58, 25.70, 17.69, 16.02 (Fig. 14b). This result is consistent with the (E)-β-farnesol nuclear magnetic data reported by Simionatto (Simionatto et al., 2007).

Compound 7:

Compound 7 was alpha-bisabolol with a retention time of 12.72 min on GC-MS. ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.42 - 5.31 (m, 1H), 5.12 (m, 1H), 1.67 (s, 3H), 1.64 (s, 3H), 1.61 (s, 3H), 1.09 ( s, 3H) (Fig. 15a). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 134.10, 131.70, 124.47, 120.45, 74.28, 42.86, 40.03, 30.95, 26.86, 25.66, 23.31, 23.21, 23.14, 21.99, 17.62 (Fig. 15b ). This result is consistent with the alpha-parmusol nuclear magnetic data reported by Miyazawa (Miyazawa et al., 1995). Compound 8:

¹ H NMR data showed that compound 8 contained one singlet methyl group (Me-12), three bimodal methyl groups (Me-13, Me-14, Me-15) and one ene methine group (H- 2) (Table 4 and Figure 16a). ¹³ C NMR and HSQC spectra confirmed that compound 8 contains 15 carbon atoms, including one sp3 hybrid quaternary carbon (C-6) and three sp ² hybrid quaternary carbons (C-3, C-9, C-10). 2 aliphatic and 2 methine groups, 4 aliphatic methylene groups, 4 methyl groups (Fig. 16b, Fig. 16c). These data show that compound 8 contains a bicyclic skeleton. The coupling relationship of ¹ H-1H COSY suggests H-1/H-2, H-4/H-5, H-13/H-7/H-8/H-9 and H-14/H-11/ H-15 (Fig. 16d). In addition, the HMBC map can see that the related signals of methyl hydrogen are Me-12 and C-2, C-3, C-4; Me-13 and C-6, C-7, C-8; Me-14 and C-10, C-11, C-15; M-15 and C-10, C-11, C-14. HMBC spectroscopy indicated a correlation between H-5 and C-1, C-6 and C-7; H-9 with C-6 and C-7; H-11 and C-9 (Fig. 16e). Thus, as shown in Figure 16f, the planar structure of Compound 8 was identified as a 5-6 membered bicyclic sesquiterpene. This result is consistent with the acoradiene results reported by Marx et al., which we named acoradiene J1 (Marx and Norman, 1975).

Table 4 NMR data for compound 8

In summary, Compound 1 (retention time is 9.65 min) and Compound 2 (retention time 11.64 min) are new backbone compounds. Compound 3 (retention time 11.27 min) is the cis-trans isomer of Compound 2. Two of the compounds were identified by GC-MS versus standard retention time and fragment ion peak alignment. The compound 4 in which the retention time is 11.11 min is nerolidol, which is widely used in industries such as flavoring and perfume production. At the same time, the compound enhances the skin's ability to absorb the drugs that are treated by skin penetration. Compound 5 with a retention time of 12.23 min is acorenol, and its biological function needs further research and development. Compound 6 (retention time 9.72 min) is farnesene which can be used as a novel aviation fuel. Compound 7 (retention time 12.72 min) is a bisabolol with antibacterial, anti-inflammatory, anti-microbial, and biological effects of treating psoriasis. It is widely used in the skin protection and skin care cosmetics manufacturing industry (Kim et al., 2016). ). Compound 8 (retention time 9.42 min) was Acoradiene and had a similar structure to Compound 5.

The compound 1-3 isolated above was subjected to nuclear magnetic identification, and finally its chemical structure was as shown in the compound 1-3 in Table 2, and named as fusarium diene, trans-grain Trans-fusagramineol and fusagramineol, the corresponding gene J1-018-A is named fusariumdiene synthase (FgFS).

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

While the embodiments of the present invention have been shown and described, the embodiments of the invention may The scope of the invention is defined by the claims and their equivalents.

Claims

An isolated polypeptide, characterized in that the isolated polypeptide has the amino acid sequence set forth in SEQ ID NO: 1.
An isolated nucleic acid, characterized in that the isolated nucleic acid is a sequence selected from the group consisting of:

(1) a nucleotide sequence encoding the isolated polypeptide of claim 1;

(2) Having the nucleotide sequence shown by SEQ ID NO: 2.
A vector comprising the isolated nucleic acid of claim 2.
A recombinant cell comprising the polypeptide of claim 1, the isolated nucleic acid of claim 2, or the vector of claim 3.
The recombinant cell according to claim 4, wherein the host cell of the recombinant cell is yeast, Escherichia coli, Bacillus subtilis or fungus.
The isolated polypeptide of claim 1, the isolated nucleic acid of claim 2, the vector of claim 3 or the recombinant cell of claim 4 or 5 for catalyzing the synthesis of a steroid or for the preparation of a catalysis The use of a catalyst for the synthesis of hydrazines.
The use according to claim 6, characterized in that the moss is sesquiterpene.
The use according to claim 7, wherein the quinone is at least one selected from the group consisting of the following.
A method for preparing a quinone, the method comprising the polypeptide of claim 1, the isolated nucleic acid of claim 2, the vector of claim 3, or the method of claim 4 or 5. The recombinant cells are carried out under the conditions present.
The method of claim 9 wherein said moss is sesquiterpene.
The method according to claim 10, wherein the steroid is at least one selected from the group consisting of the following.
The method according to claim 9, wherein the substrate of the method contains at least one of geranyl pyrophosphate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, and the product contains 2 At least one of a monoterpene of an isoprene unit, a sesquiterpene of three isoprene units, and a diterpene of four isoprene units.