Sabinene synthase, gene and application of sabinene synthase

Abstract

The invention discloses a novel sabinene synthetase, a gene and an application of the sabinene synthetase, and belongs to the technical field of biology. According to the invention, it is found for the first time that a nucleotide sequence and a coding amino acid sequence of the HbSaS gene derived from hevea brasiliensis or a plasmid containing the nucleotide sequence and the coding amino acid sequence can be used for synthesizing sabinene. The synthesized sabinene can be used for synthesizing and blending flavors, fragrances and the like, and meanwhile, the sabinene can be applied to the pharmaceutical industry due to good anti-inflammatory and antifungal activity. According to the invention, a recombinant vector and an engineering bacterium are obtained by recombining a rubber tree synthetase gene for synthesizing sabinene, a sabinene high-yield strain is successfully constructed, and the sabinene shake flask yield reaches 8mg / L. The invention provides a new synthetase gene element for biotechnology synthesis of sabinene, is beneficial to industrial application of biotechnology production of sabinene, lays a good foundation for development and utilization of rubber tree gene resources, and provides a new application strategy.

Description

Technical Field

[0001] The present invention belongs to the field of natural product biotechnology, and specifically relates to sabinene synthase genes, constructs and their applications. Background Art

[0002] Sabinene belongs to monoterpenoids, with the molecular formula C 10 H 16 , a molecular weight of 136.23, and exists in the volatile oils of various plants such as Salvia miltiorrhiza, Juniperus communis, Citrus limon, Origanum majorana, etc., endowing these plants with unique flavors, scents, medicinal physiological activities, etc. Sabinene can be used in the raw material development of the perfume and flavor industry and the formulation of high-grade perfumes and flavors; at the same time, due to its good anti-inflammatory and antifungal activities, it is also widely used in the pharmaceutical industry; due to its complex bicyclic structure, it has the advantages of high energy density, low freezing point and high flash point, and can also be used as a raw material for high-grade biofuels, with important economic value. Currently, sabinene mainly exists in a mixture in plant volatile oils. Due to its low content, it is difficult and costly to obtain by rectification, which is not conducive to the large-scale production of sabinene. The chemical synthesis process of sabinene is complex and has a negative impact on the environment. So far, due to the lack of a suitable manufacturing process, sabinene has not been commercially produced. The biosynthesis of sabinene using biotechnology has broad prospects, but currently the microbial synthesis yield of monoterpenoids is generally low. A main reason is the lack of efficient monoterpene synthases. Exploring new sabinene synthases is of great research significance for its biosynthesis and has further development value.

[0003] Currently, only several sabinene synthases have been cloned and identified from plants, and there are not many available sabinene synthase gene elements to choose from. There is no literature report on the sabinene synthase gene that has been functionally confirmed in Hevea brasiliensis. Identifying the sabinene synthase in Hevea brasiliensis through gene mining and functional characterization has important value for broadening the research and application of other small molecule compounds in Hevea brasiliensis besides natural rubber synthesis.

[0004] The present invention first clones the sabinene synthase gene from Hevea brasiliensis for the first time, which can be used for the efficient synthesis of sabinene, and determines its nucleotide sequence and amino acid sequence, filling the blank of the sabinene synthesis gene in Hevea brasiliensis in the prior art, providing a brand-new available gene element for the biosynthesis of sabinene, and being conducive to the research and application of the biosynthesis of sabinene using biotechnology. Summary of the Invention

[0005] The purpose of the present invention is, in view of the above deficiencies of the prior art, the present invention first provides an enzyme, which is the enzyme of the following a) or b):

[0006] a) An enzyme with an amino acid sequence of SEQ ID No.2;

[0007] b) A protein with rubber tree sabinene synthase activity obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No.2.

[0008] Secondly, the present invention also provides biomaterials related to the rubber tree sabinene synthase, which are any one of the following B1) to B8):

[0009] B1) A nucleic acid molecule encoding rubber tree sabinene synthase;

[0010] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0011] B3) A recombinant vector containing the nucleic acid molecule described in B1),

[0012] B4) A recombinant vector containing the expression cassette described in B2);

[0013] B5) A recombinant microorganism containing the nucleic acid molecule described in B1);

[0014] B6) A recombinant microorganism containing the expression cassette described in B2);

[0015] B7) A recombinant microorganism containing the recombinant vector described in B3);

[0016] B8) A recombinant microorganism containing the recombinant vector described in B4);

[0017] Optionally, the vector includes a pESC yeast expression vector and a transgenic plant expression vector;

[0018] Optionally, the recombinant microorganism includes an engineered bacterium or a plant cell.

[0019] The recombinant vector and the engineered bacterium provided by the present invention can be directly cultured, amplified, and expressed to obtain rubber tree β - elemene synthase, and the large amount of active rubber tree sabinene synthase obtained can be used for the production of sabinene.

[0020] Preferably, the nucleic acid molecule is the nucleic acid molecule shown in any one of the following 1) or 2) or 3) or 4):

[0021] 1) A DNA molecule or cDNA molecule whose coding sequence is the DNA molecule in SEQ ID No.1 in the sequence listing;

[0022] 2) A DNA molecule whose nucleic acid sequence is the DNA molecule in SEQ ID No.1 in the sequence listing;

[0023] 3) A cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the enzyme described in claim 1;

[0024] 4) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the enzyme recited in claim 1.

[0025] Secondly, the present invention also provides the use of the sabinene synthase of Hevea brasiliensis in the preparation of sabinene.

[0026] The present invention also provides the use of the above biological materials in the preparation of the sabinene synthase of Hevea brasiliensis, or the construction of transgenic plants, or the production of sabinene.

[0027] In addition, the present invention also provides a primer pair for amplifying a nucleic acid molecule fragment encoding the sabinene synthase of Hevea brasiliensis;

[0028] Preferably, the amplification primer pair is: the forward primer is as shown in SEQ ID NO.3, and the reverse primer is as shown in SEQ ID NO.4.

[0029] The present invention also provides a method for producing sabinene, which uses the above-mentioned sabinene synthase of Hevea brasiliensis to catalyze geranyl pyrophosphate (GPP) to obtain sabinene.

[0030] The present invention also provides the use of the sabinene synthase of Hevea brasiliensis or the above biological materials in the raw material development of essence and fragrance or the formulation of high-grade essence and fragrance.

[0031] Finally, the present invention also provides a biosynthetic product for the development or formulation of essence and fragrance, and the product comprises the sabinene synthase of Hevea brasiliensis or the above biological materials.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] (1) The present invention has first discovered a synthase gene derived from Hevea brasiliensis that can efficiently synthesize sabinene. This gene can synthesize sabinene, has good enzyme specificity, and the target product has high purity, and can be used as an aromatic compound in the essence and fragrance industry and the pharmaceutical industry.

[0034] (2) The present invention has obtained a recombinant vector and an engineered bacterium by recombining the sabinene synthase gene of Hevea brasiliensis that synthesizes sabinene, and has successfully constructed a sabinene-producing strain with a shake flask yield of up to 8 mg / L, laying an important foundation for the industrial application of sabinene. Description of the Drawings

[0035] Figure 1 is the GC-MS detection result of the fermentation product of the engineered strain YHbSAS constructed by the present invention. Detailed Embodiments

[0036] To make the objectives, technical solutions and advantages of the present invention more clear, the following further describes in detail the specific embodiments of the present invention in conjunction with specific embodiments and the accompanying drawings. For those not specifying specific technologies or conditions in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0037] The DNA polymerase, restriction endonuclease and plasmid extraction kit were all purchased from Takara Biotechnology (Beijing) Co., Ltd.; the RNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the DNA gel extraction kit and homologous recombination kit were purchased from Nanjing Novozymes Biotechnology Co., Ltd.; the pTOPO-Blunt Simple vector was purchased from Beijing Aidlab Biotechnologies Co., Ltd., and the yeast expression plasmid pESC was purchased from Novagen; the specific gene primer pair P1 / P2 was synthesized by Wuhan Kingcare Bioengineering Co., Ltd.

[0038] Detection method of sabinene: GC-MS detection was carried out using the Thermo TRACE GC Ultra system and the TSQ 9000 system. The GC detection program was set as follows: the initial oven temperature was 50°C and lasted for 1 minute; then it was increased to 280°C at a rate of 15°C / minute and held for 1 minute; then it was increased to 300°C at a rate of 20°C / minute and held for 2 minutes. Volatile samples were injected at 240°C, and the MS transfer temperature was maintained at 270°C. Compounds were determined by comparing with standards or by comparing with the NIST (National Institute of Standards and Technology) database and retention index. The standard sabinene was purchased from Shanghai TargetMol Co., Ltd., and the CAS number was 3387-41-5.

[0039] YPD medium: The medium components contained 2% glucose (Sinopharm), 2% tryptone (Angel Yeast), and 1% yeast extract (Angel Yeast);

[0040] The yeast strain YZL141, the construction method of which is recorded in Shi Bin et al., “Systematic Metabolic Engineering of Saccharomyces cerevisiae for Lycopene Overproduction.” Journal of agricultural and food chemistry vol.67, 40(2019): 11148-11157. doi:10.1021 / acs.jafc.9b04519.

[0041] Rubber tree cDNA: RNA extracted from rubber tree latex is reverse transcribed into cDNA. This process is generally carried out according to conventional conditions, such as those described in the "Molecular Cloning Laboratory Manual (4th Edition)" (Chinese version) published by Science Press, or according to the conditions recommended by the manufacturer. This is an experimental procedure well known to those skilled in the art and is not limited to this invention.

[0042] The nucleotide sequence of the rubber tree sabinene synthase gene is shown in SEQ ID NO.1.

[0043] The amino acid sequence of the rubber tree sabinene synthase provided by the present invention is shown in SEQ ID NO.2.

[0044] Application of rubber tree sabinene synthase in the production of sabinene. The rubber tree sabinene synthase can catalyze the synthesis of sabinene using geranyl pyrophosphate as a substrate, and can be applied to the flavor and fragrance industry.

[0045] The present invention provides a method for producing sabinene, which can utilize the rubber tree sabinene synthase of the present invention to catalyze farnesyl pyrophosphate to obtain sabinene.

[0046] The present invention also provides a recombinant vector containing a rubber tree sabinene synthase gene, which is recombined into an expression vector to construct a biosynthesis module. This allows for the convenient and rapid utilization of the rubber tree sabinene synthase gene, allowing for rapid and large-scale production of target genes or target proteins. In the present invention, the expression vector is preferably a pESC yeast expression vector.

[0047] The present invention also provides an engineered bacterium containing a rubber tree sabinene synthase gene. The engineered bacterium comprises the rubber tree sabinene synthase gene and a host cell. The nucleotide sequence of the rubber tree sabinene synthase gene is shown in SEQ ID NO.1.

[0048] By introducing the rubber tree sabinene synthase gene into a host cell, the target gene and target protease can be rapidly and abundantly obtained. The biosynthetic module composed of the engineered bacteria avoids the tedious operation of PCR amplification from the rubber tree genome when using the target gene. The host cell is preferably the yeast strain YZL141.

[0049] The present invention also provides plant cells or transgenic plants introduced with the aforementioned rubber tree sabinene synthase gene. The nucleotide sequence of the rubber tree sabinene synthase gene is shown in SEQ ID NO. 1. Using methods well known to those skilled in the art, a plant expression vector is constructed using the rubber tree gene and introduced into plant cells or callus tissue via Agrobacterium, protoplast generation, or a gene gun to regenerate transgenic plants that synthesize and produce sabinene.

[0050] Example 1

[0051] (1) Gene cloning

[0052] The applicant analyzed the genome of rubber trees and discovered a rubber tree monoterpene sabinene synthase gene, whose amino acid sequence is shown in SEQ ID NO.1 and whose nucleotide sequence is shown in SEQ ID NO.2.

[0053] The rubber tree latex samples were collected from the Reyan 7-33-97 clone that was routinely tapped at the experimental farm of the Chinese Academy of Tropical Agricultural Sciences in Hainan Province. Total RNA was extracted using the Total RNA Extraction Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd., and then cDNA was synthesized using the Fermentas cDNA First Strand Reverse Transcription Kit. Gene cloning was performed from the latex cDNA template using RT-PCR with the primer pair P1-P2 (see Table 1). The PCR reaction system used was (30 μL): 12.5 μL of water, 10 μL of 2x PrimerSTARMax Premix, 1 μL each of the forward and reverse primers (10 μM), and 0.5 μL of cDNA template. The PCR reaction conditions were: 3 min at 95 °C, 10 s at 98 °C, 20 s at 58 °C, 2 min at 72 °C, for 35 cycles, 10 min at 72 °C, and hold at 16 °C. Then, the DNA fragment was recovered using the Gel Extraction Kit from Nanjing Novizan Biotech Co., Ltd., ligated onto the pTOPO-Blunt Simple vector, transformed into DH5α Escherichia coli competent cells for ampicillin resistance screening, and after verification by colony PCR, it was sent to a sequencing company for sequencing confirmation. After sequencing confirmation, the target gene was obtained.

[0054] Table 1

[0055]

[0056] (2) Construction of recombinant expression vector

[0057] After re-PCR cloning and gel recovery of the target gene vector with correct sequencing, it was ligated with the yeast linearized expression vector using the homologous recombination kit from Nanjing Novizan Biotech Co., Ltd. (37 °C, 30 min), transformed into DH5α Escherichia coli competent cells for screening, and after verification by colony PCR, the yeast expression vector containing the correct target gene was obtained and named pHbSAS1.

[0058] (3) Construction of engineering strains and functional characterization

[0059] ① Preparation of yeast competent cells

[0060] The yeast YZL141 strain was cultured overnight and then transferred to 50 mL of fresh YPD medium and cultured at 30 °C until the OD600 was about 0.6. After centrifugation at 500 g for 5 min, it was suspended in TE / LiAC solution, and after high-speed centrifugation for 5 min, the cell pellet was resuspended in 1 mL of TE / LiAC solution to prepare the competent cells.

[0061] ② Yeast competent cell transformation

[0062] The above expression vector pHbSaS1 was transformed into YZL141 yeast competent cells by the PEG-LiAc transformation method, placed at 30 °C for 30 min, heat-shocked at 42 °C for 10 min, centrifuged for 5 min, and then spread on an SD-URA screening plate. After static culture at 30 °C for 3 d, colony PCR verification was performed using primer pairs P1-P2 respectively. The positive bacteria transformed with YZL141 were named YHbSAS.

[0063] ③ Functional characterization

[0064] The cryopreserved YHbSAS strain was inoculated into 50 mL of SD-URA defective medium (added with 1% galactose), cultured at 30 °C for 3 d, centrifuged at 4000 rpm for 5 min to collect the cells, and after extraction, transferred to a sample bottle for GC-MS detection. The detection results of the YHbSAS strain are as Figure 1 shown. The main product peak was identified as sabinene by comparing the retention time and fragments with the sabinene standard. The rubber tree-derived coding gene contained in this YHbSAS strain was named HbSaS.

[0065] ④ Fermentation evaluation

[0066] After determining the gene function, the YHbSAS strain was inoculated into 50 mL of YPD medium in a similar method, added with 1% galactose and 1% IPM, cultured at 30 °C for 3 d, centrifuged at 4000 rpm for 8 min to collect the organic phase, and the detection sample was prepared for GC-MS detection and quantification. The shake flask yield of YHbSAS was 8 mg / L.

[0067] Example 2

[0068] To evaluate the potential value of sabinene as a fragrance and flavoring agent, a perfumer analyzed the fragrance of this molecule. Sabinene has a woody, fresh pine fragrance, and an aroma that provides respiratory comfort. When sabinene is heated, white smoke is produced along with an enhanced odor, indicating its potential as a component for fragrance formulation and its application in products such as e-cigarettes.

[0069] Without conflict, the above embodiments and the features in the embodiments in this article can be combined with each other.

[0070] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An enzyme, which is the enzyme of a) or b) below: a) An enzyme with an amino acid sequence of SEQ ID No. 2; b) A protein with the activity of heptyl synthase of rubber tree, which is obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID No.

2.

2. A biological material related to the enzyme described in claim 1, which is any one of the following B1) to B8): B1) A nucleic acid molecule encoding the enzyme described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), B4) A recombinant vector containing the expression cassette described in B2); B5) A recombinant microorganism containing the nucleic acid molecule described in B1); B6) A recombinant microorganism containing the expression cassette described in B2); B7) A recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4); Optionally, the vector includes a pESC yeast expression vector and a transgenic plant expression vector; Optionally, the recombinant microorganism includes an engineered bacterium or a plant cell.

3. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule is the nucleic acid molecule shown in any one of the following 1) or 2) or 3) or 4): 1) A DNA molecule or cDNA molecule with a coding sequence of SEQ ID No. 1 in the sequence listing; 2) A DNA molecule with a nucleic acid sequence of SEQ ID No. 1 in the sequence listing; 3) A cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the enzyme described in claim 1; 4) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the enzyme described in claim 1.

4. Use of the enzyme described in claim 1 in the preparation of heptyl.

5. Use of the biological material described in claim 2 in the preparation of heptyl synthase of rubber tree or the construction of transgenic plants or the production of heptyl.

6. A primer pair for amplifying a nucleic acid molecule fragment encoding the enzyme described in claim 1.

7. The primer pair according to claim 6, wherein The amplification primer pair is: the forward primer is as shown in SEQ ID NO. 3, and the reverse primer is as shown in SEQ ID NO.

4.

8. A production method of sabinene, characterized in that, Using the heptyl synthase of rubber tree described in claim 1 to catalyze geranyl pyrophosphate (GPP) to obtain heptyl.

9. Use of the enzyme described in claim 1 or the biological material described in claim 2 in the development of raw materials for essence and fragrance or the formulation of high-grade essence and fragrance.

10. A biosynthetic product for the development or formulation of essence and fragrance, which includes the enzyme described in claim 1 or the biological material described in claim 2.

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