CN117467626A - Beta-olivine synthetase, gene and application thereof - Google Patents
Beta-olivine synthetase, gene and application thereof Download PDFInfo
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- CN117467626A CN117467626A CN202311421193.3A CN202311421193A CN117467626A CN 117467626 A CN117467626 A CN 117467626A CN 202311421193 A CN202311421193 A CN 202311421193A CN 117467626 A CN117467626 A CN 117467626A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
The invention discloses a novel beta-olivine (beta-maleiene) synthetase, a gene and application thereof, belonging to the technical field of biology. The invention discovers for the first time that the nucleotide sequence and the coding amino acid sequence of HbMaS gene derived from rubber tree Hevea brasiliensis or the plasmid containing the nucleotide sequence and the coding amino acid sequence can be used for synthesizing beta-olivine, and can be used for synthesizing and preparing essence, spice and the like. The invention obtains recombinant vector and engineering bacteria by recombining the rubber tree synthetase gene for synthesizing beta-olivine, and successfully constructs beta-olivine high-yield strain, the beta-olivine shake flask yield is as high as 10mg/L, which lays an important foundation for the industrial application of beta-olivine for the highest yield reported at present. The invention lays a good foundation for developing and utilizing the rubber tree gene resources and provides a new application strategy.
Description
Technical Field
The invention belongs to the technical field of natural product biology, and particularly relates to a beta-olivine synthase gene, a construct and application thereof.
Background
The terpenoid is a huge natural product family formed by polymerizing isoprene structural units singly or in a plurality of ways, and certain terpenoids have better physiological activity and very important application value. The research and excavation of new synthetase gene resources of terpenoids have important significance for the biotechnological synthesis of terpenoids.
Beta-olivine belongs to sesquiterpene compounds, and exists in volatile oil of various plants such as artemisia annua, mugwort, ginseng, nardostachys root and the like, so that the special flavor, fragrance, medicinal physiological activity and the like of the plants are endowed. The beta-olivine can also be used for raw material development and high-grade essence and spice preparation in the essence and spice industry, and has important economic value. The beta-olivine is mainly mixed in the plant volatile oil at present, and because the content is low, the rectification obtaining difficulty and the cost are high, the research on synthesizing the beta-olivine by using the biotechnology is less, and the novel synthetase for excavating the beta-olivine has important research significance on the biosynthesis of the beta-olivine and has further development value.
The beta-olivine synthetase gene with function confirmation in the rubber tree is not reported in the literature, and the identification of the rubber tree beta-olivine (beta-maleiene) synthetase by gene excavation and function characterization has important value for the research and application of widening other small molecular compounds except natural rubber synthesis of the rubber tree.
The beta-olivine synthetase gene is cloned from the rubber tree for the first time, can be used for efficiently synthesizing the beta-olivine, determines the nucleotide sequence and the amino acid sequence of the beta-olivine, fills the blank of the beta-olivine synthesized gene in the rubber tree in the prior art, and provides a gene element for the biotechnological synthesis of the beta-olivine.
Disclosure of Invention
The object of the present invention is to address the above-mentioned disadvantages of the prior art by first providing an enzyme which is either a) or b) as follows:
a) An enzyme having the amino acid sequence of SEQ ID No. 2;
b) The protein with rubber tree beta-olivine synthetase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2.
Secondly, the present invention also provides a biological material related to the rubber tree beta-olivine synthase, which is any one of the following B1) to B8):
b1 A nucleic acid molecule encoding a rubber tree beta-olivine synthase;
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);
optionally, the vector comprises a pESC yeast expression vector and a transgenic plant expression vector;
optionally, the recombinant microorganism comprises an engineered bacterium or a plant cell.
The recombinant vector and engineering bacteria provided by the invention can be directly cultured, amplified and expressed to obtain the rubber tree beta-olivine synthetase, and a large amount of obtained active rubber tree beta-olivine synthetase can be used for producing beta-olivine.
Preferably, the nucleic acid molecule is a nucleic acid molecule as set forth in 1) or 2) or 3) or 4) or 5) below:
1) The coding sequence is a DNA molecule or a cDNA molecule of SEQ ID No.1 in a sequence table;
2) The nucleic acid sequence is a DNA molecule of SEQ ID No.1 in a sequence table;
3) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) or 2) and encoding the enzyme of claim 1;
4) Hybridizing under stringent conditions to the nucleotide sequence defined in 1) or 2) and encoding a cDNA molecule or a genomic DNA molecule of the enzyme according to claim 1.
Secondly, the invention also provides application of the rubber tree beta-olivine synthetase in preparing beta-olivine.
The invention also provides application of the biological material in preparing rubber tree beta-olivine synthetase, constructing transgenic plant or producing beta-olivine.
In addition, the invention also provides a primer pair for amplifying the nucleic acid molecule fragment encoding the rubber tree beta-olivine synthetase;
preferably, the amplification primer pair is: the forward primer is shown as SEQ ID NO.3, and the reverse primer is shown as SEQ ID NO. 4.
The invention also provides a production method of the beta-olivine, which utilizes the rubber tree beta-olivine synthetase to catalyze farnesyl pyrophosphate to obtain the beta-olivine.
The invention also provides application of the rubber tree beta-olivine synthetase or the biological material in raw material development of essence and perfume or preparation of advanced essence and perfume.
Finally, the invention also provides a biosynthetic product for the development or formulation of fragrances and perfumes, said product comprising a rubber tree beta-olivine synthase or a biomaterial as described above.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The invention discovers the synthetase gene which is derived from rubber tree and can efficiently synthesize beta-olivine for the first time, and the synthetase gene can mainly synthesize beta-olivine, and can be used as a potential aromatic compound to be applied to the flavor and fragrance industry.
(2) According to the invention, the rubber tree beta-olivine synthetase gene for synthesizing the beta-olivine is recombined to obtain the recombined vector and engineering bacteria, and the successfully constructed beta-olivine production strain has a shake flask yield of up to 10mg/L, so that an important foundation is laid for the industrial application of the beta-olivine for the highest shake flask yield reported at present.
Drawings
FIG. 1 shows the GC-MS detection results of the engineering strain SRBT5 fermentation product constructed by the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
DNA polymerase, restriction endonuclease and plasmid extraction kit were all purchased from Bao Ri doctor materials technology (Beijing) Co., ltd; the RNA extraction kit was purchased from the company of the chemical technology of the root of heaven (beijing); DNA gel recovery kit and homologous recombination kit were purchased from Nanjing Norvigator Biotechnology Co., ltd; pTOPO-Blunt Simple vector was purchased from Beijing Edley Biotechnology Co., ltd, and yeast expression plasmid pESC was purchased from Novagen; specific gene primer pair P1/P2 was synthesized by Wohan Jin Kairui Bio-engineering Co.
The detection method of the beta-olivine comprises the following steps: GC-MS detection was performed using a Thermo TRACE GC Ultra system and a TSQ 9000 system. The GC detection procedure was set as follows: the initial oven temperature was 50 ℃ for 1 minute; then raising the temperature to 280 ℃ at a speed of 15 ℃/min and keeping the temperature for 1 min; then, the temperature was raised to 300℃at a rate of 20℃per minute, and the mixture was kept for 2 minutes. The volatile samples were injected at 240℃and the MS transfer temperature was maintained at 270 ℃. Compounds were determined by comparison of NIST (National Institute of Standards and Technology) database and retention index.
YPD medium: the medium composition contained 2% glucose (Country), 2% tryptone (Angel Yeast), 1% Yeast extract (Angel Yeast);
yeast strain YZL141, the construction method of which is described 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.
Yeast strain JCR27, the construction method of which is described in Siemon, thomas et al, "Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10 (14) -diene as Building Block." Journal of the American Chemical Society vol.142,6 (2020): 2760-2765.Doi:10.1021/jacs.9b12940.
Rubber tree cDNA: RNA extracted from the rubber tree latex is then reverse transcribed into cDNA, and the process is usually performed according to conventional conditions such as those described in the publication of the scientific Press (fourth edition) or according to the conditions suggested by the manufacturer, which are well known to those skilled in the art, and the present invention is not limited thereto.
The nucleotide sequence of the rubber tree beta-olivine synthetase gene is shown as SEQ ID NO. 1.
The amino acid sequence of the rubber tree beta-olivine synthetase provided by the invention is shown as SEQ ID NO. 2.
Use of rubber tree beta-olivine synthase for the production of beta-olivine. The rubber tree beta-olivine synthetase of the invention can catalyze and synthesize beta-olivine by taking farnesyl pyrophosphate as a substrate, and can be applied to the flavor and fragrance industry.
The method for producing beta-olivine can utilize the rubber tree beta-olivine synthetase to catalyze farnesyl pyrophosphoric acid to obtain beta-olivine.
The invention also provides a recombinant vector containing the rubber tree beta-olivine synthase gene, the rubber tree beta-olivine synthase gene is recombined in an expression vector to construct a biosynthesis module, so that the simple, convenient and rapid utilization of the rubber tree beta-olivine synthase gene can be realized, and a large amount of target genes or target proteins can be obtained. In the present invention, the expression vector is preferably a pESC yeast expression vector.
The invention also provides engineering bacteria containing the rubber tree beta-olivine synthase gene, wherein the engineering bacteria comprise the rubber tree beta-olivine synthase gene and host cells, and the nucleotide sequence of the rubber tree beta-olivine synthase gene is shown as SEQ ID NO. 1.
The beta-olivine synthetase gene of the rubber tree is led into host cells, thus a large amount of target genes and target protease can be obtained quickly, and a biosynthesis module formed by engineering bacteria avoids the complicated operation of PCR amplification from the genome of the rubber tree when the target genes are used. The host cell is preferably a yeast YZL141 strain or a yeast JCR27 strain.
The invention also provides a plant cell or a transgenic plant which is introduced with the rubber tree beta-olivine synthase gene, and the nucleotide sequence of the rubber tree beta-olivine synthase gene is shown as SEQ ID NO. 1. The method is well known to those skilled in the art, and the transgenic plants are regenerated by constructing plant expression vectors from the rubber tree gene, introducing the vectors into plant cells and calli by means of Agrobacterium, protoplast, gene gun, etc., to synthesize and produce beta-olivine.
Example 1
(1) Gene cloning
The applicant has analyzed the genome of rubber tree to find one rubber tree sesquiterpene beta-olivine synthetase gene with amino acid sequence shown in SEQ ID No.1 and nucleotide sequence shown in SEQ ID No. 2.
The rubber gum emulsion sample is collected from the hot grinding 7-33-97 clone of routine rubber cutting in the national academy of sciences of Tropical agriculture, hainan province. The extraction was performed using total RNA extraction kit from the company of the biochemical technology of tengen (beijing), followed by the synthesis of cDNA using the first strand reverse transcription kit of fermantas cDNA. Gene cloning was performed from latex cDNA templates using RT-PCR using primer pairs P1-P2 (see Table 1). The PCR reaction system used was (30. Mu.L): 12.5. Mu.L of water, 2x PrimerSTAR Max Permix 10. Mu.L of forward and reverse primers (10. Mu.M) each 1. Mu.L of cDNA template, 0.5. Mu.L. The PCR reaction system is as follows: 95℃for 3min,98℃for 10s,58℃for 20s,72℃for 2min,35 cycles, 72℃for 10min,16 ℃. And then, recovering DNA fragments by using a glue recovery kit of Nanjing Norvezan biotechnology limited company, connecting the DNA fragments to a pTOPO-Blunt Simple carrier, carrying out ampicillin resistance screening in the competent E.coli of transformed DH5 alpha, and carrying out sequencing confirmation by a sequencing company after bacterial liquid PCR verification, thereby obtaining the target gene after sequencing confirmation.
TABLE 1
(2) Recombinant expression vector construction
And (3) after sequencing and recovering the target gene vector again by PCR, connecting the target gene vector with a yeast linearization expression vector by adopting a homologous recombination kit of Nanjinopran biotechnology limited company (37 ℃ for 30 min), screening the target gene vector in the competent of transforming DH5 alpha escherichia coli, and obtaining the yeast expression vector containing the correct target gene after PCR verification of bacterial liquid, wherein the yeast expression vector is named pHbT1.
(3) Construction and functional characterization of engineering strains
(1) Yeast competent preparation
Yeast YZL141 strain and JCR27 strain are cultured overnight and transferred into 50mL of fresh YPD culture medium, cultured at 30 ℃ until OD600 is about 0.6, suspended in TE/LiAC solution after 500g centrifugation for 5min, and then subjected to high-speed centrifugation for 5min to collect the strain to be suspended in 1mL of TE/LiAC solution, thus obtaining the competence.
(2) Yeast competent transformation
Transferring the expression vector pHbT1 into YZL141 and JCR27 yeast competence through a PEG-LiAc conversion method, standing at 30 ℃ for 30min, carrying out heat shock at 42 ℃ for 10min, centrifuging for 5min, coating on an SD-URA screening plate, carrying out stationary culture at 30 ℃ for 3d, carrying out colony PCR verification by using primer pairs P1-P2 and P3-P4 respectively, and naming positive bacteria for converting YZL141 as SRBT5. The positive bacteria transformed with JCR27 were designated as JSRCT 5.
(3) Functional characterization
The SRBT5 strain stored at low temperature is inoculated into 50mL SD-URA defect culture medium (1% galactose is added), cultured for 3d at 30 ℃, centrifuged at 4000rpm for 5min to collect the bacterial cells, and the bacterial cells are transferred into a sample bottle for GC-MS detection after extraction. As shown in FIG. 1, the detection result of the SRBT5 strain shows that the main product is identified aS beta-olivine (beta-maleiene) by NIST database comparison, the byproduct is alpha-guljunene (alpha-guljunene), and the coding gene of the SRBT5 strain from the rubber tree is named Hb m aS.
(4) Fermentation evaluation
After the gene function is determined, the corresponding strain JSBCT 5 with higher main product is inoculated into a 50mLYPD culture medium by adopting a similar method, 1% galactose and 0.5% -1% IPM are added, after 3d culture at 30 ℃, the organic phase is collected by centrifugation at 4000rpm for 8min, and a detection sample is prepared for GC-MS detection and quantification. The shake flask yield of JSRCT 5 was 10mg/L.
Example 2
To evaluate the potential value of β -olivine as a fragrance and flavoring, the aroma of the molecule was analyzed by a cosmest, with a spicy, mild, respiratory comfort aroma. When the beta-olivine is heated, white smoke is generated with enhanced smell, which indicates that the beta-olivine has potential to be used as a component of spice formulation and applied to products such as electronic cigarettes.
The embodiments described above and features of the embodiments herein may be combined with each other without conflict.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (10)
1. An enzyme, which is an enzyme of the following a) or b):
a) An enzyme having the amino acid sequence of SEQ ID No. 2;
b) The protein with rubber tree beta-olivine synthetase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2.
2. The enzyme-related biomaterial of claim 1, which is any one of the following B1) to B8):
b1 A nucleic acid molecule encoding the enzyme 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 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);
optionally, the vector comprises a pESC yeast expression vector and a transgenic plant expression vector;
optionally, the recombinant microorganism comprises an engineered bacterium or a plant cell.
3. The biomaterial according to claim 2, characterized in that: the nucleic acid molecule is as shown in the following 1) or 2) or 3) or 4) or 5):
1) The coding sequence is a DNA molecule or a cDNA molecule of SEQ ID No.1 in a sequence table;
2) The nucleic acid sequence is a DNA molecule of SEQ ID No.1 in a sequence table;
3) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) or 2) and encoding the enzyme of claim 1;
4) Hybridizing under stringent conditions to the nucleotide sequence defined in 1) or 2) and encoding a cDNA molecule or a genomic DNA molecule of the enzyme according to claim 1.
4. Use of the enzyme of claim 1 for the preparation of β -olives.
5. Use of the biomaterial of claim 2 for the preparation of a rubber tree beta-olivine synthase or for the construction of transgenic plants or for the production of beta-olives.
6. A primer pair for amplifying a fragment of a nucleic acid molecule encoding the enzyme of claim 1.
7. The primer pair of claim 6, wherein the amplification primer pair is: the forward primer is shown as SEQ ID NO.3, and the reverse primer is shown as SEQ ID NO. 4.
8. A process for producing β -olives, characterized in that β -olives are obtained by catalyzing farnesyl pyrophosphate with the rubber tree β -olives synthase according to claim 1.
9. Use of the enzyme of claim 1 or the biomaterial of claim 2 in the development of a perfume raw material or the formulation of a higher perfume.
10. A biosynthetic product for the development or formulation of fragrances, the product comprising the enzyme of claim 1 or the biomaterial of claim 2.
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