CN116286767A

CN116286767A - Beta-caryophyllene synthetase, gene, strain and application

Info

Publication number: CN116286767A
Application number: CN202310291537.7A
Authority: CN
Inventors: 石彬; 邓小敏; 林维扬; 余承焰; 朱伶俐; 朱薇
Original assignee: Wuhan Software Engineering Vocational College Wuhan Open University
Current assignee: Wuhan Software Engineering Vocational College Wuhan Open University
Priority date: 2023-03-21
Filing date: 2023-03-21
Publication date: 2023-06-23
Anticipated expiration: 2043-03-21
Also published as: CN116286767B

Abstract

The invention discloses beta-caryophyllene synthetase, a gene, a strain and application. The invention discovers for the first time that the enzyme with the amino acid sequence shown in SEQ ID NO.1, which is derived from rubber tree Hevea brasiliensis, is the synthetase of beta-caryophyllene, and the synthetase or the coding gene thereof or the recombinant plasmid and the recombinant strain containing the coding gene thereof can be used for synthesizing or producing the beta-caryophyllene. The invention provides a beta-caryophyllene high-yield strain, which is obtained by introducing a coding gene of beta-caryophyllene synthase into a microorganism which overexpresses one or more genes in a mevalonate pathway and a farnesyl pyrophosphate synthase gene, and the yield of a fermentation tank (5L steel tank) of the beta-caryophyllene high-yield strain is up to 6.2g/L. The prepared beta-caryophyllene can be used for preparing perfume, essence and medical intermediates.

Description

Beta-caryophyllene synthetase, gene, strain and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to beta-caryophyllene synthetase, a gene, a strain and application.

Background

Beta-caryophyllene belongs to a member of a terpenoid family, is a sesquiterpene, has spicy, banksia rose, citrus, camphor and mild clove fragrance, and mainly exists in plants such as ginseng, clove flowers and the like. Beta-caryophyllene was approved by state GB2760-1996 as a food flavor for use. At present, the method is mainly used for preparing the edible essence such as clove, pepper, nutmeg, orange, herb and the like, and can also be used for synthesizing more valuable spices such as acetyl caryophyllene and the like. In addition to being used as a perfume substance, the research on the biological activity of the beta-caryophyllene shows that the beta-caryophyllene has various biological activities such as anticancer, anti-inflammatory, antioxidation, nerve protection and the like. Beta-caryophyllene not only has anticancer activity by itself, but also can remarkably improve the anticancer activity of other chemotherapeutics by increasing the permeability of cell membranes. For example, β -caryophyllene increases the permeability of paclitaxel to cell membranes, increasing its inhibitory activity on colon adenocarcinoma cell DLD-1 by a factor of 10. The main production mode of the beta-caryophyllene at present is that the beta-caryophyllene is obtained from clove oil through vacuum fractionation, the biotechnological synthesis of the beta-caryophyllene is still in a starting stage, the yield is low, the industrialization level cannot be reached far, and the research on the biosynthesis of the beta-caryophyllene has important research significance and has further development value.

Disclosure of Invention

The invention aims at providing beta-caryophyllene synthetase, gene, strain and application aiming at the defects of the prior art.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the first object of the invention is to provide a beta-caryophyllene synthase HbBaS, the amino acid sequence of which is shown as SEQ ID NO. 1.

A second object of the present invention is to provide an application of a β -caryophyllene synthase gene HbBACS in the synthesis or production of β -caryophyllene, wherein the base sequence of the β -caryophyllene synthase gene HbBACS is identical to the nucleotide sequence of SEQ ID NO:2 has a nucleotide sequence having an identity of 95% or more.

Further, the base sequence of the beta-caryophyllene synthase gene HbBACS is shown as SEQ ID NO: 2.

The third object of the present invention is to provide a recombinant expression vector comprising the above-mentioned beta-caryophyllene synthase gene HbBACS.

The fourth object of the present invention is to provide a method for constructing the recombinant expression vector, comprising the following steps: adding restriction sites to the gene HbBACS of any one of claims 2 to 3, and then performing double restriction with restriction enzymes Sal I and Xho I, respectively, to obtain a gene HbBACS fragment having a sticky end; the commercial expression vector pRS426 of the saccharomyces cerevisiae is subjected to double enzyme digestion by restriction enzymes Pme I and Not I respectively to obtain a vector pRS426 fragment with a sticky end; and connecting the gene HbBACS fragment with the sticky end and the vector pRS426 fragment with the sticky end through T4 DNA ligase to obtain the beta-caryophyllene synthase recombinant expression vector pHbBACS.

It is a fifth object of the present invention to provide a recombinant expression strain comprising the recombinant expression vector described above.

Further, the host cell of the recombinant expression strain is Saccharomyces cerevisiae.

The sixth object of the present invention is to provide a method for preparing the recombinant expression strain, wherein the recombinant expression vector pHbBACS of beta-caryophyllene synthetase is linearized through an enzyme cleavage site; the linearized recombinant expression vector pHbBACS is introduced into a host cell to obtain a recombinant expression strain.

A seventh object of the present invention is to provide a method for producing β -caryophyllene, culturing the recombinant expression strain as described above, and obtaining β -caryophyllene from the culture.

An eighth object of the present invention is to provide the use of the above-mentioned β -caryophyllene for the preparation of fragrances or perfumes.

Compared with the prior art, the technical scheme provided by the invention has the beneficial effects that:

(1) The invention discovers the beta-caryophyllene synthetase derived from rubber tree and the coding gene thereof for the first time, and the enzyme can efficiently synthesize the product beta-caryophyllene.

(2) The constructed beta-caryophyllene high-yield strain has a shake flask yield of 126mg/L and a fermentation tank (5L steel tank) yield of 6.2g/L, thus laying a solid foundation for the industrial application of the beta-caryophyllene for the highest yield reported at present.

Drawings

FIG. 1 is a graph showing the results of GC-MS detection of the JBS1 strain products of the present invention;

FIG. 2 is a diagram of a rubber tree latex used in the present 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.

Taq DNA polymerase, restriction enzyme, plasmid extraction kit and DNA extraction kit were all purchased from Takara company; the DNA gel recovery kit was purchased from nuvazan company; homologous recombination kits were purchased from assist san Co; expression plasmid pRS426 was purchased from ATCC; the specific gene primer pair P1/P2 was synthesized by Jin Kairui.

The detection method of the beta-caryophyllene comprises the following steps: the organic phase of the two-phase fermentation culture was collected, centrifuged at 3500rpm for 10 minutes to remove cell debris, and diluted with n-hexane to an appropriate range. The samples were then subjected to gas chromatography mass spectrometry (GC-MS) detection analysis, using a TSQ QUANTUM XLS mass spectrometer (Thermo Fisher Scientific, thermo, MA, USA), and the concentration of beta-caryophyllene was detected and quantified using a TR-5MS column (30 m 0.25mm 0.25 μm). The GCMS program was set as follows: the temperature was initially maintained at 50 ℃ for 1 minute, then increased to 280 ℃ at a rate of 15 ℃/minute for 1 minute, then increased to 300 ℃ at a rate of 20 ℃/minute for 2 minutes. Compounds were determined by comparison with standard β -caryophyllene from Sigma-Aldrich (St.Louis, MO, U.S.A.) or by comparison of NIST (National Institute of Standards and Technology) database and retention index;

YPD medium: the medium fraction contained 2% glucose (Country), 2% tryptone (Angel Yeast FP 318), 1% Yeast extract (Angel Yeast FM 888)

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 AEnabled 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 rubber tree latex (as shown in FIG. 2) is then reverse transcribed into cDNA, and the process is usually performed under 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.

Example 1

(1) Recombinant vector construction

The applicant analyzes the genome of the rubber tree and discovers a terpene synthase HbBaS, the amino acid sequence of which is shown as SEQ ID NO.1, and the nucleotide sequence of which is shown as SEQ ID NO. 2.

The specific gene primer pair P1/P2 (shown in Table 1) is designed, rubber tree cDNA is used as a template, a specific gene fragment is obtained through PCR amplification by using Prime STAR MAX PCR enzyme of Takara company, the fragment is recovered by using a Novain gum recovery kit, then the fragment is connected to a yeast commercial expression vector pRS426 (Sal I/Xho I) through a homologous recombination method by using a holy Corp homologous recombination kit, and after sequencing and confirmation, a yeast expression vector containing the specific gene HbBACS is obtained and named pHbBACS.

Table 1.

(2) Strain construction

The pHbBACS plasmid was transformed into the Yeast strain YZL141 competence by PEG/LiAC method, spread on SD-URA screening plate, cultured for 3d, and then colony PCR verification was performed using the Noruzbac PCR reagent using the P1 and P2 primers, and the positive bacteria was named YHbBACS. The strain YHbBACS is a plasmid which firstly strengthens the MVA pathway based on Saccharomyces cerevisiae CEN.PK2-1D, overexpresses the speed-limiting enzyme tHMG1 of the MVA pathway and is transformed into a specific gene HbBACS.

Meanwhile, the HbBACS gene fragment is recovered by utilizing the enzyme digestion pHbBACS of PmeI, the fragment is introduced into the competent yeast strain JCR27 by a PEG/LiAC method, and the positive bacteria are named as JBS1 after the PCR verification of the yeast colony. Compared with CEN.PK2-1D strain, the strain JBS1 strengthens MVA pathway, over-expresses the whole MVA pathway, and over-expresses HbBACS genes on the basis, wherein the copy numbers of the genes in the strain JBS1 are ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20, hbBACS=2, 2 and 1.

(3) Shaking flask fermentation

The YHbBACS strain was inoculated into 50mL of YPD medium supplemented with 1% galactose, 1% IPM was added, and after 3d incubation at 30℃the organic oil phase of the coating was collected by centrifugation at 8000rpm for 10min and diluted with n-hexane to the appropriate range for GC-MS detection. The main peak is compared with the retention time of a beta-caryophyllene standard substance and MS data, so that the strain YHbBACS can be determined to synthesize beta-caryophyllene, the gene HbBACS is a beta-caryophyllene synthase gene, and the shake flask yield is 18mg/L; the small peak product is compared with NIST database, and the product is alpha-humulone.

The JBS1 strain was similarly fermented for 72 hours after adding 10% isopropyl myristate (IPM) cover, centrifuged to obtain an organic oil phase, and diluted with n-hexane to an appropriate range for GC-MS detection. The yield of the sesquiterpene product β -caryophyllene increased from 18mg/L of strain YHbBACS to 126mg/L of strain JBS1 by about 7-fold.

The strain JBS1 thus constructed was fed-batch fermented with the addition of a covering agent during the fermentation to effect in situ extraction, the covering agent being IPM isopropyl myristate, as described in reference (van Hoek, P.; de Hulster, E.; van Dijken, J.P.; ink, J.T. commercial capability in high-cell-density fed-batch cultures of baker's yeast. Biotechnol. Bioeng.2000,68, 517-523.). The dissolved oxygen is controlled to be more than 20 percent in the fermentation process, the pH value is 5, the glucose concentration is 1-2g/L, and the ethanol concentration is less than 5 g/L. Finally, on a fermentation tank (5L fermentation tank), the yield of the sesquiterpene product beta-caryophyllene reaches 6.2g/L.

Example 2

To evaluate the potential value of β -caryophyllene as a fragrance and flavoring, the aroma in this molecule was analyzed by a cosmonautic engineer, with β -caryophyllene having spicy, woody, citrus, camphorous, mild clove aromas. When β -caryophyllene is heated, white smoke is produced with an enhanced odor, indicating its potential for use as a flavor ingredient and food flavoring.

Those skilled in the art know that beta-caryophyllene has been specified by national standard GB 2670-1996 as a permissible edible spice which can be used for daily chemicals of spicy type and the flavoring of edible essence, and for the coordination of tobacco essence, and the beta-caryophyllene has a better effect on increasing the burnt sweet smell.

Example 3

The organic covering phase obtained by fermenting and collecting the recombinant saccharomyces cerevisiae constructed by the new beta-caryophyllene synthase gene of the invention is used for distinguishing different components in a rectification way, the purity of the purified beta-caryophyllene can reach more than 95 percent, the purified beta-caryophyllene can be used as a medical intermediate by fermenting and collecting the purified beta-caryophyllene and can be used as a raw material medicine for continuously synthesizing other medicines,

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

1. A beta-caryophyllene synthase HbBaS, characterized by: the amino acid sequence of the beta-caryophyllene synthetase HbBaS is shown as SEQ ID NO. 1.

2. An application of a beta-caryophyllene synthase gene HbBACS in synthesis or production of beta-caryophyllene, which is characterized in that the base sequence of the beta-caryophyllene synthase gene HbBACS is identical with the base sequence of SEQ ID NO:2 has a nucleotide sequence having an identity of 95% or more.

3. The use according to claim 2, wherein the base sequence of the β -caryophyllene synthase gene HbBACS is as set forth in SEQ ID NO: 2.

4. A recombinant expression vector comprising the gene HbBACS of any one of claims 2-3.

5. The method for constructing a recombinant expression vector according to claim 4, comprising the steps of: adding restriction sites to the gene HbBACS of any one of claims 2 to 3, and then performing double restriction with restriction enzymes Sal I and Xho I, respectively, to obtain a gene HbBACS fragment having a sticky end; the commercial expression vector pRS426 of the saccharomyces cerevisiae is subjected to double enzyme digestion by restriction enzymes Pme I and Not I respectively to obtain a vector pRS426 fragment with a sticky end; and connecting the gene HbBACS fragment with the sticky end and the vector pRS426 fragment with the sticky end through T4 DNA ligase to obtain the beta-caryophyllene synthase recombinant expression vector pHbBACS.

6. A recombinant expression strain comprising the recombinant expression vector of claim 5.

7. The recombinant expression strain of claim 6, wherein the host cell of the recombinant expression strain is saccharomyces cerevisiae.

8. A method of preparing a recombinant expression strain according to claim 6 or 7, comprising the steps of: linearizing a recombinant expression vector pHbBACS of the beta-caryophyllene synthetase through enzyme cleavage sites; the linearized recombinant expression vector pHbBACS is introduced into a host cell to obtain a recombinant expression strain.

9. A method for producing beta-caryophyllene is characterized in that: culturing the recombinant expression strain of claim 6 or 7, and obtaining β -caryophyllene from the culture.

10. Use of a β -caryophyllene according to claim 9 for the preparation of a perfume or essence or pharmaceutical intermediate.