WO2022222213A1 - ENGINEERING BACTERIA FOR PRODUCING β-ELEMENE, CONSTRUCTION METHOD THEREFOR, AND APPLICATION - Google Patents

Abstract

Provided is recombinant bacteria for producing β-elemene. Also provided are a method for constructing the recombinant bacteria and a method for producing β-elemene by using the recombinant bacteria. The recombinant bacteria express Solidago canadensis Germacrene A Synthase (ScGAS) and introduce recombinant plasmids containing atoB, idi, and ispA of E. coli and ERG13, tHMG1, ERG12, ERG8, and MVD1 of Saccharomyces cerevisiae. After optimizing same under fermentation conditions, the yield of β-elemene reaches 156.94 mg/L.

Description

A kind of β-elemene-producing engineering bacteria and its construction method and use technical field

The invention relates to the technical fields of metabolic engineering, synthetic biology and biopharmaceuticals, in particular to a construction method and application of a β-elemene-producing strain.

Background technique

The elemene compounds with β-elemene as the main component are national class II non-cytotoxic anti-tumor drugs. β-Elemene has the functions of inducing tumor cell apoptosis, inhibiting its proliferation, metastasis and active immune protection. It can be used clinically alone or in combination with other chemotherapy drugs to treat various cancers. Therefore, it has good medicinal value and application prospect.

The β-elemene currently on the market is mainly extracted from the traditional Chinese medicine Curcuma Radix. The obtaining method has high cost and low purity, while the conventional chemical total synthesis method has cumbersome steps, harsh reaction conditions, low yield and unfriendly environment, which limit the supply and application of β-elemene. Therefore, finding a new economical and feasible mass production technology for β-elemene is of great significance for the popularization and application of this type of drug.

The development of synthetic biology provides a new method for the large-scale production of natural products with low content, complex structure and high application value in nature. Taking advantage of the advantages of fast growth, short cycle, low cost, and clear genetic background, which is conducive to genetic modification, there have been successful examples of rapid and large-scale acquisition of intermediates and final products by constructing recombinant cells and synthetic pathways for heterologous recombinant natural products. : The biosynthetic pathway of artemisinin was constructed in Saccharomyces cerevisiae, and the yield of its precursor artemisinic acid reached 25 g/L (Paddon, C.J., et al. Nature 496.7446(2013):528.).

β-Elemene is a common class of sesquiterpenoids in plants, but no β-elemene synthase has been cloned yet. β-Elemene is rearranged from gemene A through cope. , while gemene A is obtained from the substrate farnesenyl pyrophosphate FPP catalyzed by gemene A synthase. Research and development of a rapid, high-yield, and environmentally friendly preparation method for β-elemene has become an important topic to be studied urgently.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a β-elemene-producing strain and a construction method thereof. In the present invention, the recombinant plasmid of Farnesenyl Pyrophosphate (FPP), which is the precursor of sesquiterpenoids, is constructed and the recombinant plasmid of gemene A synthase ScGAS derived from Solidago canadensis is constructed to obtain the recombinant plasmid containing the above-mentioned Escherichia coli engineering strain of recombinant plasmid, and by optimizing temperature, inducer concentration, induction time and bacterial liquid concentration during induction, β-elemene can be prepared quickly and with high yield.

To achieve the above object, the present invention adopts the following technical solutions:

A recombinant bacterium for producing beta-elemene, the recombinant bacterium expresses gemene A synthase ScGAS, acetoacetyl-CoA thiolase atoB, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13 , Truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, pyrophosphate mevalonate decarboxylase MVD1, pyrophosphate isovalonate Enlipid isomerase idi and farnesenyl pyrophosphate synthase ispA.

Further, the amino acid sequence of the gemene A synthase ScGAS is shown in SEQ ID NO.2.

Further, the nucleotide sequence of the gemene A synthase ScGAS is as shown in SEQ ID NO.1 or as shown in SEQ ID NO.3,

Further, the nucleotide sequence of the acetoacetyl-CoA thiolase atoB is as shown in SEQ ID NO.4, and the nucleotide sequence of 3-hydroxy-3-methylglutaryl-CoA synthase ERG13 is as shown in SEQ ID NO.4 The nucleotide sequence of truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 shown in ID NO.5 is shown in SEQ ID NO.6, and the nucleotide sequence of mevalonate kinase ERG12 The sequence is as shown in SEQ ID NO.7, the nucleotide sequence of phosphomevalonate kinase ERG8 is as shown in SEQ ID NO.8, and the nucleotide sequence of pyrophosphate mevalonate decarboxylase MVD1 is as shown in SEQ ID NO. 9, the nucleotide sequence of pyrophosphate prenyl isomerase idi is as shown in SEQ ID NO.10, and the nucleotide sequence of farnesenyl pyrophosphate synthase ispA is as shown in SEQ ID NO.11 .

Further, the recombinant bacteria are recombinant Escherichia coli or recombinant yeast.

Further, the expression vector of the gemene A synthase ScGAS is pGEX-4T1 vector.

Further, the recombinant bacteria express acetoacetyl-CoA thiolase atoB, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA Reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, pyrophosphate mevalonate decarboxylase MVD1, pyrophosphate prenyl isomerase idi and farnesenyl pyrophosphate synthase ispA The expression vector is pACYCDuet-1 vector.

Further, the acetoacetyl-CoA thiolase atoB, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 The genes linked to the MCS1 site of the multiple cloning site of pACYCDuet-1, the mevalonate kinase ERG12, phosphomevalonate kinase ERG8, pyrophosphate mevalonate decarboxylase MVD1, pyrophosphate isoamyl The genes for the constitutive enzyme idi and farnesenyl pyrophosphate synthase ispA were ligated into the MCS2 site of pACYCDuet-1.

Another aspect of the present invention provides the construction method of the above-mentioned recombinant bacteria producing β-elemene, which mainly comprises the following steps:

(1) Link the gemene A synthase ScGAS gene into the pGEX-4T1 vector, transform competent cells, pick positive clones, and extract the recombinant plasmid pGEX-4T1-ScGAS;

(2) Acetoacetyl-CoA thiolase atoB gene, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13 gene, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 gene, mevalonate kinase ERG12 gene, phosphomevalonate kinase ERG8 gene, pyrophosphate mevalonate decarboxylase MVD1 gene, pyrophosphate prenyl isomerase idi gene and farnesenyl pyrophosphate synthase. The enzyme ispA gene was linked to the pACYCDuet-1 vector, transformed into competent cells, positive clones were picked, and the recombinant plasmid pACYCDuet-FPP was extracted;

(3) The pGEX-4T1-ScGAS recombinant plasmid obtained in step (1) and the pACYCDuet-FPP obtained in step (2) were co-transformed into competent cells, and positive clones were picked to obtain a recombinant strain producing β-elemene.

Further, the competent cell in step (3) is E.coil BL21(DE3).

The invention provides a method for producing β-elemene by utilizing the above-mentioned recombinant bacteria producing β-elemene, and the method mainly comprises the following steps:

(1) Under the culturing conditions of rotating speed 50～300rpm and temperature 20～32℃, cultivate the above-mentioned β-elemene-producing recombinant bacteria, when the concentration A ₆₀₀ of the recombinant bacteria liquid is 0.2～2, add IPTG inducer to the end The concentration is 0.01-1.0mM, and the culture is continued to 12-120h;

(2) using an organic solvent to extract the β-elemene in the fermentation broth, and collecting the organic phase by centrifugation, to obtain the final product.

Further, the culture medium is liquid LB medium.

Further, the organic solvent is one or a mixture of two or more of ethyl acetate, hexane, petroleum ether or chloroform.

Further, the volume ratio of the organic solvent to the fermentation broth is 2:1 to 1:20.

The beneficial effects that the present invention has with respect to the prior art are as follows:

1. The β-elemene-producing engineering bacteria constructed in the present invention have the characteristics of high yield, high purity, low cost, and no pollution, and are suitable for industrial production of β-elemene.

2. The β-elemene-producing engineering bacteria of the present invention can produce β-elemene as high as 156.94 mg/L after the fermentation conditions are optimized.

Description of drawings

In order to describe the embodiments of the present invention more clearly, the accompanying drawings related to the embodiments will be briefly introduced below.

Figure 1 Schematic diagram of the recombinant plasmid pACYCDuet-FPP.

Figure 2. Corresponding graph of the production of recombinant strains and β-elemene.

Figure 3 β-elemene production in orthogonal experimental combinations of co-expressing pGEX-4T1-ScGAS and pACYCDuet-FPP engineering bacteria.

Detailed ways

The present invention will be described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Obviously, the examples in the following description are only part of the examples of the present invention. Under the premise of creative work, obtaining other similar embodiments all fall within the protection scope of the present invention.

In the following examples, the adopted pGEX-4T1 vector and pACYCDuet-1 were purchased from Shanghai Sangon Biotechnology Co., Ltd., and E.coli Trans-1 and E.coil BL21 (DE3) competent cells were purchased from Shanghai Yuanye Biology. Technology Co., Ltd.

Example 1 Construction of recombinant plasmid pGEX-4T1-ScGAS

According to the gemene A synthase ScGAS derived from Solidago canadensis in the NCBI database, its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. Sangon Bioengineering (Shanghai) Co., Ltd. carried out optimization, gene synthesis and sequencing based on E. coli codons, and the codon-optimized nucleotide sequence was obtained as shown in SEQ ID NO.3, and inserted into pBlueScript II SK ( +) vector, constitute the pBlueScript II SK(+)-ScGAS recombinant plasmid.

The pBlueScript II SK(+)-ScGAS recombinant plasmid and pGEX-4T1 plasmid were double digested with EcoR I and Sal I respectively (20μl total digestion system, 10×Quic. Cut Green Buffer 2μl, EcoR I and Sal I 1 μl each, plasmid pBlueScript II SK(+)-ScGAS or pGEX-4T1 8 μl each, ddH ₂ O 8 μl), run 1% agarose gel electrophoresis, cut the gel and recover, ligated into the pGEX-4T1 vector (10 μl ligation total In the system, 5 μl of Solution I, 1 μl of pGEX-4T1 linearized vector, 4 μl of ScGAS fragment, reacted at 16°C for 3 hours), transformed E.coli Trans-1 competent cells, coated with LB/Amp plate containing 100 mg/L, 37 Incubate overnight at ℃, pick transformants for colony PCR verification (20 μl PCR system, 10×ExTaq Buffer 2 μl, dNTP 2 μl, ScGAS-F and ScGAS-R 1 μl each, bacterial broth 1 μl, ExTaq enzyme 0.3 μl, ddH ₂ O 12.7 μl PCR reaction conditions: First, denaturation at 98°C for 5min; secondly, denaturation at 98°C for 30sec, annealing at 58°C for 30sec, and extension at 72°C for 2min, for 32 cycles; and finally at 72°C for 15min extension; the primer sequences are shown in Table 1), and the plasmids from the positive colonies are extracted. pGEX-4T1-ScGAS recombinant plasmid.

Table 1. Primer Sequence Listing

Example 2 Construction of recombinant plasmid pACYCDuet-FPP

2.1 Obtaining the full-length gene sequence

The whole genome sequences of Escherichia coli Trans-1 and Saccharomyces cerevisiae S288C were extracted. According to the atoB, ERG13, tHMG1, ERG12, ERG8, MVD1, idi, ispA gene sequences published on NCBI, primers (primers No. 7-22, the sequence is shown in Table 1) were used to amplify atoB, idi, and , ispA, using yeast genomic DNA as a template to amplify ERG13, tHMG1, ERG12, ERG8, MVD1, using high-fidelity enzyme PrimeSTAR ^GXL (Takara) for amplification (50μl PCR system, 5× PrimeSTAR Buffer 10μl, dNTP 4μl, primer-F 1 μl of primer-R and 1 μl of template, 0.5 μl of PrimeSTAR enzyme, 32.5 μl of ddH ₂ O; PCR reaction conditions: First, denature at 98°C for 5min; 32 cycles; finally, extended at 68°C for 15 min), after running 1% agarose gel electrophoresis, after gel cutting and recovery, the recovered product was subjected to A-tail reaction with ExTaq enzyme, and after ligation with pMD18-T vector, transformed into E. coli Trans-1 competent cells, coated with LB/Amp plates containing 100 mg/L, cultured overnight at 37°C, picked transformants for colony PCR verification, positive clones were sent to Shanghai Sangon Biological Co., Ltd. for sequencing, and the correct colonies were sequenced , after the plasmid is extracted, it can be used as a template for subsequent vector construction.

2.2 Using the principle of overlapping PCR to construct operons A and B

The operon A contains the genes atoB, ERG13, and tHMG1, and the operon B contains the genes ERG12, ERG8, MVD1, idi, and ispA. Using the plasmids containing atoB, ERG13, and tHMG1 in step 2.1 as templates, PCR amplification was carried out with the high-fidelity enzyme PrimeSTAR ^GXL , and the forward and reverse primers (23-28 primers, sequences are shown in Table 1) of the respective genes were used to carry out the first round. PCR reactions were performed to amplify the above three genes respectively (PCR reaction conditions were firstly denatured at 98°C for 5 min; secondly, denatured at 98°C for 10 sec, annealed at 55-60°C for 5 sec, and extended at 68°C for 1-2 min, for 15 cycles; finally, re-extension at 68°C 15min), then use 1 μl of the above PCR products as templates, use primers No. 23 and No. 28 to carry out the second round of PCR reaction (PCR reaction conditions are firstly denatured at 98°C for 5min; then denatured at 98°C for 10sec, annealed at 55-60°C for 5sec, Extend at 68°C for 1-2 min, 32 cycles; finally, extend at 68°C for 15 min), run 1% agarose gel electrophoresis, and recover by cutting the gel, which is operon A. The construction process of operon B is similar to that of operon A. Only different primers are used to amplify different genes. The first round of PCR uses primers 29-38 to amplify ERG12, ERG8, MVD1, idi, and ispA genes, respectively, and the second round uses 29. and primer 38.

2.3 Using the OK Clon DNA ligation kit (Hunan Aikerui Bioengineering Co., Ltd.), see the kit instructions for the specific operation method, and homologously recombine the operon A to the Sal I of the multiple cloning site 1 (MCS2) of the pACYCDuet-1 vector. site, homologous recombination of operon B into the Xho I site of multiple cloning site 2 of the pACYCDuet-1 vector, to obtain the pACYCDuet-FPP recombinant plasmid.

Example 3 Construction of high-yielding β-elemene engineering bacteria

3.1 Use plasmid pGEX-4T1 or pGEX-4T1-ScGAS and pACYCDuet-1 or pACYCDuet-FPP to co-transform E.coil BL21(DE3) competent, coat LB/Amp&Cm resistant plate, after overnight culture, pick a single clone for use The universal primers M13-F&M13-R on the pGEX-4T1 vector and the universal primers Cm-F&Cm-R on the pACYCDuet-1 vector (primers 3-6, see Table 1) were used for colony PCR identification, and the positive clones were respectively pGEX -4T1/pACYCDuet-FPP/BL21, pGEX-4T1-ScGAS/pACYCDuet-FPP/BL21, pGEX-4T1-ScGAS/pACYCDuet-1/BL21, pGEX-4T1/pACYCDuet-1/BL21 recombinant strains.

3.2 The four recombinant strains obtained in the above step 3.1 were respectively inoculated into 2 mL liquid LB medium containing Amp&Cm antibiotics, and cultured overnight at 180 rpm in a 37°C constant temperature shaking incubator. The next day, it was expanded into 50 mL of liquid LB medium containing Amp&Cm antibiotics at a ratio of 1:50, and continued to culture in a constant temperature shaking incubator at 37 °C until the bacterial concentration A600 was about 2, and 10 μL of IPTG with a concentration of 0.5M was added. 10ml of n-dodecane solution was incubated in a constant temperature shaking incubator at 28°C for 48h at 180rpm. Cool the fermentation broth to room temperature, distribute equal volumes into three 50 mL centrifuge tubes, add 20 mL of ethyl acetate to each centrifuge tube, seal with parafilm, shake, mix and extract, and ultrasonicate for 5 minutes; centrifuge at 12,000 rpm for 10 minutes at room temperature , collect the supernatant into a round-bottomed flask; spin to no ethyl acetate on a rotary evaporator at 28°C, 50 rpm, collect n-dodecane; add an appropriate amount of anhydrous Na ₂ SO ₄ to remove the moisture in the organic phase, and centrifuge again to collect The supernatant is the fermentation product.

3.3 Filter the fermentation product of step 3.2 with a 0.22 μm organic filter, dilute it 200 times with ethyl acetate, add nonyl acetate with a final concentration of 20 mg/L as an internal reference, and use GC-MS to detect the sample. The peak area ratio of β-elemene was calculated (as shown in Figure 2). GC-MS detection conditions: quartz capillary column HP-5MS (30m×0.25mm×0.25μm); heating program: 80°C, hold for 3min; 10°C/min to 210°C, hold for 1min. Carrier gas: high-purity helium, the flow rate is set to 1mL/min; the inlet and interface temperatures are set to 250°C and 280°C respectively; the injection volume is 1 μL; the ion source EI; the electron energy 70eV; the ion source temperature 250°C; scanning Mass range 35～550amu; solvent delay 6.5min.

The results showed that the yield of β-elemene in the recombinant bacteria expressing only pGEX-4T1-ScGAS was 49.21 mg/L (Fig. 2, ScGAS B), while the recombinant bacteria co-expressing pGEX-4T1-ScGAS and pACYCDuet-FPP The yield of β-elemene was 146.88 mg/L (Fig. 2, ScGAS D), a 2.98-fold increase in yield. Therefore, the recombinant strain co-expressing pGEX-4T1-ScGAS and pACYCDuet-FPP can be used as an engineered strain for mass production of β-elemene.

Example 4 Optimization of fermentation conditions for high-yielding β-elemene engineering bacteria

The pGEX-4T1-ScGAS/pACYCDuet-FPP/BL21 engineered bacteria obtained in step 3.1 were activated to optimize fermentation conditions. The main factors affecting the yield of fermentation products include the concentration of bacteria when IPTG is added, the culture temperature, the concentration of IPTG used and the induction time. Three different experimental conditions (as shown in Table 2) were selected to design L ₉ (3 ⁴ ) orthogonal experiments. Table (as shown in Table 3), and used the range analysis method of orthogonal experiment to analyze the influence of 4 factors on the yield of β-elemene. The samples to be tested were prepared according to method 3.2, the changes in the content of β-elemene in the samples were detected by method 3.3, and the effects of various factors on the yield of β-elemene were calculated and evaluated.

The primary and secondary relationship results of each factor on β-elemene production determined by the range analysis method of the orthogonal experiment are shown in Table 4. Under different fermentation conditions, the production of β-elemene is shown in Figure 3. The final determination The fermentation conditions were that IPTG was added when the A600 of the recombinant bacteria was 0.5, and the final concentration of IPTG was 0.1 mM. When fermented in a shake flask at 28 °C for 72 h, the yield of β-elemene reached 156.94 mg/L.

Table 2. Orthogonal Factor Levels

Table 3. Orthogonal experimental conditions

Table 4. The relationship between the yield of β-elemene and various factors

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.

Claims (10)

1. A recombinant bacterium for producing beta-elemene, characterized in that the recombinant bacterium expresses gemene A synthase ScGAS, acetoacetyl-CoA thiolase atoB, 3-hydroxy-3-methylglutaryl coenzyme A synthase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, pyrophosphomevalonate decarboxylase MVD1, Isopentenyl pyrophosphate isomerase idi and farnesenyl pyrophosphate synthase ispA.
2. The recombinant bacteria according to claim 1, wherein the amino acid sequence of the gemene A synthase ScGAS is as shown in SEQ ID NO.2.
3. The recombinant bacteria according to claim 1, wherein the nucleotide sequence of the gemene A synthase ScGAS is as shown in SEQ ID NO.1 or as shown in SEQ ID NO.3, and the acetoacetyl-CoA The nucleotide sequence of thiolase atoB is shown in SEQ ID NO.4, and the nucleotide sequence of 3-hydroxy-3-methylglutaryl-CoA synthase ERG13 is shown in SEQ ID NO.5, truncated The nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 is shown in SEQ ID NO.6, and the nucleotide sequence of mevalonate kinase ERG12 is shown in SEQ ID NO.7 , the nucleotide sequence of phosphomevalonate kinase ERG8 is as shown in SEQ ID NO.8, the nucleotide sequence of pyrophosphate mevalonate decarboxylase MVD1 is as shown in SEQ ID NO.9, isopentenyl pyrophosphate The nucleotide sequence of lipoisomerase idi is shown in SEQ ID NO.10, and the nucleotide sequence of farnesenyl pyrophosphate synthase ispA is shown in SEQ ID NO.11.
4. The recombinant bacteria according to claim 1, wherein the expression vector of the gemene A synthase ScGAS is a pGEX-4T1 vector; the acetoacetyl-CoA thiolase atoB, 3-hydroxy-3-methyl Glutaryl-CoA synthase ERG13, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, mevalonate kinase ERG12, phosphomevalonate kinase ERG8, mevalonate pyrophosphate The expression vector of acid decarboxylase MVD1, isopentenyl pyrophosphate isomerase idi and farnesenyl pyrophosphate synthase ispA is pACYCDuet-1 vector.
5. The recombinant bacteria according to claim 4, wherein the acetoacetyl-CoA thiolase atoB, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13, truncated 3-hydroxy-3 - The gene for methylglutaryl-CoA reductase tHMG1, the mevalonate kinase ERG12, phosphomevalonate kinase ERG8, pyrophosphomevalonate decarboxylase, is linked to the multiple cloning site MCS1 of pACYCDuet-1 The genes for MVD1, prenyl pyrophosphate isomerase idi, and farnesenyl pyrophosphate synthase ispA were ligated into the multiple cloning site MCS2 of pACYCDuet-1.
6. The construction method of the described recombinant bacteria of claim 4 or 5, is characterized in that, mainly comprises the following steps:

   (1) Link the gemene A synthase ScGAS gene into the pGEX-4T1 vector, transform competent cells, pick positive clones, and extract the recombinant plasmid pGEX-4T1-ScGAS;

   (2) Acetoacetyl-CoA thiolase atoB gene, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13 gene, truncated 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1 gene, mevalonate kinase ERG12 gene, phosphomevalonate kinase ERG8 gene, pyrophosphate mevalonate decarboxylase MVD1 gene, pyrophosphate prenyl isomerase idi gene and farnesenyl pyrophosphate synthase. The enzyme ispA gene was linked to the pACYCDuet-1 vector, transformed into competent cells, positive clones were picked, and the recombinant plasmid pACYCDuet-FPP was extracted;

   (3) The pGEX-4T1-ScGAS recombinant plasmid obtained in step (1) and the pACYCDuet-FPP obtained in step (2) were co-transformed into competent cells, and positive clones were picked to obtain a recombinant strain producing β-elemene.
7. The construction method according to claim 6, wherein the competent cell in step (3) is E.coil BL21 (DE3).
8. Utilize the method for producing β-elemene by the recombinant bacteria described in any one of claim 1-5, it is characterized in that, mainly comprises the following steps:

   (1) under the culturing condition of rotating speed 50～300rpm, temperature 20～32 ℃, cultivate the recombinant bacteria described in any one of claim 1-5, when the recombinant bacteria liquid concentration A ₆₀₀ is 0.2～2, add IPTG to induce The final concentration was 0.01-1.0 mM, and the culture was continued for 12-120 h;

   (2) using an organic solvent to extract the β-elemene in the fermentation broth, and collecting the organic phase by centrifugation, to obtain the final product.
9. The method according to claim 8, wherein the culture medium is a liquid LB medium.
10. The method according to claim 8 or 9, wherein the organic solvent is one or more mixtures of ethyl acetate, hexane, petroleum ether or chloroform, the organic solvent and the fermentation broth are The volume ratio is 2:1～1:20.

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