A strain and method for producing beta-elemene

Abstract

The present invention discloses a strain and method for producing β-elemene, and belongs to the field of bioengineering technology. The present invention constructs a recombinant cell expressing farnesol kinase, farnesyl phosphate kinase, polyphosphate kinase 2, and a combination of recombinant cells of germarene A synthase, and utilizes the recombinant cell or the combination of recombinant cells to catalyze farnesol to synthesize β-elemene. The substrate of the present invention is cheap, easily available, and the present invention can obtain 17g / L β-elemene by adding 20g / L farnesol reaction for 24h. Therefore, the present invention has good industrial application prospects and is conducive to the large-scale production of β-elemene.

Description

Technical Field

[0001] The invention relates to a strain and a method for producing beta-elemene, belonging to the technical field of bioengineering. Background Art

[0002] β-elemene belongs to the sesquiterpenoid family of compounds. Among the four isomers of elemene (α-elemene, β-elemene, γ-elemene, and δ-elemene), it is the most important substance with anticancer effects. In 1981, Chinese researchers first reported that β-elemene, extracted from the volatile oil of Curcuma zedoaria, a traditional Chinese herbal medicine for promoting blood circulation and removing blood stasis, possessed antitumor activity (Shi Jihui. Experimental Pharmacological Studies on the Volatile Oil of Curcuma zedoaria [J]. Dalian Institute of Medical Sciences, 1981). In 1995, elemene injection, containing β-elemene as its main ingredient, was approved by the government as a Class II new anticancer drug for clinical use. β-elemene has shown promising results in the treatment of malignant serous effusions, brain metastasis of lung cancer, lung cancer, gastric cancer, superficial bladder cancer, primary liver cancer, and brain malignancies. Research has shown that the anti-tumor effects of β-elemene differ from those of chemotherapy drugs in that it possesses the dual effects of inhibiting tumor cell proliferation and enhancing immune function. As research continues into the biological activities and metabolic characteristics of β-elemene, more and more biological activities of β-elemene are being discovered. Therefore, further rational development and utilization of β-elemene for human use will become a research hotspot.

[0003] The preparation method of β-elemene at present mainly includes the following two kinds: 1) separation and extraction of natural plant volatile oil, Chinese invention patents CN102432419A, CN102432420A are separated and extracted from the volatile oil of Eupatorium adenophorum and Lantana camara respectively. However, this method has many feedings, is time-consuming, and has low purity. 2) chemical synthesis, international patents AU7111598A, WO2006016912A2 have realized etc. and realized the stereoselective total synthesis of elemene, but chemical synthesis needs to consume a large amount of organic solvents, is costly, and has a large impact on the environment. The bioenzymatic method has the advantages of low cost, mild reaction conditions, and little environmental pollution. As people pursue green environmental protection, the bioenzymatic method is increasingly attracting people's attention.

[0004] In 2013, Li Haifeng et al. proposed using farnesyl pyrophosphate (FPP) produced by Escherichia coli's own metabolism as a substrate and then overexpressing β-elemene synthase via a plasmid vector to produce β-elemene. However, after 20 hours of induction at 30°C, the yield of β-elemene was only 128μg / L (Chinese invention patent CN201310280524.6). In 2016, Xie Tian et al. expressed the sesquiterpene synthase from Curcuma longa in Escherichia coli, purified it, and catalyzed FPP in vitro to produce β-elemene. However, the conversion rate was only 34.5%, and the yield was only 0.345μg (Chinese invention patent CN201610867872.7). In addition, the FPP substrate is expensive, and the production cost is too high. Summary of the Invention

[0005] β-elemene has technical problems in production, such as high cost, low yield, and environmental pollution. The present invention uses farnesol as a substrate to synthesize β-elemene, thereby increasing the yield of β-elemene synthesized by biological or enzymatic methods.

[0006] The present invention provides a method for synthesizing β-elemene using farnesol as a substrate. First, farnesol kinase catalyzes farnesol to produce farnesyl phosphate (FP). Farnesyl phosphate kinase further catalyzes farnesyl phosphate to produce farnesyl pyrophosphate (FPP). FPP is then catalyzed by germarene A synthase to produce germarene A. Germarene A is then converted to β-elemene in vitro through a Cope rearrangement. During this process, farnesol kinase uses ATP as a coenzyme, which is hydrolyzed to ADP. Farnesyl phosphate kinase can use ATP or CTP as a coenzyme, which is hydrolyzed to ADP or CDP. Polyphosphate kinase 2 (PPK2) is also present in the catalytic system. This enzyme can use inorganic polyphosphate to regenerate ADP or CDP into ATP and CTP, thereby ensuring the continued progress of the reaction.

[0007] The present invention provides the use of farnesol kinase, farnesyl phosphate kinase, germarene A synthase and polyphosphate kinase 2 in synthesizing beta-elemene.

[0008] In one embodiment, the application is to use farnesol as a substrate and farnesol kinase, farnesyl phosphate kinase, germarene A synthase and polyphosphate kinase 2 or microbial cells overexpressing farnesol kinase, farnesyl phosphate kinase, germarene A synthase and polyphosphate kinase 2 as catalysts to catalyze the synthesis of β-elemene.

[0009] In one embodiment, the overexpression is to express one or more genes encoding farnesol kinase, farnesyl phosphate kinase, germarene A synthase and polyphosphate kinase 2 through a vector, or to integrate the genes into the host genome for expression.

[0010] In one embodiment, the overexpression is to use one vector to co-express the genes of the above four enzymes; or to use multiple vectors to co-express the genes of the above four enzymes, each vector expressing at least one enzyme gene, and each vector expressing a different gene.

[0011] In one embodiment, the overexpression of the microorganism is to express 1 to 4 enzyme genes on one vector: one vector expresses 1 enzyme gene; or one vector co-expresses 4 enzyme genes; or one vector co-expresses 2 to 3 enzyme genes, wherein the gene encoding farnesol kinase and the gene encoding farnesyl phosphate kinase are on one vector.

[0012] In one embodiment, when the genes encoding the four enzymes are connected to the vector, each gene is preceded by a T7 promoter and an RBS binding site, and each gene is followed by a T7 terminator.

[0013] In one embodiment, the vector includes but is not limited to pETDuet-1, pACYDuet-1, pRSFDuet-1 and pCDFduet-1.

[0014] In one embodiment, the recombinant cell uses Escherichia coli as a host, including but not limited to Escherichia coli BL21 (DE3).

[0015] In one embodiment, the farnesol kinase is derived from Arabidopsis thaliana, Capsella rubella, Eutrema salsugineum, or Camelina sativa. Alternatively, the amino acid sequence of the farnesol kinase is a sequence with NCBI accession numbers NP_200664.1, XP_006280848.1, XP_006401067.1, or XP_010443595.1. Alternatively, the nucleotide sequence of the farnesol kinase is a sequence with NCBI accession numbers NM_125242.4, XM_006280786.2, XM_006401004.2, or XM_010445293.2.

[0016] In one embodiment, the farnesyl phosphate kinase is derived from Arabidopsis thaliana, Brassica napus, or Camelina sativa. Alternatively, the amino acid sequence of the farnesyl phosphate kinase is the sequence with NCBI accession numbers BAD43853.1, XP_013680590.1, or XP_010503534.1. Alternatively, the nucleotide sequence of the farnesol kinase is the sequence with NCBI accession numbers AK176090.1, XM_013825136.2, or XM_010505232.1.

[0017] In one embodiment, the polyphosphate kinase 2 is derived from Agrobacterium tumefaciens or Ralstonia eutropha. Alternatively, the amino acid sequence of the polyphosphate kinase 2 is the sequence with NCBI accession numbers AAK86947.2 and YP_725721. Alternatively, the nucleotide sequence of the polyphosphate kinase 2 is the sequence with NCBI accession numbers AE007869.2, REGION: 1132974..1135172, and NC_008313.1, Region: 1317773..1318885.

[0018] In one embodiment, the germarene A synthase is derived from Helianthus annuu, Lactucasativa, Mycobacterium kansasii, or Rhodococcus erythropolis. Alternatively, the amino acid sequence of the germarene A synthase is a sequence with NCBI accession numbers AAY41422.2, AAM11627.1, KEP43283.1, or GCB53675.1. Alternatively, the nucleic acid sequence of the germarene A synthase is a sequence with NCBI accession numbers DQ016668, AF489965, JNDJ01000040.1REGION:133397..134536, or BHXB01000001.1REGION:94040..94993.

[0019] The present invention also provides a combination of recombinant cells capable of synthesizing β-elemene using farnesol as a substrate; the combination of recombinant cells consists of recombinant cells that overexpress one or more of farnesol kinase, farnesyl phosphate kinase, polyphosphate kinase 2 and germarene A synthase, and each recombinant cell does not repeat the expression with other recombinant cells.

[0020] In one embodiment, the combinatorial overexpression of the recombinant cell is to co-express the genes of four enzymes using multiple vectors, each vector expresses at least one enzyme gene, and the genes expressed by each vector are different.

[0021] In one embodiment, the combined overexpression of the recombinant cell is a vector that co-expresses 1 to 4 enzyme genes: one vector expresses 1 enzyme gene, for a total of 4 vectors; or one vector co-expresses 4 enzyme genes; or one vector co-expresses 2 to 3 enzyme genes, for two vectors, wherein the gene encoding farnesol kinase and the gene encoding farnesyl phosphate kinase are on one vector.

[0022] In one embodiment, when the genes encoding the four enzymes are connected to the vector, each gene is preceded by a T7 promoter and an RBS binding site, and each gene is followed by a T7 terminator.

[0023] In one embodiment, the vector includes but is not limited to pETDuet-1, pACYDuet-1, pRSFDuet-1 and pCDFduet-1.

[0024] In one embodiment, the recombinant cell or combination of recombinant cells is hosted by Escherichia coli, such as Escherichia coli BL21 (DE3).

[0025] In particular, the present invention also provides a recombinant cell capable of synthesizing β-elemene using farnesol as a substrate. The recombinant cell expresses genes encoding farnesol kinase, farnesyl phosphate kinase, polyphosphate kinase 2, and germarene A synthase. The recombinant cell uses Escherichia coli as a host, pRSFDuet-1 as a vector to express the genes encoding farnesol kinase and farnesyl phosphate kinase, and pETDuet-1 as a vector to express the genes encoding polyphosphate kinase 2 and germarene A synthase. Each gene is preceded by a T7 promoter and an RBS binding site, and followed by a T7 terminator.

[0026] The present invention also provides a method for producing β-elemene by whole-cell catalysis, which utilizes the recombinant cell or a combination of recombinant cells of the present invention as a whole-cell catalyst and uses farnesol as a substrate to synthesize β-elemene.

[0027] In one embodiment, the whole-cell catalyst is prepared by culturing and propagating recombinant cells or a combination of recombinant cells, allowing the recombinant cells or the combination of recombinant cells to express the four enzymes, and then collecting the recombinant cells. When using the whole-cell catalyst, in addition to providing a substrate, it is necessary to maintain an appropriate temperature and pH, and, if necessary, provide coenzymes or nutrients to help the whole-cell catalyst better perform its catalytic function.

[0028] In one embodiment, the whole-cell conversion production system includes a cell wet weight of 1-200 g / L, farnesol 1-100 g / L, ATP 0-1 g / L, CTP 0-1 g / L, sodium hexametaphosphate 300 g / L, pH 5.0-9.0; and the reaction is carried out at 15-40° C. for 1-48 hours.

[0029] Beneficial effects

[0030] The invention constructs a genetically engineered bacterium capable of enhancing the expression of four enzymes for use in the production of beta-elemene. The invention uses farnesol as a substrate, which is relatively cheap and easily available.

[0031] The present invention uses a reasonable expression strategy to express farnesol kinase and farnesyl phosphate kinase while also expressing polyphosphate kinase 2, thereby achieving coenzyme regeneration of ATP and CTP, effectively ensuring the continuous progress of the enzyme-catalyzed reaction, reducing production costs, and increasing the yield of β-elemene. A maximum of 17 g / L of β-elemene can be obtained by adding 20 g / L of farnesol and reacting for 24 hours. DETAILED DESCRIPTION

[0032] 1. Strains and plasmids involved in the present invention

[0033] The pRSFDuet-1, pETDuet-1, pCDFDuet-1, pACYDuet-1 plasmids and Escherichia coli BL21 (DE3) were purchased from Novagen.

[0034] 2. Construction of multi-gene co-expression system and cell culture

[0035] There are currently many methods for co-expressing multiple genes in Escherichia coli (for example, the method described in the article "Strategies for co-expressing multiple genes in Escherichia coli, Chinese Journal of Biotechnology, 2012, 32(4):117-122"). The present invention uses the method described in Liu Xianglei's doctoral thesis (Synthetic Biology Technology for Transforming Escherichia coli to Produce Shikimic Acid and Resveratrol, 2016, Shanghai Institute of Pharmaceutical Industry) to construct recombinant Escherichia coli. In the following examples, when co-expressing multiple genes, each gene contains the T7 promoter and RBS binding site of Escherichia coli BL21 (DE3), and each gene is followed by a T7 terminator. Theoretically, because each gene is preceded by T7 and RBS, the expression intensity of the gene is not greatly affected by the order of gene arrangement on the plasmid. The constructed plasmid is heat-transfected into Escherichia coli competent cells and coated on a monoclonal antibody or mixed antibiotic solid plate. Positive transformants are screened to obtain recombinant Escherichia coli.

[0036] Cell culture: According to the classic recombinant E. coli culture and induced expression protocol, the recombinant E. coli was transferred to LB fermentation medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L) at a volume ratio of 2%. 600 After the p-value reached 0.6-0.8, IPTG was added to a final concentration of 0.4 mM and cultured at 20°C for 8 h to induce expression. After the induction of expression, the cells were collected by centrifugation at 4°C, 8000 rpm / min for 20 min.

[0037] 3. Selection of relevant enzymes

[0038] Polyphosphate kinase 2

[0039] The gene atppk encoding polyphosphate kinase 2 from Agrobacterium tumefaciens was selected. The sequence of the gene atppk is the sequence with accession number AE007869.2 REGION: 1132974..1135172 on NCBI, and the corresponding amino acid sequence is AAK86947.2;

[0040] The gene reppk encoding polyphosphate kinase 2 from Ralstonia eutropha was selected. The sequence of the gene reppk is the sequence with accession number NC_008313.1Region:1317773..1318885 on NCBI, and the corresponding amino acid sequence is YP_725721.

[0041] 4. Sample Detection and Analysis

[0042] The β-elemene content was determined according to the literature (Metabolic engineering of Saccharomyces cerevisiae for production of germacrene A, aprecursor of beta-elemene. J Ind Microbiol Biotechnol (2017) 44:1065–1072). Farnesol has a low solubility. The conversion process in the present invention involves the addition of an excess of substrate, which dissolves during the reaction. At high concentrations, the product precipitates during the reaction. The product was diluted with ethanol before measurement.

[0043] Farnesol kinase and farnesyl phosphate kinase activities were determined according to the literature: L. Thai, J. Waechter, Farnesolis utilized for isoprenoid biosynthesis in plant cells via farnesylpyrophosphate formed by successive monophosphorylation reactions, Proc Natl Acad Sci US A. 96(23)(1999)13080-13085.

[0044] Germarene A synthase activity was determined according to the literature Metabolic engineering of Saccharomyces cerevisiae for production of germacrene A, a precursor of beta-elemene, J Ind Microbiol Biotechnol (2017) 44:1065–1072.

[0045] Specific enzyme activity (U / mg) is defined as the units of enzyme activity per mg of enzyme. One unit of enzyme activity (U) is defined as the amount of enzyme required to generate 1 μmol of product in 1 min.

[0046] 5. Cope rearrangement: refer to https: / / baike.baidu.com / item / Cope%E9%87%8D%E6%8E%92 / 6776025.

[0047] Example 1: Screening and expression of farnesol kinase

[0048] Farnesol kinase is widely distributed in plants. We synthesized the farnesol kinases atfolk, crfolk, esfolk, and csfolk from the NCBI farnesol kinase gene information from Arabidopsis thaliana, Capsella rubella, Eutrema salsugineum, and Camelina sativa, respectively. The amino acid sequences are listed under NCBI accession numbers NP_200664.1, XP_006280848.1, XP_006401067.1, and XP_010443595.1, while the nucleotide sequences are listed under NCBI accession numbers NM_125242.4, XM_006280786.2, XM_006401004.2, and XM_010445293.2.

[0049] The synthesized genes were separately connected to the pRSFDuet-1 vector and induced to express in Escherichia coli BL21 (DE3) to obtain four recombinant Escherichia coli.

[0050] Induction expression method: The recombinant E. coli was inoculated into LB fermentation medium at a volume ratio of 2%. 600 When the pH reaches 0.6-0.8, IPTG is added to a final concentration of 0.4 mM and cultured at 20°C for 8 hours to induce expression. After induction, the cells are harvested by centrifugation at 8000 rpm for 20 minutes at 4°C. The enzyme is purified using a Histag tag after cell lysis, and the activity of the purified enzyme is measured.

[0051] When farnesol was used as substrate, the specific enzyme activities of the enzymes expressed by the farnesol kinase genes atfolk, crfolk, esfolk, and csfolk were 152, 143, 161, 179, and 88 U / mg, respectively.

[0052] Example 2: Screening and expression of farnesyl phosphate kinase

[0053] Farnesyl phosphate kinases are widely distributed in plants. Farnesyl phosphate kinases atfylk, bnfylk, and csfylk were fully synthesized based on the farnesyl phosphate kinase gene information from Arabidopsis thaliana, Brassica napus, and Camelina sativa on NCBI. The amino acid sequences are from NCBI accession numbers BAD43853.1, XP_013680590.1, and XP_010503534.1, respectively. The nucleotide sequences are from NCBI accession numbers AK176090.1, XM_013825136.2, and XM_010505232.1. The synthesized genes were individually ligated into the pRSFDuet-1 vector to generate three recombinant Escherichia coli. Expression and purification were performed using the same methods as in Example 1.

[0054] When farnesyl phosphate is used as substrate, the specific enzyme activities of the enzymes expressed by the farnesyl phosphate kinase genes atfylk, bnfylk, and csfylk are 66, 54, and 87 U / mg, respectively, when ATP is used as coenzyme; when CTP is used as coenzyme, the specific enzyme activities of the enzymes expressed by the farnesyl phosphate kinase genes atfylk, bnfylk, and csfylk are 48, 46, and 66 U / mg, respectively.

[0055] Example 3: Screening and expression of gemmaene A synthase

[0056] Germaene A synthase is widely distributed in various organisms. Full synthesis of the germarene A synthase genes hagas, lsgas, mkga, and regas was performed based on the NCBI accession numbers AAY41422.2, AAM11627.1, KEP43283.1, and GCB53675.1, respectively. Nucleic acid sequences are from NCBI accession numbers DQ016668, AF489965, JNDJ01000040.1REGION:133397..134536, and BHXB01000001.1REGION:94040..94993. The synthesized genes were ligated into pETDuet-1 to obtain four types of recombinant E. coli. The expression and purification were carried out in the same manner as in Example 1.

[0057] When farnesyl pyrophosphate was used as substrate, the specific enzyme activities of the enzymes expressed by the germarene A synthase genes hagas, lsgas, mkgas, and regas were 88, 66, 87, and 68 U / mg, respectively.

[0058] Example 4: Construction of recombinant Escherichia coli expressing four enzymes simultaneously

[0059] Recombinant E. coli construction:

[0060] Polyphosphate kinase 2 can catalyze the conversion of ADP to ATP using inorganic phosphate. Polyphosphate kinase 2 can catalyze the conversion of ADP or CDP to ATP or CTP using inorganic phosphate. As shown in Table 1, four plasmids, pETDuet-1, pACYDuet-1, pRSFDuet-1, and pCDFduet-1, were selected. The genes encoding the four enzymes were ligated to the same plasmid, or separately to two plasmids (each expressing 2-3 genes), or to four plasmids. Each gene was preceded by the T7 promoter and RBS binding site of Escherichia coli BL21 (DE3), and each gene was followed by a T7 terminator. The constructed recombinant plasmids were transformed into Escherichia coli BL21, and positive transformants were screened using mixed antibiotic plates based on the resistance genes on the different plasmids, resulting in recombinant E. coli with enhanced expression of the four genes.

[0061] Recombinant E. coli was induced for expression. After induction, the cells were collected and incubated in a 100 mL reaction system containing 30 g / L cell wet weight, 20 g / L farnesol, 0.5 g / L ATP, 0 g / L CTP, and 30 g / L sodium hexametaphosphate at pH 7.0 at 30°C for 24 hours. Farnesol has a low solubility, so it was dissolved during conversion. After conversion, the solution was diluted with ethanol and dissolved. The concentration of β-elemene in the reaction solution was determined by gas chromatography. The results are shown in Table 1.

[0062] Table 1

[0063]

[0064]

[0065] Example 5: In vitro synthesis of β-elemene using four enzymes

[0066] The atppk gene encoding polyphosphate kinase 2 from Agrobacterium tumefaciens, along with three genes: atfolk, csfylk, and hagas, were ligated into the pETDuet-1 vector to obtain four recombinant vectors. The four recombinant vectors were then transformed into Escherichia coli BL21 to obtain recombinant Escherichia coli expressing the four enzymes. Four pure enzymes were expressed and purified using the same method as in Example 1. A 100 mL reaction system was then added with 2 mg of each of the four pure enzymes, 20 g / L farnesol, 0.5 g / L ATP, 0.5 g / L CTP, and 30 g / L sodium hexamethaphosphate at pH 7.0. The reaction was carried out at 30°C for 24 hours. After Cope rearrangement, the β-elemene concentration in the reaction solution was determined by gas chromatography to be 16 g / L.

[0067] Example 6: In vitro synthesis of β-elemene using four enzymes

[0068] The gene encoding polyphosphate kinase 2 (reppk) from Ralstonia eutropha, along with three genes (atfolk, csfylk, and hagas) were ligated into the pETDuet-1 vector to generate four recombinant vectors. These recombinant vectors were then transformed into Escherichia coli BL21 to produce recombinant E. coli expressing each of the four enzymes. The four pure enzymes were expressed and purified using the same method as in Example 1. A 100 mL reaction system was then added with 2 mg of each of the four pure enzymes, 20 g / L farnesol, 0.5 g / L ATP, 0 g / L CTP, and 30 g / L sodium hexamethaphosphate at pH 7.0. The reaction was carried out at 30°C for 24 hours. After Cope rearrangement, the β-elemene concentration in the reaction solution was determined by gas chromatography to be 14 g / L.

[0069] Example 7: Synthesis of β-elemene using four enzyme cells

[0070] The four genes atfolk, atfylk, atppk, and hagas were ligated into the pETDuet-1 vector to obtain four recombinant vectors. The four recombinant vectors were then transformed into Escherichia coli BL21 to obtain recombinant Escherichia coli expressing each of the four enzymes. The recombinant E. coli was induced to express the enzymes using the same method as in Example 1. Then, 20 g / L of each of the four whole cells, 20 g / L of farnesol, 0.5 g / L of ATP, 0 g / L of CTP, and 30 g / L of sodium hexamethaphosphate were added to a 100 mL reaction system at pH 7.0. The reaction was carried out at 30°C for 24 hours. After Cope rearrangement, the β-elemene concentration in the reaction solution was determined by gas chromatography to be 13 g / L.

[0071] Example 8: Synthesis of β-elemene using recombinant Escherichia coli whole cells

[0072] The following two recombinant bacteria were constructed: Escherichia coli BL21(DE3) / Escherichia coli BL21(DE3) / pRSFDuet-1-atfolk-atfylk (named E1) and Escherichia coli BL21(DE3) / pETDuet-1-atppk-hagas (named E2).

[0073] According to the method described in Example 1, E1 and E2 were induced to express separately, and then the cells were collected. In a 100 mL reaction system, the wet weight of E1 cells was 30 g / L, the wet weight of E2 cells was 30 g / L, farnesol was 20 g / L, ATP was 0.5 g / L, CTP was 0.5 g / L, sodium hexametaphosphate was 30 g / L, and the pH was 7.0. The reaction was carried out at 30°C for 24 hours. After Cope rearrangement, the β-elemene concentration in the reaction solution was determined by gas chromatography to be 14 g / L.

[0074] Example 9: Synthesis of β-elemene using recombinant Escherichia coli whole cells

[0075] Recombinant Escherichia coli BL21(DE3) / pRSFDuet-1-lsgas-atppk (designated E7) and Escherichia coli BL21(DE3) / pCDFDuet-1-esfolk-bnfylk (designated E8) were constructed. Expression of the recombinant bacteria was induced according to the method described in Example 1, and the cells were then collected. In a 100 mL reaction system, the wet weight of E7 cells was 30 g / L, the wet weight of E8 cells was 40 g / L, farnesol was 20 g / L, ATP was 0.5 g / L, CTP was 0.5 g / L, and sodium hexamethaphosphate was 30 g / L. The reaction was carried out at 30°C for 24 hours. After Cope rearrangement, the β-elemene concentration in the reaction solution was determined by gas chromatography to be 14 g / L.

[0076] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of farnesol kinase, farnesyl phosphate kinase, germarene A synthase and polyphosphate kinase 2 in the synthesis of β-elemene, characterized in that: β-elemene is synthesized using farnesol as a substrate and a catalyst; the catalyst is (a) or (b): (a) Farnesol kinase, farnesyl phosphate kinase, germarene A synthase, and polyphosphate kinase 2; (b) Microbial cells overexpressing farnesol kinase, farnesyl phosphate kinase, germarene A synthase, and polyphosphate kinase 2.

2. The use according to claim 1, characterized in that The overexpression is to express the genes encoding farnesol kinase, farnesyl phosphate kinase, germarene A synthase and polyphosphate kinase 2 through a vector, or to integrate them into the host genome for expression.

3. The use according to claim 2, characterized in that The overexpression is to use one vector to co-express the genes of farnesol kinase, farnesyl phosphate kinase, germarene A synthase and polyphosphate kinase 2; or to use multiple vectors to co-express the genes of farnesol kinase, farnesyl phosphate kinase, germarene A synthase and polyphosphate kinase 2, each vector expresses at least one enzyme gene, and the genes expressed by each vector are different.

4. The use according to claim 2 or 3, characterized in that The vectors include but are not limited to pETDuet-1, pACYDuet-1, pRSFDuet-1 and pCDFduet-1.

5. A combination of recombinant cells capable of synthesizing β-elemene using farnesol as a substrate, comprising recombinant cells that overexpress one or more of farnesol kinase, farnesyl phosphate kinase, polyphosphate kinase 2, and germarene A synthase, respectively, so that the combination of recombinant cells simultaneously expresses farnesol kinase, farnesyl phosphate kinase, polyphosphate kinase 2, and germarene A synthase; and each recombinant cell does not duplicate expression with other recombinant cells.

6. The recombinant cell combination according to claim 5, characterized in that The recombinant cell uses Escherichia coli as a host bacterium, including Escherichia coli BL21.

7. A method for producing β-elemene by whole-cell catalysis, characterized in that: The method utilizes the combination of recombinant cells according to claim 5 or 6 as a whole-cell catalyst and uses farnesol as a substrate to synthesize β-elemene.

8. The method according to claim 7, wherein The whole-cell catalytic production system includes a cell wet weight of 1-200 g / L, farnesol 1-100 g / L, ATP 0.5-1 g / L, CTP 0.5-1 g / L, sodium hexametaphosphate 300 g / L, pH 5.0-9.0; and a reaction temperature of 15-40° C. for 1-48 hours.

9. Use of the recombinant cell combination according to claim 5 or 6 in producing β-elemene, or a product containing β-elemene, or a substance with β-elemene as a precursor.

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