An engineered bacterium producing β-elemene and its application
By constructing recombinant genetically engineered bacteria and using a lettuce-derived germarene A synthase mutant to optimize the biosynthesis of β-elemene in Escherichia coli, the problems of difficult separation and purification and high cost in existing technologies were solved, achieving efficient production.
Patent Information
- Application Number
- CN202210818797.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-12
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2042-07-12
AI Technical Summary
In the existing technology, the acquisition of β-elemene mainly relies on separation and extraction from Curcuma aromatica, which has the problems of difficult separation and purification, low yield and high cost, hindering its large-scale production and application.
A recombinant genetically engineered bacterium containing a mutant of the lettuce-derived germarene A synthase gene was constructed. The lettuce-derived germarene A synthase was expressed in Escherichia coli, and the culture medium and fermentation conditions were optimized to improve the biosynthesis efficiency of β-elemene.
Efficient biosynthesis of β-elemene was achieved in Escherichia coli, with a yield of 126.39 mg/L and a significantly improved space-time yield, laying the foundation for the industrial production of β-elemene.
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Figure CN115975893B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, relates to an engineered bacterium for producing β-elemene and its application, and particularly relates to a lettuce-derived germarene A synthase and the preparation of its mutant strain and the efficient biosynthesis of β-elemene. Background Art
[0002] β-Elemene, a sesquiterpenoid isolated from the traditional Chinese medicine Curcuma aromatica, has broad application prospects due to its potent antitumor activity, broad spectrum of action, mild toxicity, and resistance to drug resistance. Currently, β-elemene is primarily isolated and extracted from Curcuma aromatica. However, traditional production methods are difficult to separate and purify, resulting in low yields and high costs, severely hindering the large-scale production and application of β-elemene. With the advancement of synthetic biology, the use of microbial cell factories to biosynthesize natural pharmaceuticals has become a research hotspot, offering new approaches for the production of β-elemene.
[0003] β-Elemene is a sesquiterpene belonging to the sesquiterpenoid family. Generally, plant-derived sesquiterpenes are based on isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) as their basic structural components. IPP and DMAPP are primarily synthesized in plants via the mevalonate pathway (MVA) and the methylerythritol 4-phosphate pathway (MEP). Under the catalysis of isopentenyltransferase, one molecule of IPP and one molecule of DMAPP undergo head-to-tail condensation to form geranyl pyrophosphate (GPP). GPP then condenses with one molecule of IPP to form farnesyl pyrophosphate (FPP). Germaene A synthase, acting on FPP, produces germanene A, which undergoes a Cope rearrangement to produce β-elemene. Currently, a germanene A biosynthetic pathway has been constructed in yeast cells, enabling the production of β-elemene. However, yeast fermentation requires a long time; the shortest fermentation time, approximately 72 hours, yields 309.8 mg / L, with a space-time yield of 4.20 mg / L / h. After 144 hours of induction culture, the yeast engineered bacteria constructed by Zhang et al. achieved a yield of 469 mg / L, with a space-time yield of 3.26 mg / Lh.
[0004] Currently, the biosynthesis of β-elemene reported in domestic and foreign literature is mostly carried out in Saccharomyces cerevisiae. There are no reports on the complete biosynthesis of β-elemene in Escherichia coli. However, Escherichia coli has great advantages in the total biosynthesis of active ingredients of traditional Chinese medicine due to its rapid growth, simple genetic manipulation, and simple and mature fermentation methods. Summary of the Invention
[0005] The first object of the present invention is to address the deficiencies of the prior art and provide a recombinant bacterium that contains or expresses genes for enzymes related to the biosynthetic pathways of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), as well as farnesyl pyrophosphate synthase, and a lettuce-derived germacene A synthase gene mutant.
[0006] Preferably, the nucleotide sequence of the gene encoding the lettuce-derived germarene A synthase gene is shown in SEQ.ID NO. 1. The amino acid sequence of the lettuce-derived germarene A synthase gene is shown in SEQ.ID NO. 2.
[0007] Preferably, the lettuce-derived germaiene A synthase mutant is obtained by subjecting at least one of positions 229, 243, 364, 392, and 410 in the amino acid sequence of the lettuce-derived germaiene A synthase BL21 to site-directed mutagenesis, wherein the I at position 364 is mutated to K, the T at position 410 is mutated to S, the T at position 392 is mutated to A, the T at position 392 is mutated to V, the A at position 229 is mutated to S, and the S at position 243 is mutated to N.
[0008] Preferably, the lettuce-derived germaiene A synthetase mutant is obtained by mutating the 364th I to K and the 410th T to S in the amino acid sequence of the lettuce-derived germaiene A synthetase.
[0009] The second object of the present invention is to provide a recombinant genetically engineered bacterium, characterized in that the recombinant vector constructed by the gene encoding the germarene A synthase from lettuce, the pBbA5c-MM recombinant vector, is transformed into Escherichia coli BL21 (DE3) star;
[0010] The recombinant vector constructed by the lettuce-derived germarene A synthetase encoding gene is obtained by connecting the lettuce-derived germarene A synthetase mutant with a pET28a vector.
[0011] Preferably, the nucleotide sequence of the gene encoding the lettuce-derived germarene A synthase gene is shown in SEQ.ID NO. 1. The amino acid sequence of the lettuce-derived germarene A synthase gene is shown in SEQ.ID NO. 2.
[0012] Preferably, the lettuce-derived germarene A synthetase mutant is obtained by site-directed mutation of at least one of positions 38, 58, 229, 243, 364, 392, 410 and 492 in the amino acid sequence of the lettuce-derived germarene A synthetase.
[0013] Preferably, the lettuce-derived germaiene A synthetase mutant is obtained by mutating the 364th I to K and the 410th T to S in the amino acid sequence of the lettuce-derived germaiene A synthetase.
[0014] The third object of the present invention is to provide the use of the above-mentioned recombinant genetically engineered bacteria in the preparation of germaceene A and β-elemene.
[0015] As a preferred application, the specific method is to culture the above-mentioned recombinant genetically engineered bacteria in SBMSE medium to an OD of 600 =0.6-1.0, add IPTG with a final concentration of 0.4 mM and induce culture for 18 hours to obtain a culture solution containing germacene A; and use n-dodecane to cover and collect the volatile germacene A product, which is converted into β-elemene after high temperature.
[0016] To achieve the above object, the technical solution adopted by the present invention is:
[0017] The invention provides a recombinant vector containing a gene encoding the lettuce-derived germarene A synthase or a mutant thereof.
[0018] The recombinant vector constructed by the gene encoding the lettuce-derived germacene A synthase or mutant described in the above technical solution, wherein: the recombinant vector is pET28a-LTC2 I364K-T410S, which is obtained by connecting the lettuce-derived germacene A synthase encoding gene shown in SEQ.ID NO.1 with the pET28a vector.
[0019] The recombinant genetically engineered bacteria prepared by transformation with the recombinant vector described in the above technical solution are E. coli BL21(DE3)star MM, obtained by transforming the recombinant vectors pBbA5c-MM and pET28a-LTC2 I364K-T410S into E. coli BL21(DE3)star. Expression of the recombinant vector pBbA5c-MM in E. coli cells can produce FPP, a precursor of germarene A synthase.
[0020] The use of the germaiene A synthase and mutants described in the above technical solution in the preparation of germaiene A and β-elemene. The use described in the above technical solution, wherein the key component LTC2 is modified using site-directed mutagenesis to improve its catalytic activity. Mutation sites were selected based on literature research and homologous sequence comparison results, and 10 mutants were obtained. The single mutations T410S, T392A, T392V, A229S, S243N, and I364K catalyzed β-elemene production in vitro that was 1.27-fold, 1.11-fold, 1.12-fold, 1.08-fold, 1.10-fold, and 1.21-fold higher than that of the wild type. Next, the LTC2 double mutation vector was constructed. The β-elemene yields of T392A-T410S, S243N-T410S and I364K-T410S after induction culture in SBMSN medium for 18 h were 73.60 mg / L, 98.20 mg / L and 126.39 mg / L, respectively, which were 1.10 times, 1.47 times and 1.89 times the yield of the wild type.
[0021] The present invention also provides an application of the above-mentioned β-elemene engineered strain, which produces β-elemene by inducing the expression of the LTC2 mutant enzyme I364K-T410S and the exogenous MVA biosynthetic pathway in the engineered strain. Specifically, the engineered bacteria E. coli BL21 (DE3) star LTC2 I364K-T410S+pBbA5c-MM are activated and then transferred to SBMSN medium for cultivation until the OD600 is 0.6-1.0, IPTG is added for induction at a final concentration of 0.4 mM, fermented at 25°C for 18 hours, and 10% n-dodecane is added to enrich the product. After the fermentation is completed, the upper layer product is directly collected by centrifugation, and the yield reaches 126.39 mg / L. The engineered bacteria of the present invention have the highest spatiotemporal yield reported so far and are suitable for the industrial production of β-elemene. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Biosynthetic pathway of β-elemene precursor in Escherichia coli and composition of recombinant plasmid
[0023] Figure 2 Optimization of Escherichia coli host bacteria and culture medium
[0024] Figure 3 Comparison of β-elemene production and fermentation OD between LTC2 and its mutants DETAILED DESCRIPTION
[0025] The present invention will be further analyzed below with reference to specific embodiments.
[0026] Figure 1 This is a schematic diagram of the biosynthetic pathway of β-elemene precursor in Escherichia coli and the composition of the recombinant plasmid.
[0027] Example 1 Screening of engineered bacterial strains and culture media for high-yield FPP precursors
[0028] E. coli BL21(DE3), E. coli BW25113, E. coli JM109(DE3), E. coli BL21 txB(DE3) and E. coli BL21 Star(DE3) were selected as host cells, and competent cells were prepared by the CaCl2 method. The pBbA5c-MM plasmid (E. coli expression vector pBbA5c-MevT(CO)-MBIS(CO,ispA)) was introduced into the above-mentioned different E. coli host cells by the heat shock method. Positive clones were screened for chloramphenicol resistance (25 μg / mL) to obtain MM engineered bacteria.
[0029] The successfully constructed MM engineered bacteria were expressed in three different culture media (SBMSN medium, LB medium, and YM9 medium) under IPTG induction, and their ability to biosynthesize FPP was determined. In E. coli, pyrophosphatase exists, and FPP is further hydrolyzed to produce farnesol. Therefore, the ability of the engineered bacteria to produce FPP was inferred by measuring the production of farnesol. Figure 2 Results showed that MM engineered strains grown in different hosts produced varying amounts of farnesol when induced in SBMSN medium. For example, MM-BL21 produced 4.88-fold and 3.34-fold more farnesol in LB and YM9 medium, respectively. MM-BL21 star produced even more farnesol in SBMSN, reaching 7.83 mg / L after 18 hours of shake flask fermentation. Therefore, BL21 star was selected as the expression strain and SBMSN as the medium for subsequent screening.
[0030] The SBMSN medium formula is: peptone 12 g, yeast extract 24 g, KH2PO4 1.7 g, K2HPO4 11.42 g, MgCl2·6H2O 1 g, ammonium oxalate 1.42 g, Tween-80 2 g, dissolved in ddH2O, dilute to 1 L, and filter sterilize with a 0.22 μm filter membrane;
[0031] YM9 medium formula: yeast extract 2g, Na2HPO4 6g, KH2PO4 3g, NaCl 0.5g, NH4Cl 1g, MgSO4 1mM, CaCl2 0.1 Mm, dissolved in ddH2O, dilute to 1L, and filter sterilize with a 0.22μm filter membrane;
[0032] LB medium was prepared as usual.
[0033] Example 2 Construction and Analysis of LTC2 Mutant Enzymes
[0034] Through literature review and homologous sequence alignment, site-directed mutagenesis was performed at positions 38 (threonine), 58 (leucine), 229 (alanine), 243 (serine), 364 (isoleucine), 392 (threonine), 410 (threonine), and 492 (isoleucine) of LTC2 using the Fast Mutagenesis System kit. The mutagenesis primers are shown in Table 1.
[0035] Take 20 μL of PCR product, add 1 μL of DMT enzyme, mix thoroughly, and digest at 37°C for 1 hour. Take 5 μL of DMT enzyme digestion product and add it to 50 μL of E. coli DMT competent cells, mix gently, and let it stand on ice for 30 minutes; heat shock in a water bath at 42°C for 1 minute, remove immediately, and continue to place on ice for 2 minutes; add 500 μL of antibiotic-free LB medium, shake and culture at 37°C, 200 rpm for 1 hour; centrifuge at 5000 rpm for 1 minute, discard the supernatant to a final volume of 200 μL; evenly spread it on LB solid medium containing kanamycin resistance (50 μg / mL), incubate inverted at 37°C overnight to screen for positive clones, and confirm them as mutant plasmids after PCR identification. The mutated plasmids were transformed into BL21 (DE3) for induced expression, extract the crude enzyme solution, add FPP as a substrate, and react at 30°C, 280 rpm for 2 hours. At the same time, samples were collected from the headspace using solid-phase microextraction, and the products were analyzed by GC after the reaction. According to the yield calculation, T410S, T392A, T392V, A229S, S243N and I364K were beneficial to improving the yield of β-elemene produced by FPP catalyzed by LTC2, which was 1.27 times, 1.11 times, 1.12 times, 1.08 times, 1.10 times and 1.21 times that of the wild type.
[0036] Based on this, double mutations were further constructed, resulting in four double mutant genes: LTC2-S243N-T410S, LTC2-I364K-T410S, LTC2-T392A-T410S, and LTC2-T392V-T410S. The mutant plasmids were transformed into BL21(DE3) cells for induced expression. The crude enzyme was extracted and reacted with FPP as a substrate at 30°C, 280 rpm, for 2 hours. Headspace samples were collected using solid-phase microextraction. After the reaction, the products were analyzed by GC. Yield calculations showed that LTC2-T392A-T410S and LTC2-S243N-T410S significantly increased the yield of β-elemene produced by LTC2-catalyzed FPP, respectively; the yields were 1.40-fold and 1.09-fold higher than those of the wild-type.
[0037] Table 1 Primers used in site-directed mutagenesis of LTC2
[0038]
[0039]
[0040] 3 Analysis of β-elemene production by engineered bacteria carrying LTC2 mutant enzyme
[0041] The PET-28a-LTC2-T410S, PET-28a-LTC2-T392A, PET-28a-LTC2-T392V, PET-28a-LTC2-S243N, PET-28a-LTC2-I364K, PET-28a-LTC2-T392A-T410S, PET-28a-LTC2-T392V-T410S, PET-28a-LTC2-S243N-T410S, and PET-28a-LTC2-I364K-T410S plasmids were transformed into E. coli containing the pBbA5c-MM plasmid using the heat shock method. Positive clones were identified using universal primers T7 and T7 in BL21star(DE3) strains by colony PCR. Cultures containing germarene A were then induced and fermented in SBMSN medium for 18 hours to obtain culture fluids. The volatile germarene A product was collected by overlaying with n-dodecane and converted to β-elemene after high-temperature treatment above 260°C. The yield of β-elemene produced by shake-flask fermentation was determined. GC analysis revealed that MM-LTC2-LTC2-I364K-T410S S-BL21 star (126.39 mg / L) exhibited increased β-elemene production compared to the wild-type MM-LTC2-BL21 star (66.71 mg / L). The engineered strain with the highest β-elemene production, MM-LTC2-I364K-T410S-BL21 star, achieved 126.39 mg / L, 1.89 times that of the wild-type strain.
[0042] Figure 3 Comparison of β-elemene production and fermentation OD of LTC2 and its mutants.
[0043] This study uses Escherichia coli as a host to construct an engineered bacterium for the heterologous biosynthesis of β-elemene. By optimizing the host cells and culture medium to increase the supply of the precursor FPP, and by screening and mutagenesis of the key enzyme, germarene A synthase, the yield of β-elemene biosynthesized in E. coli was increased to 126.39 mg / L. Further fermentation in tanks will yield even higher β-elemene yields, laying the foundation for the efficient synthesis of β-elemene, an active anticancer ingredient in traditional Chinese medicine.
Claims
1. A recombinant bacterium, characterized in that The invention relates to an Escherichia coli BL21 (DE3) star that contains or expresses genes for enzymes related to the biosynthetic pathways of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), as well as farnesyl pyrophosphate synthase and a mutant of a lettuce-derived germarene A synthase gene. The mutant of the lettuce-derived germarene A synthase is obtained by mutating the I at position 364 to K and the T at position 410 to S in the amino acid sequence of the lettuce-derived germarene A synthase. The amino acid sequence of the gene encoding the lettuce-derived germarene A synthase gene is shown in SEQ.ID NO.
2.
2. A recombinant genetically engineered bacterium, characterized in that The recombinant vector pBbA5c-MM constructed from the gene encoding the germarene A synthase from lettuce was transformed into Escherichia coli BL21 (DE3) star; The recombinant vector constructed by the lettuce-derived germarene A synthase encoding gene is obtained by connecting a lettuce-derived germarene A synthase mutant with a pET28a vector; the lettuce-derived germarene A synthase mutant is obtained by mutating the I at position 364 to K and the T at position 410 to S in the amino acid sequence of the lettuce-derived germarene A synthase. The amino acid sequence of the gene encoding the lettuce-derived germarene A synthase gene is shown in SEQ.ID NO.
2.
3. A recombinant genetically engineered bacterium according to claim 2, characterized in that The nucleotide sequence of the gene encoding the lettuce-derived germarene A synthase gene is shown in SEQ.ID NO.
1.
4. Use of the recombinant genetically engineered bacteria according to claim 2 or 3 in the preparation of germaceene A and β-elemene.
5. The use according to claim 4, characterized in that The recombinant genetically engineered bacteria according to claim 2 or 3 are cultured in SBMSE medium to an OD 600 =0.6-1.0, IPTG was added for induction culture to obtain a culture solution containing germacene A, and the volatile germacene A product was collected by covering with n-dodecane, and β-elemene was generated after high temperature conversion.