An alkenyl synthase derived from Fischerella thermalis and its applications
By constructing the alfalfaene synthetase gene vector from Fischerella thermolis and expressing it in E. coli, the complex problem of alfalfaene chemical synthesis is solved, and efficient biosynthesis and industrial production are achieved.
Patent Information
- Application Number
- CN202211165529.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Priority Date
- 2022-03-31
- Filing Date
- 2022-09-23
- Publication Date
- 2025-07-22
- Estimated Expiration
- 2042-09-23
AI Technical Summary
In the prior art, the chemical synthesis method of alfalfaene is complex and is not suitable for large-scale production. There are few studies on enzyme catalysts and the products are not specific, making it difficult to achieve efficient biosynthesis.
The recombinant vector was constructed with the alfalfaene synthase gene from Fischerella thermolis and transformed with recombinant genetically engineered bacteria. The pure enzyme was prepared using the E. coli expression system, and alfalfaene was generated by bio-transforming farnesyl pyrophosphate. The reaction conditions were mild and the selectivity was high.
High-efficiency biosynthesis of alfalfaene was achieved in E. coli, with a yield of 0.25mg·L-1·h-1, green and pollution-free, suitable for industrial production.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and relates to a carotenoid synthase, a gene, a vector, an engineered bacterium and an application derived from Fischerella thermalis, and particularly relates to a carotenoid synthase, a gene, a vector, an engineered bacterium derived from Fischerella thermalis and an application thereof for preparing carotenoid. Background Art
[0002] Carotenoid is a sesquiterpene volatile component, and may synthesize seco-sativene through oxidative cleavage between C-4 and C-5. The latter has various antifungal activities. At the same time, seco-sativene structure analogues helminthosporol (C 15 H 24 O2) and helminthosporic acid (C 15 H 24 O3) have growth-promoting activities, drechslerine A (C 14 H 24 O2) exhibits strong neuraminidase inhibitory activity, bipolenin G (C 15 H 24 O3) and 11-hydroxylhelminthosporol (C 15 H 24 O3) exhibit obvious anti-NO production activities. Therefore, carotenoid is a potential active compound.
[0003] At present, the synthesis of carotenoid mainly relies on chemical synthesis, and the chemical synthesis method has a complex process and is not conducive to large-scale production. Using the enzyme-catalyzed method for the biosynthesis of carotenoid has certain advantages, such as mild reaction conditions, high selectivity and high product specificity. And the primary prerequisite for enzyme catalysis is the acquisition of high-quality biocatalysts. At present, there is little research on carotenoid synthase, and the products are not specific, mostly generated as by-products. Therefore, studying the cloning and expression of carotenoid synthase genes with high catalytic efficiency and product specificity is helpful for the biosynthesis of carotenoid, and at the same time, it can further utilize metabolic engineering or biosynthesis-related technologies for the cell factory production of carotenoid and seco-sativene. The present invention has important significance. Summary of the Invention
[0004] The present invention aims to provide a germacrene synthase, a gene thereof, and an application thereof in catalyzing the biosynthesis of germacrene from farnesyl pyrophosphate (FPP), in view of the fact that there is little research on germacrene synthase. The germacrene synthase belongs to sesquiterpene synthase, has good activity in catalyzing the generation of germacrene from the substrate FPP, and the only product is germacrene.
[0005] The first object of the present invention is to disclose a germacrene synthase derived from Fischerella thermalis.
[0006] The second object of the present invention is to disclose a gene encoding the germacrene synthase derived from Fischerella thermalis.
[0007] The third object of the present invention is to disclose a recombinant vector constructed from the gene encoding the germacrene synthase derived from Fischerella thermalis.
[0008] The fourth object of the present invention is to disclose a recombinant genetically engineered bacterium prepared by transforming the above recombinant vector.
[0009] The fifth object of the present invention is to disclose the application of the gene encoding the germacrene synthase from the above source in the preparation of the germacrene synthase derived from Fischerella thermalis.
[0010] The sixth object of the present invention is to disclose the application of the germacrene synthase derived from Fischerella thermalis in the preparation of germacrene.
[0011] To achieve the above objects, the technical solution adopted by the present invention is as follows:
[0012] A germacrene synthase derived from Fischerella thermalis, wherein the amino acid sequence of the enzyme is shown as SEQ.ID NO.2.
[0013] The gene encoding the germacrene synthase derived from Fischerella thermalis according to the above technical solution, wherein the nucleotide sequence of the encoding gene is shown as SEQ.ID NO.1.
[0014] The recombinant vector constructed from the gene encoding the germacrene synthase derived from Fischerella thermalis according to the above technical solution, wherein: the recombinant vector is FtTPS-pET28a, which is obtained by ligating the gene encoding the germacrene synthase shown as SEQ.ID NO.1 with the pET28a vector.
[0015] The recombinant genetically engineered bacterium prepared by transforming the recombinant vector described in the above technical solution, wherein: the recombinant genetically engineered bacterium is E.Coil BL21 codon plus / pET 28a / FtTPS, which is obtained by transforming the recombinant vector FtTPS-pET28a into E.CoilBL21 codon plus competent cells.
[0016] Use of the albaflavenone synthase-encoding gene derived from Fischerella thermalis described in the above technical solution in the preparation of albaflavenone synthase derived from Fischerella thermalis.
[0017] The use described in the above technical solution, wherein the use is: transforming the recombinant vector FtTPS-pET28a containing the albaflavenone synthase-encoding gene of Fischerella thermalis into Escherichia coli E.Coil BL21 codonplus, and culturing the obtained recombinant genetically engineered bacterium E.Coil BL21 codon plus / pET 28a / FtTPS on LB medium containing 50 mg / mL kanamycin at 37 °C for 5-7 h until OD 600 = 0.6-0.8, adding IPTG with a concentration of 0.05-0.2 mM, inducing expression at 25 °C for 12-15 h, separating and purifying the induced culture solution to obtain cell bodies containing recombinant albaflavenone synthase, and obtaining albaflavenone synthase by affinity chromatography separation using a nickel column.
[0018] Use of the albaflavenone synthase derived from Fischerella thermalis described in the above technical solution in the preparation of albaflavenone.
[0019] The use described in the above technical solution, wherein the use is: using the purified enzyme obtained by purifying the supernatant after ultrasonic disruption of the wet cell bodies obtained by induced culture of the genetically engineered bacterium containing the albaflavenone synthase-encoding gene of Fischerella thermalis as a catalyst, using farnesyl pyrophosphate as a substrate, and forming a reaction system in a buffer solution with a pH of 7.0-7.5 under the action of dithiothreitol, MgCl2 and glycerol, and performing a biotransformation reaction at 30 °C, separating and purifying the reaction solution to obtain albaflavenone.
[0020] The use described in the above technical solution, wherein in the reaction system, the dosage of the catalyst is 50-100 μg / mL, the final concentration of the substrate is 2-5 μg / mL, the final concentrations of dithiothreitol and MgCl2 are 1-3 mM and 10-30 mM respectively, and the final volume concentration of glycerol is 10%.
[0021] The application according to the above technical solution, wherein the catalyst is prepared by the following method: inoculating a genetically engineered bacterium encoding alfa - farnesene synthase derived from Fischerella thermalis in an LB liquid medium containing 50 μg / mL kanamycin, culturing overnight in a shaker at 37 °C; then inoculating the culture solution into an LB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 1% by volume, culturing at 37 °C until the OD 600 reaches 0.6 - 0.8, adding IPTG with a final concentration of 0.05 - 0.2 mM, inducing at 25 °C for 12 - 15 h, then centrifuging to collect wet cells; resuspending the wet cells with a phosphate buffer at pH 7.0 - 7.4, ultrasonically disrupting the cells, centrifuging to take the supernatant, filtering with a 0.22 μm cellulose acetate filter membrane, subjecting the filtrate to nickel column affinity chromatography, collecting the target component to obtain pure enzyme.
[0022] The present invention provides an alfa - farnesene synthase (i.e., FtTPS) derived from Fischerella thermalis with the amino acid sequence shown in SEQ.ID NO.2. It has been experimentally proven that the protein with the above structure belongs to a sesquiterpene synthase and can catalyze FPP to generate a certain amount of alfa - farnesene. It is not difficult to imagine that without changing the protein properties, appropriately changing the amino acid sequence still has the properties of the alfa - farnesene synthase of the present invention. For example, a conservative variant polypeptide of the amino acid sequence SEQ ID NO.2, or its active fragment, or its derivative.
[0023] The present invention provides a gene encoding alfa - farnesene synthase (i.e., FtTPS gene) derived from Fischerella thermalis, and the nucleotide sequence of the encoding gene is shown in SEQ ID No.1.
[0024] The present invention provides a recombinant vector containing the gene encoding alfa - farnesene synthase of Fischerella thermalis. Further, the recombinant vector is prepared by the following method: ligating the gene encoding alfa - farnesene synthase with the pET28a vector to obtain a ligation product FtTPS - pET28a, which is a recombinant vector containing the gene encoding alfa - farnesene synthase of Fischerella thermalis. Transforming the recombinant vector FtTPS - pET28a into E.coli DH5α competent cells, culturing in an antibiotic - free LB medium at 37 °C and 180 rpm for 1 h in a shaker, centrifuging to take the precipitate and then coating it on an LB plate containing 50 mg / mL kanamycin, culturing overnight and then picking monoclonal colonies for PCR and extracting the recombinant vector, thus obtaining a recombinant vector containing the gene encoding alfa - farnesene synthase of Fischerella thermalis.
[0025] The present invention also provides a recombinant genetically engineered bacterium containing the above-mentioned Fischerella thermalis albaene synthase-encoding gene or recombinant vector. The recombinant genetically engineered bacterium is prepared by the following method: The recombinant vector FtTPS-pET28a is transferred into E.Coil BL21 codonplus competent cells by heat shock method, and positive clones are screened on an LB solid plate containing 50 μg / mL kanamycin to obtain the engineered bacterium E.Coil BL21 codon plus / pET 28a / FtTPS.
[0026] The present invention provides an application of the above-mentioned Fischerella thermalis albaene synthase-encoding gene in the preparation of recombinant albaene. Specifically, the application is as follows: A recombinant vector containing the Fischerella thermalis albaene synthase-encoding gene is transformed into Escherichia coli BL21 codon plus, and the obtained recombinant genetically engineered bacterium is cultured at 37 °C for 5 - 7 h to OD 600 = 0.6 - 0.8 in an LB medium containing 50 mg / mL kanamycin, IPTG with a concentration of 0.05 - 0.2 mM is added, and induced expression is carried out at 25 °C for 12 - 15 h. The induced culture solution is separated and purified to obtain cell bodies containing the recombinant albaene synthase gene, and pure albaene synthase is obtained by affinity chromatography separation using a nickel column.
[0027] The present invention provides an application of the albaene synthase derived from Fischerella thermalis in the preparation of albaene. The application is as follows: Using the pure enzyme purified from the supernatant after ultrasonic disruption of the wet cell bodies obtained by induced culture of the genetically engineered bacterium containing the albaene synthase-encoding gene derived from Fischerella thermalis as a catalyst, with farnesyl pyrophosphate (FPP) as a substrate, in the presence of dithiothreitol, MgCl2 and dilute glycerol (i.e., glycerol), a reaction system is formed in a buffer solution with a pH of 7.0 - 7.5, and a biotransformation reaction is carried out at 30 °C. After 2 hours, solid-phase microextraction is used to extract in the headspace of the reaction flask, the extraction time is 30 min, the oscillation frequency is 280 rpm, and the extraction temperature is 30 °C. In the reaction system, the dosage of the catalyst is 50 - 100 μg / mL, the final concentration of the substrate is 2 - 5 μg / mL, the final concentrations of dithiothreitol and MgCl2 are 1 - 3 mM and 10 - 30 mM respectively, and the final volume concentration of glycerol is 10%.
[0028] The engineered E. coli BL21 Star / pET28a / MM-FtTPS containing the albaflavenone synthase encoding gene from Fischerella thermalis and the recombinant plasmid pBbA5c-M–M (pBbA5cMevT(CO)–MBIS(CO,ispA)) prepared by the present invention reaches a yield of 24 mg / L within 96 h after induced culture.
[0029] The present invention has the following beneficial effects:
[0030] The present invention verifies the function of the albaflavenone synthase from Fischerella thermalis and invents and applies the method for synthesizing albaflavenone in Escherichia coli. It is found that the albaflavenone synthase from Fischerella thermalis can catalyze the formation of a large amount of albaflavenone from FPP in the prokaryotic expression system, and albaflavenone is the only product. After fermentation, the production rate in Escherichia coli reaches 0.25 mg·L -1 ·h -1 , which is the highest reported yield of albaflavenone. The engineered bacteria of the present invention produce albaflavenone in a green, pollution-free and efficient manner, and are suitable for industrial production. Description of the Drawings
[0031] Figure 1 SDS-PAGE analysis of the expression results of the recombinant FtTPS engineered bacteria induced by IPTG; where M: marker; 1, bacterial liquid before induction; 2, bacterial liquid after induction; 3, supernatant part after induction; 4, precipitate part after induction; 5, impurity protein; 6, target protein.
[0032] Figure 2 GC-MS diagram of the product of the FtTPS catalytic reaction; where a is the full-scan mode of GC-MS, b is a partial enlarged view of a, c is the GC-MS analysis diagram of albaflavenone, and d is a partial enlarged view of c.
[0033] Figure 3 FtTPS fermentation time-yield curve. Detailed Embodiments
[0034] To facilitate the understanding of the technical solution of the present invention, the following further describes a kind of albaflavenone synthase, gene, vector, engineered bacteria and application from Fischerella thermalis of the present invention in combination with specific embodiments.
[0035] Example 1: Preparation of an albaflavenone synthase, gene, vector, and engineered bacteria from Fischerella thermalis:
[0036] 1. Construction of a recombinant vector and engineered bacteria containing the target gene FtTPS
[0037] The FtTPS gene coding sequence (SEQ ID NO.1), with NdeI and XhoI restriction site sequences added to both ends after codon optimization, was fully synthesized by Hangzhou Qingke Biotechnology Co., Ltd. Subsequently, it was double-digested and ligated downstream of the T7 promoter of pET-28a to construct the FtTPS-pET28a expression vector. This vector was transformed into E.Coil BL21 codon plus competent cells by heat shock method, and positive clones were screened on LB solid plates containing 50 μg / mL kanamycin to obtain the engineered strain E.Coil BL21 codon plus / pET 28a / FtTPS.
[0038] 2. Induced expression and purification of FtTPS protein:
[0039] The engineered strain E.Coil BL21 codon plus / pET 28a / FtTPS obtained in the first step was cultured overnight at 37 °C in an LB liquid medium containing 100 mL of 50 μg / mL kanamycin. 10 mL of the overnight cultured bacterial solution was poured into 1 L of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C until the OD 600 reached 0.6 - 0.8, then IPTG was added with a final concentration of 0.05 - 0.2 mM, and induced at 25 °C for 12 - 15 h. After that, the cells were collected by centrifugation, 5 g of wet cells were resuspended in 10 - 25 ml of phosphate buffer with Ph 7.0 - 7.4, and the cells were ultrasonically disrupted. The supernatant was taken by centrifugation and filtered through a 0.22 μm cellulose acetate filter membrane. According to the product manual, the filtrate was purified by nickel column (Qiagen, Germany) affinity chromatography to obtain the FtTPS recombinant enzyme solution. The electrophoresis pattern is shown in Figure 1 as shown. The result shows that FtTPS is expressed in a soluble form in Escherichia coli.
[0040] Example 2: Catalytic property analysis of recombinant enzyme FtTPS
[0041] Using the FtTPS recombinant enzyme solution prepared by the method of Example 1 (concentration: 50 mg / L, volume: 10 μL) as a catalyst, add pH 7.5 Tris-HCl buffer, 1 - 3 μg FPP, 20 - 30 μL of 100 mM MgCl2 solution, 1.5 - 2 μL of 1 M DTT, and 100 μL of dilute glycerol (glycerol) to form a reaction system of 0.5 - 1 mL. Stir well at 30 °C for 120 min. Meanwhile, use headspace-solid phase microextraction technology to adsorb the reaction products. After the reaction is completed, take out a Fischerella thermalis-derived sclarene synthase extraction head and inject it into a gas chromatograph for qualitative analysis of the products. Chromatographic conditions: Select a GC-2010 Shimadzu gas chromatograph; the chromatographic column is HP-5; carrier gas: N2, purge flow rate is 3 mL / min, splitless; initial column oven temperature is 40 °C, hold for 2 minutes, then increase the temperature to 220 °C at a rate of 7 °C / min, hold for 5 minutes; injection port temperature is 250 °C; detector temperature is 250 °C. Mass spectrometry data is collected in the full scan mode from 45 - 500. Compare the products with the NIST database: The largest main product peak, sclarene, appears at 16.10 min. According to the peak area calculation, sclarene accounts for 100% (as Figure 2 shown).
[0042] Example 3: Analysis of the in vivo enzyme catalytic time yield of recombinant enzyme FtTPS
[0043] Co-transform the recombinant plasmid pBbA5c-M–M (pBbA5cMevT(CO)–MBIS(CO,ispA) preserved in this experiment and the plasmid FtTPS-pET28a prepared by the method of Example 1 into E. coli BL21 Star competent cells by heat shock method. Culture at 37 °C and 200 rpm for 1 h in LB medium without resistance. Centrifuge and discard part of the supernatant, and perform positive clone screening on an LB solid plate containing 25 μg / mL chloramphenicol and 50 μg / mL kanamycin simultaneously to obtain the engineered strain E.CoilBL21 Star / pET 28a / MM-FtTPS.
[0044] Culture the engineered strain E.Coil BL21 Star / pET 28a / MM-FtTPS in SBMSN liquid medium containing 25 μg / mL chloramphenicol and 50 μg / mL kanamycin simultaneously at 37 °C until OD 600= 0.6 - 0.8, add IPTG to a final concentration of 0.4 - 1 mM, and at the same time add n-dodecane in a volume ratio of 1 / 10 to the upper layer. Induce at 25°C for 12, 24, 36, 48, 60, 72, 84, 96 h in sequence, and take n-hexane to dilute it in a volume ratio of 1:20 - 1:50 (n-dodecane:n-hexane). Inject the prepared sample into a gas chromatograph for qualitative analysis of the product. The chromatographic conditions are the same as in Example 2. (As Figure 3 shown).
[0045] As described above, only the preferred embodiments of the present invention are provided, and there is no restriction on the present invention in any formal or substantial form. Those skilled in the art, within the scope of the technical solution of the present invention, may make some minor changes, modifications and equivalent changes in evolution by using the technical content disclosed above, which are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims
1. An Fischerella thermalis application of the sourced sclarene synthase in the preparation of sclarene, characterized in that The application is as follows: Using Fischerella thermalis the alkenyl synthase from Fischerella thermalis as a catalyst, and using farnesyl pyrophosphate as a substrate, in the presence of dithiothreitol, MgCl2 and glycerol, a reaction system is formed in a buffer solution with a pH of 7.0 - 7.5, and a biotransformation reaction is carried out at 30°C. The reaction solution is separated and purified to obtain alkenyl; the Fischerella thermalis amino acid sequence of the alkenyl synthase from Fischerella is as shown in SEQ ID NO.
2.
2. The application according to claim 1, characterized in that, In the reaction system, the dosage of the catalyst is 50-100 µg / mL, the final concentration of the substrate is 2-5 µg / mL, the final concentrations of dithiothreitol and MgCl2 are 1-3 mM and 10-30 mM respectively, and the final volume concentration of glycerol is 10%.