Germaene A synthase mutant and its application

Abstract

The present invention belongs to the field of enzyme engineering technology, and specifically relates to a mutant of germaene A synthase and its application. The present invention obtains a series of chicory-derived germaene A synthase (CiGASlo) mutants by computer-aided design, constructs CiGASlo mutants by point mutation PCR method, further expresses recombinant protein and purifies, verifies enzyme activity by in vitro enzymatic reaction, and thus screens out CiGASlo mutants with significantly improved relative enzyme activity. The present invention can provide a CiGASlo mutant with significantly improved catalytic activity, which is helpful to promote and apply in the industrial production process of sesquiterpene compounds such as β-elemene. The present invention has the advantages of mild reaction conditions, high stereoselectivity, simple catalyst treatment steps, and environmental friendliness, and reduces equipment requirements, significantly reduces production costs, and shows a wide range of application prospects in industrial applications.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a germarene A synthase mutant and application thereof. Background Art

[0002] β-elemene is a sesquiterpenoid compound extracted from the natural Chinese herbal medicine Curcuma zedoaria (Zingiber officinale). It exhibits broad-spectrum anti-tumor effects and is listed alongside paclitaxel, camptothecin, vinblastine, indirubin, and homoharringtonate as an important botanical anti-tumor drug. Because β-elemene contains three chiral carbon atoms in its molecular structure, it theoretically should have eight stereoisomers. However, only four have been reported to date. Of these, the enantiomers (+)-β-elemene and (-)-β-elemene exhibit clear pharmacological activity.

[0003] Compared to obtaining terpenoids through plant extraction, microbial fermentation has become a promising alternative for industrial application. Model microorganisms such as Saccharomyces cerevisiae and Escherichia coli, widely used and excellent cell factories, possess a biosynthetic pathway for farnesyl diphosphate (FPP), a key intermediate of β-elemene. However, due to the lack of germacrene A synthase (GAS, EC 4.2.3.23), they are unable to catalyze FPP into germacrene A, the precursor of β-elemene. Therefore, screening for GAS with excellent catalytic performance has become a primary task in constructing β-elemene microbial cell factories.

[0004] Germaene A synthase belongs to the cleavage enzyme family. Two naturally occurring GASs catalyze the conversion of FPP to the enantiomers (+)-germaene A and (-)-germaene A, respectively. (+)-germaene A can undergo a Cope rearrangement to form the pharmacologically active (-)-β-elemene. Germaene A synthase (CiGASlo) from chicory (Cichorium intybus L.) has been shown to catalyze the conversion of FPP to (+)-germaene A. However, naturally occurring CiGASlo has low enzymatic activity, hindering the construction of microbial cell factories and purification of the target product, severely restricting its industrial application. Therefore, screening and engineering CiGASlo mutants with enhanced relative enzymatic activity could pave the way for the industrial application of FPP-catalyzed β-elemene production. Summary of the Invention

[0005] The present invention aims to overcome the defects of chicory-derived germarene A synthase in the prior art, which is used to catalyze the conversion of FPP to (+)-germarene A, such as relatively low enzyme activity, poor catalytic performance, and difficulty in adapting to industrial production. The present invention provides a germarene A synthase mutant, a recombinant vector containing a gene encoding the mutant, and a recombinant genetically engineered bacterium containing the gene encoding the mutant. The germarene A synthase mutant is applied to the industrial production process for preparing β-elemene.

[0006] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions:

[0007] A germarene A synthase mutant is obtained by site-directed mutagenesis of the amino acid sequence shown in SEQ ID NO. 1, wherein the mutation site is any one of the following:

[0008] W305A, D337H, L415W, D477H, E485Y, R488A, P44W, L309M, A421P, N429Y, R474L, D478S, E485A, Q490W, S491Y, A492P, Y553F.

[0009] Preferably, a mutant of germacene A synthase is derived from the amino acid sequence shown in SEQ ID NO. 1 by site-directed mutagenesis, wherein the mutation site is any one of the following:

[0010] W305A, D337H, L415W, D477H, E485Y, R488A.

[0011] A recombinant vector comprising the coding sequence of the germarene A synthase mutant described above.

[0012] A recombinant genetically engineered bacterium, comprising the recombinant vector described above.

[0013] Preferably, the recombinant genetically engineered bacteria uses Escherichia coli as an expression host.

[0014] Use of the germacene A synthase mutant described above in the preparation of sesquiterpene compounds.

[0015] Preferably, the germarene A synthase mutant is used in the preparation of β-elemene.

[0016] Preferably, the application is:

[0017] The wet bacteria obtained by fermentation culture of a recombinant genetically engineered bacterium containing a gene encoding a mutant of germarene A synthase or the pure enzyme liquid extracted after ultrasonic disruption of the wet bacteria is used as a catalyst, and farnesyl pyrophosphate is used as a substrate to carry out a catalytic reaction to synthesize germarene A, and further produce β-elemene.

[0018] Preferably, an organic solvent is required to extract the product in the catalytic reaction system, and the organic solvent is any one of n-hexane, n-pentane and dodecane.

[0019] Preferably, the pH of the catalytic reaction system is 6.8-7.4.

[0020] Therefore, the present invention has the following beneficial effects:

[0021] (1) The present invention obtains a series of chicory-derived germarene A synthase (CiGASlo) mutants by computer-aided design, constructs CiGASlo mutants by point mutation PCR, further expresses and purifies the recombinant protein, and verifies the enzyme activity by in vitro enzymatic reaction, thereby screening CiGASlo mutants with significantly improved relative enzyme activity;

[0022] (2) The present invention can provide a CiGAS10 mutant with significantly improved catalytic activity, which is helpful for promoting its application in the industrial production process of sesquiterpenoid compounds such as β-elemene;

[0023] (3) The present invention has the advantages of mild reaction conditions, high stereoselectivity, simple catalyst treatment steps, and environmental friendliness. It also has reduced equipment requirements and significantly reduces production costs, showing broad application prospects in industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a nucleic acid gel image of the CiGAS1o mutants W305A, D337H, L415W, D477H, E485Y, and R488A in Example 1.

[0025] Figure 2 This is an SDS-PAGE image of the protein purification of CiGAS1o mutants W305A, D337H, L415W, D477H, E485Y, and R488A in Example 3.

[0026] Figure 3 This is the GC chart of the β-elemene standard in Example 4.

[0027] Figure 4 This is the GC chart of the product produced by FPP catalyzed by the CiGAS1o mutant in Example 4.

[0028] Figure 5 This is the MS chart of the β-elemene standard in Example 4.

[0029] Figure 6 This is the MS graph of the product produced by FPP catalyzed by the CiGAS1o mutant in Example 4.

[0030] Figure 7 This is a diagram of the relative enzyme activity of the CiGAS1o mutation site in Example 5. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only a portion of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0032] Example 1: Construction of CiGAS1o mutant

[0033] First, the cDNA of wild-type chicory-derived germarene A synthase (CiGASlo) (GenBank #AF497999.1, synthesized by Shanghai Jierui Biotechnology Co., Ltd.) was constructed into the E. coli expression vector pET-28a(+) (provided by Qingke Biotechnology Co., Ltd., containing the C-terminal -6×His target gene inserted into the Nco I and Not I restriction sites). Based on the wild-type germarene A synthase (WT), the following mutants were designed using SnapGene as primer design software. The amino acid sequence of the wild-type chicory (Cichorium intybus L.) germarene A synthase is shown in SEQ ID NO.1. SnapGene software was used to design upstream and downstream primers, which were synthesized by Qingke Biotechnology Co., Ltd. The primers designed for the CiGASlo mutant were amplified by PCR (KOD One Master Mix, Shanghai Toyobo) to obtain the PCR product of the CiGASlo mutant. The DNA template in the PCR product was removed using DpnI (Thermo Scientific, FD1703) and then transformed into competent cells (DH5α) to circularize the PCR product. The transformed competent cell suspension was plated on LB solid medium containing 50 μg / mL kanamycin (Kan) and cultured at 37°C for 15 hours. Single colonies were selected and the CiGAS1o mutant plasmid was extracted using a plasmid extraction kit. DNA sequencing confirmed that the CiGAS1o mutant plasmid had the correct sequence. The primer sequences for the CiGAS1o mutant are shown in Table 1 below.

[0034] Table 1: CiGAS1o mutant sites and primer sequences

[0035]

[0036]

[0037]

[0038] PCR amplification of target gene:

[0039] Reaction system: KOD One Master Mix, upstream primer 1 μL (10 ppm), downstream primer 1 μL (10 ppm), plasmid template 1 μL (50 ng), add ddH2O system to 50 μL.

[0040] PCR amplification conditions:

[0041] (1) Pre-denaturation: 98°C for 5 min;

[0042] (2) Denaturation: 98°C for 30 seconds; Annealing: 60°C for 30 seconds; Extension: 72°C for 90 seconds; 30 cycles in total;

[0043] (3) Post-extension: 72°C for 10 min;

[0044] (4) Store at 4℃.

[0045] After the PCR amplification is completed, the amplified product is detected and verified by 0.9% agarose gel electrophoresis. Figure 1 shown. Figure 1 The nucleic acid gel images of the CiGAS1o mutants W305A, D337H, L415W, D477H, E485Y, and R488A in this example show that the amplified product is a single band with a size of approximately 7030 bp, which is consistent with the expected value.

[0046] Example 2: Construction of recombinant genetically engineered bacteria

[0047] Preparation of competent cells E. coli BL21 (DE3): Streak the E. coli BL21 (DE3) bacterial solution onto an antibiotic-free LB solid plate and culture at 37°C overnight; on the second day, pick a single colony from the plate and inoculate it into a test tube containing LB medium and culture it at 37°C overnight; on the third day, inoculate it into a shake flask containing 40 mL LB medium and culture it at 37°C, 180 rpm, for 1-2 hours until the bacterial solution OD reaches 0. 600 The pH value is between 0.4 and 0.6; place the shake flask in an ice bath for about 10 minutes, centrifuge at 4°C and 5000 rpm for 10 minutes, remove the supernatant in a clean bench and retain the bacteria; add about 20 mL of pre-sterilized and pre-cooled 0.1 M CaCl2 solution, resuspend and place in an ice bath for 30 minutes; centrifuge at 5000 rpm for 10 minutes, add 1 mL of pre-cooled solution containing 0.1 M CaCl2 and 30% glycerol, resuspend in an ice water bath, and distribute into 1.5 mL sterile centrifuge tubes (100 μL per tube), and store in a -80°C ultra-low temperature refrigerator for use.

[0048] The obtained recombinant vector pET-28a(+)-CiGASlo and its mutant plasmids were respectively transformed into E. coli BL21(DE3) competent cells, spread on LB solid plates containing 50 μg / mL Kan, and cultured inverted in a 37°C constant temperature incubator overnight; single colonies were picked from the plates and inoculated into LB test tubes, cultured at 37°C for 8-12 hours, and stored in a -80°C refrigerator to obtain the recombinant genetically engineered bacteria of each mutant.

[0049] Example 3: Protein expression and purification

[0050] (1) Culture of bacteria

[0051] The recombinant genetically engineered bacteria containing the coding sequence of CiGASlo and its mutants were inoculated into LB liquid medium containing Kan resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and deionized water as the solvent). A test tube containing 4 mL of LB liquid medium (containing a final concentration of 50 μg / mL Kan) was placed in a shaker at 37°C and cultured at 200 rpm for 10 to 12 hours to obtain a seed solution.

[0052] (2) Protein induced expression

[0053] In a clean bench, transfer the seed solution in the test tube to a shake flask containing 1L LB liquid medium (containing a final concentration of 50μg / mL Kan). Place the LB medium containing the seed solution in a 37℃ shaker and culture at 200rpm for 2-3 hours. 600 When the value reaches 0.6-0.8, IPTG (Sigma, 367-93-1) is added to a final concentration of 0.1-0.8 mM to induce expression at a temperature of 16-37°C. The preferred IPTG concentration is 0.2 mM, and the preferred induction temperature is 17°C. After induction for 15 hours under optimal conditions, the cells are collected by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl pH = 7.4, 150 mM NaCl, 20 mM imidazole, 10% (v / v) glycerol, 0.1% (v / v) Tween 20). Phenylmethylsulfonyl fluoride (PMSF, final concentration 1 mM) is added before disruption.

[0054] (III) Protein purification

[0055] The E. coli cells were broken with a pre-cooled high-pressure homogenizer, centrifuged at 9000 rpm for 45 min, and the supernatant was collected. 2+The NET medium was rotated and mixed at 4°C for 1 hour to allow the 6×His-containing protein to bind to the medium. The bound solution was added to a gravity column and allowed to drain naturally by gravity. The column was washed with 10 volumes of lysis buffer. The eluted fractions were then collected into 30 kDa (Millipore) ultrafiltration tubes using elution buffer (50 mM Tris-HCl pH = 7.4, 150 mM NaCl, 300 mM imidazole, 10% (v / v) glycerol, 0.1% (v / v) tween 20, 20 mM β-mercaptoethanol). The buffer was exchanged by centrifugation and the protein was stored in exchange buffer (50 mM Tris-HCl pH = 7.4, 150 mM NaCl, 10% (v / v) glycerol, 0.1% (v / v) tween 20). Protein concentration was determined using a BCA protein quantification kit (Biyuntian, P0009-5000). The concentrated protein was subjected to protein electrophoresis to detect the expression of the target protein. Figure 2 As shown, the molecular weight of the protein is approximately 67 kDa, which is consistent with the expected value. Figure 2 This is an SDS-PAGE image of the protein purification of CiGAS1o mutants W305A, D337H, L415W, D477H, E485Y, and R488A in this example.

[0056] Example 4: Germaene A synthase activity detection method and product GC-MS identification

[0057] (1) Enzyme activity determination method

[0058] The method for determining the catalytic activity of germarene A synthase on the substrate FPP is as follows:

[0059] The 0.5 mL reaction system contained the following components: 10 mM MgCl2, 50 mM Tris-HCl pH = 7.4, 20 mM β-mercaptoethanol, 10% (v / v) glycerol, 0.1% (v / v) tween 20, 1 μM recombinant enzyme pure enzyme solution and 20 μM FPP.

[0060] After mixing the reaction mixture, overlay with 0.5 mL of n-hexane and react at 30°C for 30 min. The centrifuge tube containing the reaction mixture was vigorously shaken and centrifuged at 10,000 × g for 5 min. 200 μL of the upper n-hexane organic phase was collected for GC-MS analysis. An HP-5 (30 m × 0.25 mm, 0.25 μm) column was used for GC-MS analysis. The inlet temperature was 250°C in splitless mode. The initial temperature was 50°C, held for 3 min, then increased to 220°C at a rate of 20°C / min, then slowly increased to 260°C at a rate of 5°C / min and held for 5 min.

[0061] (II) GC-MS identification of products

[0062] Using FPP as substrate, the target product was generated by catalysis of the CiGASlo mutant. The catalytic reaction system was the above enzyme activity detection reaction system. 200 μL of the upper n-hexane organic phase was taken for GC-MS determination. The gas phase column used was an HP-5MS (30 m × 0.25 mm, 0.25 μm) column. The determination results are shown in Figure 2. Figures 3 to 6 As shown, Figure 3 This is the GC chart of the β-elemene standard in this example. Figure 4 This is the GC graph of the product produced by FPP catalyzed by the CiGAS1o mutant in this example. Figure 5 This is the MS chart of the β-elemene standard in this example. Figure 6 This is the MS graph of the product produced by FPP catalyzed by the CiGAS1o mutant in this example.

[0063] from Figures 3 to 6 It can be determined that the peak time of the product generated by the CiGASlo mutant using FPP as a substrate is 7.67 min, which is consistent with the peak time of the β-elemene standard. According to the comparison and analysis of the compound data, the product with a molecular weight of 189.2 is the β-elemene product.

[0064] Example 5: Enzyme activity assay of CiGAS1o and its mutants

[0065] Based on the methods of Examples 1 to 3, CiGAS1o and its mutants were constructed and expressed, and pure enzyme solutions of WT, M1 to M28 were obtained respectively. Enzyme activity was detected based on the detection method of the activity of the germarene A synthase in Example 4. The results are as follows: Figure 7 shown. Figure 7 The relative enzyme activities of the CiGAS1o mutants are shown in Table 2 below.

[0066] Table 2: Relative enzyme activities of CiGAS1o mutants

[0067]

[0068]

[0069] from Figure 7 As shown in Table 2, there are five CiGASlo mutants whose relative activities are significantly improved compared with the original enzyme activity, including D337H (142%), L415W (146%), D477H (152%), E485Y (146%) and R488A (153%). Among them, the relative enzyme activity of the CiGASlo mutant R488A is the highest, which is 1.5 times that of the wild type.

[0070] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, based on the ideas provided by the present invention, there may be changes in the specific implementation methods, and these changes should also be considered as the scope of protection of the present invention.

Claims

1. A mutant of germacene A synthase, characterized in that: The amino acid sequence is derived from the sequence shown in SEQ ID NO.1 through site-directed mutagenesis, wherein the mutation site is R488A.

2. A recombinant vector, characterized in that: The recombinant vector contains the coding sequence of the germacene A synthase mutant according to claim 1.

3. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria contains the recombinant vector according to claim 2.

4. A recombinant genetically engineered bacterium according to claim 3, characterized in that: The recombinant genetic engineering bacteria uses Escherichia coli as an expression host.

5. Use of the germarene A synthase mutant according to claim 1 in the preparation of β-elemene.

6. The use according to claim 5, characterized in that: The applications described are: The wet bacteria obtained by fermentation culture of a recombinant genetically engineered bacterium containing a gene encoding a mutant of germanene A synthase or the pure enzyme solution extracted after ultrasonic crushing of the wet bacteria is used as a catalyst, and farnesyl pyrophosphate is used as a substrate for catalytic reaction to synthesize germanene A, and further produce β-elemene.

7. The use according to claim 6, characterized in that: An organic solvent is required to extract the product in the catalytic reaction, and the organic solvent is any one of n-hexane, n-pentane and dodecane.

8. The use according to claim 6, characterized in that: The pH of the catalytic reaction is 6.8-7.4.

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