Germacene A synthase mutant, recombinant engineering bacterium and application

Abstract

The invention relates to the technical fields of bioengineering, synthetic biology, computational biology and the like, in particular to a germacene A synthase mutant, recombinant engineering bacteria and application. The amino acid sequence of the germacene A synthase mutant ScGASY376L is as shown in SEQ ID NO. 1. The germacene A synthase mutant ScGASY376L After the germacene A synthase mutant is transferred into escherichia coli, the yield of the product beta-elemene is detected, and the yield reaches 487.46 mg / L, so that the mutant can be used for modifying engineering strains, and a powerful research basis is provided for high-yield beta-elemene.

Description

Technical Field

[0001] The invention relates to the technical fields of bioengineering, synthetic biology, computational biology and the like, and in particular to a mutant of germacene A synthase producing beta-elemene, a recombinant engineering bacterium and uses thereof. Background Art

[0002] β-elemene is the main active ingredient of anti-tumor drugs, but the current method of obtaining it is to extract it from the traditional Chinese medicine Curcuma zedoaria, which has the problems of low yield and low purity. The rise of synthetic biology has brought hope for solving this problem. β-elemene is a sesquiterpene compound. Its precursor, germaene A, is first produced by farnesyl pyrophosphate (FPP) catalyzed by germaene A synthase (GAS), and then germaene A is rearranged to form β-elemene. Germaene A synthase exists in both plants and microorganisms, but the original activity of GAS from different sources varies greatly, which determines the starting level of the product. The application of optimization strategies such as enzyme activity and metabolic flow can further increase the yield and obtain large quantities of high-purity β-elemene.

[0003] With the development of biological research, various omics data have surged, and traditional experimental methods can no longer meet the needs of large-scale gene mining, screening and verification. Sesquiterpene synthase is usually composed of more than 500 amino acids, each of which can mutate into 19 other common amino acids, which poses a huge obstacle to the activity optimization of sesquiterpene synthase. Computational biology comprehensively utilizes data analysis, mathematical modeling, virtual simulation and other technologies to identify protein coding regions through large-scale and efficient theoretical models and numerical calculations, and then analyze the genetic language rules hidden in nucleic acid sequences. Therefore, computational biology has become an important tool for researchers to screen and optimize genes. It has been widely used in many biological research fields such as peptide synthesis and small molecule design, amino acid virtual mutation and screening, protein structure prediction, protein interaction prediction, database construction and prediction of bioactive substances.

[0004] The catalytic activity of GAS that has not been optimized and modified is often relatively low, which limits its efficiency in the substrate conversion process. Low activity may be due to the structural characteristics of the enzyme or defects in the active site, resulting in insufficient substrate binding or slow reaction rate. Therefore, screening for more active GAS mutants is of great significance for obtaining efficient β-elemene synthesis strains. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a germacene A synthase mutant and its application, so as to provide a reference for the research on the production of β-elemene using germacene A synthase.

[0006] The present invention provides a germarene A synthase mutant ScGAS Y376L , and its amino acid sequence is shown in SEQ ID NO.1.

[0007] The present invention also provides a method for constructing the germacene A synthase mutant, which is carried out according to the following method:

[0008] The protein crystal with the highest homology to germaene A synthase was selected as the template protein to construct the target protein;

[0009] Construct a homology model of germaene A synthase and screen the optimal model;

[0010] Based on the optimal model screened and through molecular docking of the germarene A synthase substrate, the key amino acid residues affecting the enzyme activity were screened;

[0011] Virtual mutations were performed on the key amino acid residues obtained through screening, and mutants that could simultaneously improve protein stability and affinity were screened;

[0012] The screened mutants were subjected to in vivo enzyme activity analysis, and the mutant with the highest enzyme activity was the germacene A synthase mutant.

[0013] As a preferred embodiment of the present invention, when constructing the target protein, the protein crystal with the highest homology to the germacene A synthase is used as the template protein, the similarity and homology between the germacene A synthase and the template protein are compared, and the different amino acids between the amino acid sequence of the template protein and the germacene A synthase on both sides are deleted to improve the homology between the germacene A synthase and the template protein.

[0014] Further preferably, the primary sequence of ScGAS is obtained from the NCBI database, and the protein crystal with the highest homology is obtained as the template protein using BLAST (Basic Local Alignment Search Tool). The similarity and homology of the primary structure of ScGAS and the template protein are compared with the Align sequence to templates module of Insight II software, and the different amino acids between the amino acid sequence of ScGAS and the two sides of the template protein are deleted to improve the homology between the two sequences.

[0015] As a preferred embodiment of the present invention, the optimal model is selected according to the following steps:

[0016] Constructing a homology model of the germacene A synthase, and evaluating the Ramachandran plot, Verify 3D, and ERRAT evaluation results of the homology model; wherein the Ramachandran plot evaluation requires that the number of unreasonable amino acids is less than 5% of the total number of amino acids, the Verify 3D evaluation requires that at least 80% of the amino acid residues have a score of no less than 0.2, and the ERRAT evaluation requires that the Quality Factor is higher than 80;

[0017] The optimal model is selected based on the evaluation results.

[0018] Further preferably, the three-dimensional model of ScGAS is constructed by the MODELLER 9.2 module of Discovery Studio 2022, and the model is evaluated using an online website (http: / / servicesn.mbi.ucla.edu).

[0019] As a preferred embodiment of the present invention, screening of key amino acid residues affecting enzyme activity is carried out according to the following steps:

[0020] Find Mg-containing 2+ For known proteins, refer to the Mg in their three-dimensional structures 2+ The way it interacts with amino acids determines the Mg 2+ At the location within the germene A synthase, the ligand FPP was docked with two conserved metal ion binding motifs DDXXD and NSE / DTE, and the amino acid evolution rate was analyzed and positioned on the three-dimensional structure to find out the key amino acid residues.

[0021] As a preferred embodiment of the present invention, the process of performing virtual mutations on the key amino acid residues obtained by screening and screening mutations that can simultaneously improve protein stability and affinity is as follows: estimating the effect of amino acid residues on function through SNAP2 site-directed mutagenesis, predicting the effect of amino acid residues on enzyme activity using the Calculate Mutation Energy (Stability) and Calculate Mutation Energy (Binding) modules of the Discovery Studio software, and constructing mutants using the BuildMutants module.

[0022] The second aspect of the present invention provides a vector or a host cell containing the germacene A synthase mutant.

[0023] In a third aspect, the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium is obtained by transforming a vector containing a germacene A synthase mutant and a plasmid for synthesizing a farnesyl pyrophosphate precursor into a host strain. The recombinant engineered bacterium expresses the germacene A synthase mutant.

[0024] As a preferred embodiment of the present invention, the host strain of the recombinant engineered bacteria is Escherichia coli.

[0025] Further preferably, the recombinant engineered bacteria is constructed according to the following steps:

[0026] The germacene A synthase mutant is constructed onto the plasmid pGEX-4T-1 by homologous recombination, and then the plasmid containing the germacene A synthase mutant and the plasmid for synthesizing farnesyl pyrophosphate (FPP) precursor are co-transformed into Escherichia coli BL21 to obtain the recombinant engineered bacteria.

[0027] In a fourth aspect, the present invention provides a use of the germacene A synthase mutant, the vector, the host cell or the recombinant engineered bacteria in producing germacene A.

[0028] In a fifth aspect, the present invention provides a use of the germacene A synthase mutant, the vector, the host cell or the recombinant engineered bacteria in producing β-elemene.

[0029] In a sixth aspect, the present invention provides a method for producing β-elemene and / or germacene A, comprising the step of fermenting the recombinant engineered bacteria.

[0030] As a preferred embodiment of the present invention, the method comprises the following steps:

[0031] fermenting the recombinant engineered bacteria and collecting the fermentation product;

[0032] extracting germene A from the fermentation product;

[0033] The germaceous ene A is converted by heating to obtain the β-elemene.

[0034] More preferably, the fermentation is carried out at 200 rpm and 28°C until A 600 =0.5, IPTG and n-dodecane were added to induce fermentation for 48 hours.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] The present invention uses computational biology and other means to perform homology modeling, sequence optimization, molecular docking, virtual mutation and enzyme site-directed mutation on ScGAS from Canada solidago, constructs different protein mutants of ScGAS, and screens out a mutant ScGAS with the highest enzyme activity by detecting the yield of the product β-elemene after being transferred into Escherichia coli. Y376L , the yield of β-elemene reached 487.46 mg / L. Y376L It can be used for the transformation of engineering strains, providing a strong research basis for high production of β-elemene. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0038] Figure 1 This is the sequence alignment result of ScGAS and 4GAX.

[0039] Figure 2 This is the Laplace plot of the ScGAS.M0090 model after optimization (A) and the ribbon structure diagram after evaluation by the Profiles-3D method (B).

[0040] Figure 3 Schematic diagram of ScGAS model optimization, (A) ScGAS and tobacco 5EAS crystals superimposed; (B) Mg 2+ Binding mode within ScGAS.

[0041] Figure 4 This is the molecular docking diagram of ScGAS and FPP.

[0042] Figure 5 This is the feasibility analysis of ScGAS mutants, (A) stability and affinity of different ScGAS mutants, (B) in vivo enzyme activity analysis. DETAILED DESCRIPTION

[0043] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0044] β-elemene is the main active ingredient of anti-tumor drugs, but the current method of obtaining it is to extract it from the traditional Chinese medicine Curcuma zedoaria, which has the problems of low yield and low purity. The rise of synthetic biology has brought hope for solving this problem. β-elemene is a sesquiterpene compound. GAS first catalyzes farnesyl pyrophosphate (FPP) to produce its precursor, germaene A, which is then rearranged to form β-elemene. Germaene A synthase exists in both plants and microorganisms, but the original activity of GAS from different sources varies greatly, which determines the starting level of the product.

[0045] The catalytic activity of unoptimized and modified GAS is often relatively low, which limits its efficiency in substrate conversion. Low activity may be due to the structural characteristics of the enzyme or defects in the active site, resulting in insufficient substrate binding or slow reaction rate.

[0046] The present invention provides a germarene A synthase mutant ScGAS Y376L The amino acid sequence is shown in SEQ ID NO. 1. The mutant of the germacene A synthase can be used to obtain β-elemene with a yield of 487.46 mg / L.

[0047] The following describes the invention in conjunction with specific embodiments.

[0048] 1. Construction of target protein

[0049] When the sequence homology is greater than 50%, a high-precision model can be obtained, and the main chain atomic error is about 0.1nm, which is basically equivalent to the medium-resolution nuclear magnetic resonance (NMR) structure and the low-resolution X-ray diffraction structure. The primary sequence of ScGAS (GI: CAC36896.1) was obtained through the BLAST program. The 4GAX crystal with the highest homology was used as the template protein. The Align sequence to templates module of the Insight II software was used to compare the similarity and homology of the primary structure of ScGAS and the template protein. The difference amino acids between the template protein and the ScGAS amino acid sequence on both sides were deleted to improve the homology between the two sequences. The sequence identity (Identity) of the two is 53.0%, and the sequence similarity (Similarity) is 74.0% ( Figure 1 ), indicating that the target protein constructed using 4GAX as the template protein has a high accuracy.

[0050] 2. Homology modeling and preliminary optimization

[0051] First, the homology model of ScGAS was constructed by Discovery Studio 2022, and the model was evaluated using an online website (http: / / servicesn.mbi.ucla.edu) to examine whether the model's Ramachandran plot, Verify 3D, and ERRAT evaluation results met the requirements. The Ramachandran plot evaluation requires that unreasonable amino acids should be less than 5% of the total amino acids, the Verify 3D evaluation requires that at least 80% of the amino acid residues have a score of no less than 0.2, and the ERRAT evaluation requires a Quality Factor higher than 80. Five optimal models were selected (Table 1), and the ScGAS.M0090 model with two smaller parameters was selected based on the PDF total energy and DOPE score. Then, the model was loop refined and energy minimized using RamachandranPlot and Verify 3D. Ramachandran plot ( Figure 2 A) shows that the optimized model contains 6 unreasonable amino acid residues, accounting for 1.09% of the total amino acids. The optimized model was then evaluated by the Profiles-3D method, and the verification score was 226.54 (highest expected value: 245.497; lowest expected value: 110.474). The verification score should be higher than the lowest expected value, and the closer the model is to the highest expected value, the more reliable it is. Coloring according to the verification score, blue, white, and red correspond to high partitions, average partitions, and low partitions, respectively. The thinner the ribbon, the better the structure ( Figure 2 B).

[0052] Table 1 Optimal model for homology modeling

[0053]

[0054] 3. Further optimization and molecular docking

[0055] After ScGAS docks with FPP, FPP will bind to two magnesium ions (Mg 2+ ) and docking in a "U"-shaped conformation, it is easier to undergo 1,10-cyclization to generate germaene A. However, there is no Mg in the 4GAX crystal. 2+ , while the 3D structure of germarene A synthase is highly similar to that of another sesquiterpene synthase 5EAS (5-epi-aristolochene synthase) ( Figure 3 A), RSMD value is 2.909, referring to Mg 2+ The interaction mode with amino acids was optimized by molecular dynamics simulation to determine its position within ScGAS ( Figure 3 B). Therefore, based on the crystal structure of 5EAS in tobacco (NtEAS, PDB ID: 3M01), the ScGAS model was optimized and the two Mg 2+ It was superimposed on its active site and refined by the standard kinetic cascade module. Then molecular docking was performed, and the Lib Dock module was used to dock the ligand FPP with the active site between two conserved metal ion binding motifs ("DDXXD" and "NSE / DTE") under the CHARMM force field ( Figure 4 ), when the radius of the active sphere is 13.8 nm, FPP successfully docks with gemene A in a “U”-shaped conformation to form the final ScGAS model.

[0056] 4. Screening and site-directed mutagenesis of amino acid residues that affect enzyme activity

[0057] ScGAS was analyzed online (https: / / www.predictprotein.org). Consurf analysis estimates the evolutionary rate of amino acids based on the correlation between ScGAS and its homologous proteins and maps it to the three-dimensional structure to find the key amino acids.

[0058] The results showed that 24 amino acid residues may affect the activity of ScGAS, which are adjacent to or located in the conserved domains of DDXXD and NSE / DTE or the enzyme activity pocket. These residues include Y15, R264, W273, A298, D301, D302, D305, Y364, Y376, E379, W382, H394, T398, T401, G402, N446, H451, E454, Q455, R457, Y522, D526, L528, and K529. 456 sets of SNAP2 site-directed mutations were performed on the 24 potential key amino acids, and the Calculate Mutation Energy (Stability) and Calculate Mutation Energy (Binding) modules of Discovery Studio software were used to analyze the changes in protein thermal stability before and after mutation and the changes in affinity with the substrate FPP, so as to predict the effect of mutations on enzyme activity. The results showed that 16 groups of mutations could potentially improve both protein stability and affinity.

[0059] 5. Enzyme activity analysis

[0060] The ScGAS mutants Y376L, W382R, G402C, H451A, H451L, H451W, and D526P with the highest predicted activity were selected and transformed into Escherichia coli to detect the yield of the product β-elemene and analyze the enzyme activity of the mutants. The specific method is as follows:

[0061] The mutant was constructed into plasmid pGEX-4T-1 by homologous recombination, and the plasmid containing the mutant and the plasmid for synthesizing farnesyl pyrophosphate precursor were co-transformed into Escherichia coli BL21. A single clone was selected in 50 mL of LB liquid medium containing Amp and Chl resistance and cultured in a shaker at 200 rpm and 28°C until A 600 =0.5, add 100 μL of 0.5 mM IPTG and 20% V / V n-dodecane solution to induce fermentation for 48 hours, then cool the fermentation broth to room temperature, add an equal volume of ethyl acetate to collect the fermentation product. Dilute the product and β-elemene standard with ethyl acetate, and add an appropriate amount of nonyl acetate until the final concentration of the product or β-elemene standard is 20 mg / L, and perform GC-MS and GC detection to determine the detection conditions.

[0062] Among them, the construction process of the plasmid for synthesizing the farnesyl pyrophosphate precursor can be found in the invention patent with application number 202110443722.4 previously applied by the present invention team.

[0063] GC-MS detection conditions: quartz capillary column HP-5MS (30m×0.25mm×0.25μm).

[0064] Heating program: 80℃, hold for 3min; increase to 210℃ at 10℃ / min, hold for 1min.

[0065] Carrier gas: high-purity helium, flow rate set to 1 mL / min; injection port and interface temperatures set to 250°C and 280°C respectively; injection volume 1 μL; ion source EI; electron energy 70 eV; ion source temperature 250°C; scanning mass range 35-550 amu; solvent delay 6.5 min.

[0066] GC detection conditions: Japan Hitachi G-3900 gas chromatograph, DB-1 capillary column (0.32 mm×30 m), detector is FID (H2, 60 KPa; AIR, 50 KPa, temperature 260°C); the injection port temperature is set to 250°C, and the carrier gas (N2) flow rate is 20 mL / min.

[0067] Temperature program: initial 150°C, maintain for 6 min; increase to 220°C at 20°C / min, maintain for 2 min; injection volume is 1 μL.

[0068] During the GC-MS detection process, germaene A was converted into β-elemene due to heating.

[0069] Enzyme activity results ( Figure 5 ) It can be seen that virtual mutation greatly improved the success rate of site-directed mutation optimization of enzyme activity. The enzyme activity of 5 out of 7 mutants was significantly improved, and the screening success rate was as high as 71.4%. Y376L The production of β-elemene was the highest, reaching 487.46 mg / L, which can be used for the subsequent transformation of engineering strains.

[0070] ScGAS Y376L The amino acid sequence is shown in SEQ ID NO.1:

[0071] MAAKQVEVIRPVANYHPSLWGDQFLHYDEQEDEHVEVDQQIEILKEETRKEILASLDDPTKHTNLLKLIDVIQRLGIAYYFEHEITQALDHIYSVYGDEWNGGRTSLWFRLLRQQGFYVSCDIFNIYKLDNGSFKDS LTKDIECMLELYEAAYMRVQGEIILDEALEFTKTHLEHIAKDPLRCNNTLSRHIHEALERPVQKRLPRLDAIRYIPFYEQQDSHNKSLLRLAKLGFNRLQSLHKKELSQLSKWWKEFDAPKNLPYVRDRLVELYFWI LGVYFEPQYSRSRIFLTKTIKMAAILDDTYDIYGTYEELEIFTKAVQRWSITCMDTLPDYMKVIYKSLLDVYEEMEEIIEKDGKAYQVHYAKESMIDLVTSLMTEAKWLHEGHVPTFDEHNSVTNITGGYKMLTASS FVGMHGDIVTQESFKWVLNNPPLIKASSDISRIMNDIVGHKEEQQRKHIASSVEMYMKEYNLAEEDVYDFLKERVEDAWKDINRETLTCKDIHMALKMPPINLARVMDMLYKNGDNLKNVGQEIQDYMKSCFINPMSV

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A germarene A synthase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A vector or host cell containing the germacene A synthase mutant according to claim 1.

3. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria is obtained by transforming the vector containing the germacene A synthase mutant and the plasmid for synthesizing the farnesyl pyrophosphate precursor in claim 2 into a host strain.

4. The recombinant engineered bacterium according to claim 3, characterized in that The host strain of the recombinant engineering bacteria is Escherichia coli.

5. The recombinant engineered bacterium according to claim 4, characterized in that The recombinant engineering bacteria are constructed according to the following steps: The germacene A synthase mutant is constructed on the plasmid pGEX-4T-1, and then the plasmid containing the germacene A synthase mutant and the plasmid for synthesizing the farnesyl pyrophosphate precursor are co-transformed into Escherichia coli BL21 to obtain the recombinant engineering bacteria.

6. Use of the germacene A synthase mutant according to claim 1, the vector or host cell according to claim 2, or the recombinant engineered bacteria according to claim 3 in producing β-elemene.

7. Use of the germacene A synthase mutant according to claim 1, the vector or host cell according to claim 2, or the recombinant engineered bacteria according to claim 3 in producing germacene A.

8. A method for producing β-elemene and / or germacene A, characterized in that: The method comprises the step of fermenting the recombinant engineered bacteria according to claim 3.

9. The method according to claim 8, characterized in that The following steps are involved: fermenting the recombinant engineered bacteria and collecting the fermentation product; extracting germacene A from the fermentation product; The germaceous ene A is converted by heating to obtain the β-elemene.

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