Germaene A synthase mutant, recombinant engineering bacteria and applications

Abstract

The present invention relates to the technical fields of bioengineering, synthetic biology and computational biology, and in particular to a germacene A synthase mutant, a recombinant engineered bacterium and its use. Y376L The amino acid sequence of the germacrene A synthase mutant is shown in SEQ ID NO. 1. The germacrene A synthase mutant was transformed into Escherichia coli and the yield of the product, β-elemene, was measured, with a yield of 487.46 mg / L. This indicates that the mutant can be used for engineering strain transformation, providing a strong research foundation for high-yield β-elemene production.

Description

Technical Field

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

[0002] β-Elemene is the main active ingredient in anti-tumor drugs, but its current extraction method, from the traditional Chinese medicine Curcuma zedoaria, suffers from low yield and purity. The rise of synthetic biology has brought hope for resolving this problem. β-Elemene is a sesquiterpene compound. Germaene A synthase (GAS) catalyzes the conversion of farnesyl pyrophosphate (FPP) to its precursor, germarene A. Germaene A then rearranges to form β-elemene. Germaene A synthase is present in both plants and microorganisms, but the initial activity of GAS varies significantly between sources, determining the initial product level. Optimization strategies, such as enzyme activity and metabolic flux, can further increase yield and produce large quantities of high-purity β-elemene.

[0003] With the advancement of biological research, various omics data have surged, and traditional experimental methods are no longer sufficient for large-scale gene mining, screening, and verification. Sesquiterpene synthases are typically composed of over 500 amino acids, each of which can mutate into 19 other common amino acids, creating a significant obstacle to optimizing their activity. Computational biology utilizes a combination of data analysis, mathematical modeling, and virtual simulation techniques to identify protein coding regions through large-scale, efficient theoretical models and numerical calculations, thereby deciphering the genetic language hidden within nucleic acid sequences. Therefore, computational biology has become an important tool for researchers in gene screening and optimization. It is currently widely used in a variety of biological research fields, including peptide synthesis and small molecule design, amino acid virtual mutagenesis and screening, protein structure prediction, protein interaction prediction, database construction, and prediction of bioactive substances.

[0004] Unoptimized and unmodified GAS often exhibits relatively low catalytic activity, limiting its efficiency in substrate conversion. This low activity may stem from structural characteristics of the enzyme or defects in the active site, leading to insufficient substrate binding or slow reaction rates. Therefore, screening for more active GAS mutants is crucial for identifying strains that efficiently synthesize β-elemene. 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 germarene A synthase was selected as the template protein to construct the target protein;

[0009] Construct a homology model of germarene 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 are performed on the key amino acid residues obtained through screening, and mutants that can simultaneously improve protein stability and affinity are 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 germarene A synthase (ScGAS) is obtained from the NCBI database, and the protein crystal with the highest homology is obtained using BLAST (Basic Local Alignment Search Tool) as a template protein. The similarity and homology of the primary structure of ScGAS and the template protein are compared with the help of the Align sequence to templates module of Insight II software, and the different amino acids between the template protein and the ScGAS amino acid sequence on both sides are deleted to improve the homology between the two sequences.

[0015] As a preferred embodiment of the present invention, screening the optimal model is performed 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 greater 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 for key amino acid residues affecting enzyme activity is carried out according to the following steps:

[0020] Search for Mg-containing enzymes with high similarity to the germacene A synthase 2+ For known proteins, refer to the Mg in their three-dimensional structures. 2+ The way it interacts with amino acids determines Mg 2+ At the location within the germarene 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 mapped to the three-dimensional structure to find 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 for 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] In a second aspect, the present invention provides a vector or 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 germarene A synthase mutant and a plasmid for synthesizing a farnesyl pyrophosphate precursor into a host strain. The recombinant engineered bacterium expresses the germarene 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 was constructed onto the plasmid pGEX-4T-1 by homologous recombination, and the plasmid containing the germacene A synthase mutant and the plasmid for synthesizing farnesyl pyrophosphate (FPP) precursor were 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 germacene 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 methods to conduct homology modeling, sequence optimization, molecular docking, virtual mutagenesis and enzyme site-directed mutagenesis on the germacrene A synthase (ScGAS) from Canada solidago. Different protein mutants of ScGAS were constructed. After being transformed into Escherichia coli, the production of the product β-elemene was detected, and a mutant ScGAS with the highest enzyme activity was screened. Y376L , the yield of β-elemene reached 487.46 mg / L, and the mutant ScGAS Y376L It can be used for the transformation of engineering strains, providing a strong research basis for high-yield 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 The Laplace diagram 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 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 some embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative work all fall within the scope of protection of the present invention.

[0044] β-Elemene is the main active ingredient in anti-tumor drugs, but it is currently obtained through extraction from the traditional Chinese medicine Curcuma zedoaria, which suffers from low yield and purity. The rise of synthetic biology has brought hope for solving this problem. β-Elemene is a sesquiterpene compound. GAS catalyzes farnesyl pyrophosphate (FPP) to produce its precursor, germarene A, which then rearranges to form β-elemene. Germaene A synthase is present in both plants and microorganisms, but the original activity of GAS from different sources varies greatly, determining the initial level of the product.

[0045] Unoptimized and unmodified GAS often exhibits relatively low catalytic activity, limiting its efficiency in substrate conversion. This low activity may stem from structural characteristics of the enzyme or defects in the active site, leading to insufficient substrate binding or slow reaction rates.

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

[0047] The following describes the details 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, with a main chain atomic error of approximately 0.1nm, which is basically equivalent to a medium-resolution nuclear magnetic resonance (NMR) structure and a low-resolution X-ray diffraction structure. The primary sequence of ScGAS (GI: CAC36896.1) was obtained through the BLAST program, and the 4GAX crystal with the highest homology was used as a template protein. The Align sequence to templates module of the Insight II software was used to align the similarity and homology of the primary structure of ScGAS and the template protein, and the different 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 high accuracy.

[0050] 2. Homology modeling and preliminary optimization

[0051] First, the homology model of ScGAS was constructed using 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 required that the unreasonable amino acids should be less than 5% of the total amino acids, the Verify 3D evaluation required that at least 80% of the amino acid residues have a score of no less than 0.2, and the ERRAT evaluation required a Quality Factor higher than 80. Five optimal models were selected (Table 1). The ScGAS.M0090 model with the smallest two parameters was selected based on the PDF total energy and DOPE score. Then, the model was subjected to loop refinement and energy minimization 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 acid residues. The optimized model was then evaluated by the Profiles-3D method, with a validation score of 226.54 (highest expected value: 245.497; lowest expected value: 110.474). The validation 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 validation score, blue, white, and red correspond to high partition, average partition, and low partition, 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 more likely to undergo 1,10-cyclization to generate germaene A. However, there is no Mg in the 4GAX crystal. 2+ , and 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 (NtEAS, PDB ID: 3M01) in tobacco, the ScGAS model was optimized and the two Mg 2+ It was superimposed on its active site and refined by the standard dynamics cascade module. Then molecular docking was performed. Under the CHARMM force field, 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") ( Figure 4 ), when the active sphere radius is 13.8 nm, FPP successfully docks with gemmaene A in a “U”-shaped conformation, forming the final ScGAS model.

[0056] 4. Screening and site-directed mutagenesis of amino acid residues affecting 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 identify key amino acids.

[0058] Results revealed 24 amino acid residues that may affect ScGAS activity, located adjacent to or within the conserved DDXXD and NSE / DTE domains or the enzymatic 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. SNAP2 was subjected to 456 site-directed mutagenesis of these 24 potential key amino acids. The Calculate Mutation Energy (Stability) and Calculate Mutation Energy (Binding) modules of Discovery Studio software were used to analyze changes in protein thermal stability and affinity for the substrate FPP before and after mutation, thereby predicting the effects of the mutations on enzyme activity. The results showed that there are 16 groups of mutations that may 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 E. coli to detect the production 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 the plasmid pGEX-4T-1 by homologous recombination, and the plasmid containing the mutant and the plasmid for synthesizing the 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, 100 μL of 0.5 mM IPTG and 20% v / v n-dodecane solution was added to induce fermentation for 48 hours. The fermentation broth was then cooled to room temperature and an equal volume of ethyl acetate was added to collect the fermentation product. The product and β-elemene standard were diluted with ethyl acetate, and an appropriate amount of nonyl acetate was added to a final concentration of 20 mg / L for the product or β-elemene standard. GC-MS and GC analysis were performed 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 application number 202110443722.4 previously applied for by the present invention team.

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

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

[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: Hitachi G-3900 gas chromatograph, DB-1 capillary column (0.32 mm × 30 m), detector: FID (H2, 60 kPa; AIR, 50 kPa, temperature 260 °C); the injection port temperature was set to 250 °C, and the carrier gas (N2) flow rate was 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 mutagenesis to optimize 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 highest yield of β-elemene was achieved, 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 principles of the present invention. These improvements and modifications should also be regarded as within 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 gene encoding the germarene 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 gene encoding 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 engineered bacteria are constructed according to the following steps: The encoding gene of the germacene A synthase mutant is constructed into the plasmid pGEX-4T-1, and then the plasmid containing the encoding gene of 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 engineered bacteria.

6. Use of the germarene A synthase mutant according to claim 1, the vector or host cell according to claim 2, or the recombinant engineered bacterium 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 bacterium 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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