Anthranilate synthase mutant and use thereof

Abstract

The present disclosure relates to an Escherichia coli-derived anthranilate synthase mutant, and a method of preparing the same and use thereof. The mutant is obtained by substituting the last amino acid of a TXLLES domain in an anthranilate synthase derived from Streptomyces, enterobacteria, or Bacillus with methionine. The mutant has high resistance to feedback inhibition. Compared with the wild-type anthranilate synthase, the inhibition constant Ki for tryptophan is increased by more than 400-fold. On this basis, further substituting the serine at position 94 with asparagine can further increase the feedback inhibition resistance to 533-fold relative to the wild type. Compared with the TrpE(S40F) mutant most widely used in current industrial applications, the feedback inhibition resistance is increased by nearly two-fold. In addition, the anthranilate synthase constructed according to the present disclosure has relatively high catalytic activity at pH=6-9, thus having broad applications.

Description

Anthranilate synthase mutant and its application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anthranilate synthase mutant and application thereof. Background Art

[0002] Anthranilate synthase (AS) is a complex enzyme that catalyzes the reaction of chorismate with an amino group to produce anthranilate. It is a key enzyme in the tryptophan biosynthesis pathway. AS consists of an α-subunit, which is catalytic, and a β-subunit, which is an amino group donor. AS activity is feedback inhibited by tryptophan.

[0003] Therefore, by altering the structure or expression of anthranilate synthase through metabolic engineering, the content of tryptophan and its derivatives in plants can be increased, thereby enhancing the nutritional value and stress resistance of plants. Research on anthranilate synthase mainly focuses on its biochemical characteristics, genetic structure, regulatory mechanisms, and application prospects. The anthranilic acid mutants used for tryptophan production reported in the literature and patents have mutations at several sites, namely, serine at position 40 is changed to phenylalanine (Chen L, Chen M, Ma C, et al. Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E.coli for efficient tryptophan bioproduction[J]. Metabolic engineering, 2018, 47: 434-444.); serine at position 40 is changed to arginine (Liu SD, Wu YN, Wang TM, et al. Maltose utilization as a novel selection strategy for continuous evolution of microbes with enhanced metabolite production[J]. ACS synthetic biology, 2017, 6(12): 2326-2338.); alanine at position 63 is changed to valine (Ding D, Bai D, Li J, et al. Analyzing the genetic characteristics of a tryptophan-overproducing Escherichia coli[J]. Bioprocess and Biosystems Engineering, 2021, 44: 1685-1697. These mutations all reduce the inhibitory effect of tryptophan on enzyme activity. Among them, the S40F mutant is currently the most widely used and can promote tryptophan production.(Chen L,Chen M,Ma C,et al.Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E.coli for efficient tryptophan bioproduction[J].Metabolic engineering,2018,47:434-444..Mindt M,Ferrer L,Bosch D,et al.De novo tryptophanase-based indole production by metabolically engineered Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology,2023,107(5-6):1621-1634..Kuepper J,Dickler J,Biggel M,et al.Metabolic engineering of Pseudomonas putida KT2440 to produce anthranilate from glucose[J].Frontiers in Microbiology,2015,6:1310.).

[0004] Despite reports of these mutants, no anthranilate synthase mutant has yet been found that completely eliminates tryptophan feedback inhibition. This may be a key reason for the current industrial fermentation method for L-tryptophan production. There are two approaches to address this issue. The first is to isolate the product during the tryptophan production process and reduce tryptophan inhibition of anthranilate synthase, but this approach is costly and inefficient. A more effective approach is to mutate other potential anthranilate synthase residues through biotechnology to identify mutants that are more resistant to feedback inhibition, thereby enhancing the synthesis of anthranilate, a key intermediate in tryptophan production, and ultimately promoting tryptophan production.

[0005] Summary of the Invention

[0006] In response to the existing problems, the present invention provides an anthranilate synthase mutant, a preparation method and an application thereof. Compared with the wild type, the mutant relieves the feedback inhibition of tryptophan on the enzyme activity. The inhibition constant Ki of the mutant is increased by more than 400 times compared with the wild type anthranilate synthase and tryptophan, and the preferred embodiment increases it by more than 533 times.

[0007] The present invention provides an anthranilate synthase mutant, which is obtained by mutating the last position of the TXLLES domain of an anthranilate synthase from Streptomyces, Enterobacter, or Bacillus to methionine; more specifically, from Streptomyces coelicolor, Escherichia coli, or Bacillus subtilis, wherein X is any amino acid;

[0008] More specifically, the 40th position of the anthranilate synthase from Escherichia coli, the 48th position of the anthranilate synthase from Streptomyces coelicolor, and the 48th position of the anthranilate synthase from Bacillus subtilis are mutated to methionine;

[0009] More specifically, the 40th position of the anthranilate synthase derived from Escherichia coli and encoded by the nucleotide sequence shown in SEQ ID NO. 1 is mutated to methionine; and the 94th serine is mutated to asparagine.

[0010] Preferably, the amino acid sequence of the anthranilate synthase mutant is shown as SEQ ID NO.3, SEQ ID NO.13, SEQ ID NO.14 or SEQ ID NO.15.

[0011] The present invention provides a gene encoding the anthranilate synthase mutant, preferably the nucleotide sequence of the gene is shown in SEQ ID NO. 2. Furthermore, based on the gene in SEQ ID NO. 2, a gene encoding a more preferred anthranilate synthase mutant is disclosed, and a more preferred gene sequence is shown in SEQ ID NO. 16.

[0012] The present invention provides a recombinant vector containing the coding gene.

[0013] The present invention provides a recombinant genetic engineering bacterium containing the recombinant vector.

[0014] The present invention further provides a method for preparing an anthranilate synthase mutant, characterized in that it comprises the following steps:

[0015] S1: inserting the coding gene into a vector plasmid to obtain a recombinant plasmid;

[0016] S2: Transform the recombinant plasmid obtained in S1 into competent cells;

[0017] S3: The competent cells described in S2 are cultured to obtain monoclonal colonies, and the colonies are expanded;

[0018] S4: The colonies expanded in S3 are fermented and cultured.

[0019] S5: Method for collecting, disrupting and purifying bacteria after S4 culture.

[0020] Specifically, the vector plasmid in step S1 is pET28a, and the competent cells in step S2 are Escherichia coli cells.

[0021] The present invention also provides use of the anthranilate synthase mutant in preparing tryptophan.

[0022] The present invention further provides the use of the anthranilate synthase mutant in the synthesis of foods, pharmaceuticals, fine chemicals, health products, and cosmetics. Specifically, the mutant is used to relieve feedback inhibition of anthranilate synthase by the end product or intermediate tryptophan in the biosynthesis of foods, pharmaceuticals, fine chemicals, health products, and cosmetics containing an indole ring.

[0023] In summary, compared with the prior art, the present invention achieves the following effects: the anthranilate synthase mutant TrpE (S40M) of the present invention significantly eliminates the feedback inhibition of tryptophan on enzyme activity; has high catalytic activity at pH 6 to 9; and can therefore be used to promote the production of tryptophan and reduce production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0025] FIG1A shows the optimal conformation of the molecular docking in Example 1 of the present invention and the overlapping conformation of the TrpE-tryptophan co-crystallized molecule (PDB=1i1q) derived from Salmonella typhimurium.

[0026] FIG1B shows the conformations of the three key residues in the docking pocket of anthranilic acid and tryptophan derived from E. coli in Example 1 of the present invention.

[0027] FIG2 is a map of the recombinant plasmid constructed in Example 1 of the present invention.

[0028] FIG3 is the protein electrophoresis result of the anthranilate synthase mutant prepared in Example 2 of the present invention. The molecular weight of anthranilate synthase is about 58.4 kDa.

[0029] FIG4 shows the feedback inhibition constant Ki determination of tryptophan on TrpE (S40M), TrpE (S40M / S94N), wild type and the previously reported efficient mutant TrpE (S40F) constructed by the present invention in Example 3 of the present invention.

[0030] FIG5 is a graph showing the determination of the optimal reaction pH value of TrpE (S40M) in Example 4 of the present invention.

[0031] Figure 6 is a comparison of the amino acid sequences of anthranilate synthases from Escherichia coli and several other representative prokaryotic microorganisms using ClustalW. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0033] Example 1 Design and preparation of TrpE mutants

[0034] The gene sequence of Escherichia coli anthranilate synthase TrpE was obtained from the NCBI website as SEQ ID NO. 1. Homology modeling of E. coli anthranilate synthase mutants was performed using Swiss-Model (www.swissmodel.expasy.org / ). A docking model of tryptophan and E. coli anthranilate synthase (TrpE) was constructed using Autodock Vina software. The optimal conformation was selected by comparison with a Salmonella typhimurium TrpE-tryptophan cocrystallized molecule (PDB=1i1q). The docking model of E. coli TrpE and tryptophan was analyzed to design mutants. As shown in Figure 1A, the dark portion represents the optimal molecular docking conformation of E. coli TrpE and tryptophan, while the light portion represents the Salmonella typhimurium TrpE-tryptophan cocrystallized molecule. The tryptophan in the two conformations superimposes well, indicating a high degree of confidence in the docking conformation. Analysis of the tryptophan binding pocket revealed that three residues around it are closely associated with tryptophan: serine at position 40, glycine at position 454, and cysteine ​​at position 465. To relieve the feedback inhibition of tryptophan on TrpE, it is hoped that these three sites will be converted to larger side chains so that the residues and the tryptophan molecule will create steric hindrance, thereby inhibiting the binding of tryptophan to the allosteric center. Five mutants were initially designed: C465W, G454I, G454Y, G454F, and S40M. As described in the reference, the enzyme activities of the different mutants were tested in a buffer containing 2 g / L tryptophan. The enzyme activity was defined by the production rate of the product anthranilate (Lin X, Xu S, Yang Y, et al. Purification and characterization of anthranilate synthase component I (TrpE) from Mycobacterium tuberculosis H37Rv[J]. Protein expression and purification, 2009, 64(1): 8-15.). After screening, an optimal mutant was obtained, named TrpE (S40M), whose gene sequence is SEQ ID NO.2; and its amino acid sequence is SEQ ID NO.3.

[0035] The linearized expression vector was amplified using the pET28a plasmid as a template. The pET28a sequence is shown in SEQ ID NO. 4, and the primers used are shown in SEQ ID NO. 5 and SEQ ID NO. 6. The TrpE gene was amplified using the Escherichia coli W3110 genome as a template, using primers shown in SEQ ID NO. 7 and SEQ ID NO. 8. The TrpE gene and the linearized pET28a were recombined using the ClonExpress II One Step Cloning Kit (Vazyme) to obtain pET28a-TrpE. The sequence of pET28a-TrpE is shown in SEQ ID NO. 9. The recombinant product was transformed into Escherichia coli BL21 (DE3) competent cells, plated on LB plates containing Kan resistance, and cultured overnight at 37°C. The correctly identified pET28a-TrpE was used as a template to construct pET28a-TrpE (S40M) using Quick Change technology. The Quick Change primer sequences are shown in SEQ ID NO. 10 and SEQ ID NO. 11. After eliminating the template plasmid using Dpn I, the cells were transformed into competent BL21(DE3) strains and plated onto LB plates containing Kan resistance and cultured overnight at 37°C. Sequencing confirmed the identity of each BL21(DE3) engineered strain containing pET28a-TrpE(mutant). The map of the pET28a-TrpE(S40M) recombinant plasmid is shown in Figure 2, and the sequence of the pET28a-TrpE(S40M) recombinant plasmid is shown in SEQ ID NO. 12.

[0036] Example 2 Expression and purification of TrpE mutants

[0037] In order to evaluate the difference between the present mutant and the prior art, the widely used S40F mutant was constructed in this example with reference to the method in Example 1. 10 μl of bacterial culture was taken from a BL21 (DE3) glycerol tube containing the pET28a-TrpE (WT, S40F, S40M) plasmid and transferred to an LB test tube containing Kan, and cultured overnight at 37°C, 220 rpm; the pre-culture was inoculated into a fresh 250 ml TB shake flask at a 1% inoculum size, and cultured at 37°C, 220 rpm for 3 h to an OD600 of 0.6-0.8; IPTG was added to the culture medium at a final concentration of 0.1 mM to induce expression of TrpE and its mutant proteins, and culture was continued at 22°C, 180 rpm for 20 h; the bacterial culture was taken for SDS-PAGE to identify whether the protein was overexpressed; the cells were collected by low-temperature centrifugation at 5000 rpm for 5 min, disrupted, centrifuged, and filtered; each mutant protein was purified using a Ni-NTA affinity chromatography column using standard procedures; the cells were concentrated and desalted, replaced into enzyme storage buffer, and stored at -20°C for later use.

[0038] The results are shown in Figure 3. The three proteins expressed in this example were all efficiently expressed in E. coli. The expression of TrpE wild type, S40M mutant and S40F mutant was detected in the eluate. The above results indicate that the preparation method of the anthranilic acid mutant constructed by the present invention is effective, can be soluble expressed in E. coli, and has biological activity.

[0039] Example 3 Tryptophan inhibition curve determination

[0040] Experimental steps: Prepare a reaction buffer containing 100mM NH4Cl, 10mM MgCl, 20mM Tris-HCl, and 0.1mM EDTA, and adjust the pH to 9.0; take 50ml of the buffer to prepare an 8g / L tryptophan buffer, and adjust the pH to 9.0; mix the buffers prepared in the first two steps in proportion to obtain tryptophan buffers containing different concentrations; add 200μl of tryptophan buffers of different concentrations to a pure black opaque ELISA plate, add the mutant enzyme solution prepared in Example 2 to a final concentration of 0.1μM, and incubate at 30°C for 10min; add chorismate to a final concentration of 300μM (far excess) to start the reaction The fluorescence value at 400 nm after 320 nm excitation was measured using a fluorescence microplate reader, with detection every ten seconds. The microplate reader automatically plotted a fluorescence value versus time curve (the concentration of the reaction product anthranilic acid and the fluorescence value were linearly related within the anthranilic acid concentration range of 0-50 μM); the initial slope of the curve plotted by the microplate reader when the tryptophan concentration was 0 was defined as the enzyme activity = 1.0, and the ratio of the slope under different tryptophan concentration conditions to the slope without feedback inhibition was defined as the relative enzyme activity; the inhibition constant Ki value was calculated using the formula Y = (1-((((Et+X+(Ki*(1+(S / Km))))-(((Et+X+(Ki*(1+(S / Km))))^2)-4*Et*X)^0.5)) / (2*Et))), where Y is the enzyme activity, Et is the enzyme concentration, X is the inhibitor concentration, S is the substrate concentration, and Km is the Michaelis constant of the enzyme.

[0041] The test results are shown in Figure 4. The feedback inhibition constant (Ki) of tryptophan on S40M is 0.4579 g / L, which is over 400-fold higher than that of the wild-type (Ki = 0.0011 g / L). Furthermore, the S40M constructed in the present invention has been shown to have stronger resistance to feedback inhibition than the most widely used TrpE mutant, S40F. The Ki of tryptophan inhibition is approximately 1.7 times that of S40F (Ki = 0.2752 g / L), suggesting broad prospects for the industrial production of tryptophan.

[0042] Example 4 Effect of pH on TrpE (S40M)

[0043] The enzyme activity of the TrpE(S40M) mutant was measured in buffer systems with different pH values ​​(5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9) to determine its optimal pH. In this example, the pH of the reaction buffer was adjusted with hydrochloric acid and aqueous ammonia, and the enzyme activity was evaluated using the reaction and detection methods described in Example 3. As shown in Figure 5, TrpE(S40M) exhibited high enzyme activity at pH values ​​between 6 and 9, with the optimal pH for TrpE(S40M) being 7.5. These results demonstrate that TrpE(S40M) exhibits high enzyme activity under neutral and slightly alkaline conditions.

[0044] Example 5: Anthracene synthase mutants in other prokaryotic microorganisms

[0045] The amino acid sequences of anthranilate synthases from Escherichia coli and several other representative prokaryotic microorganisms were aligned using ClustalW. As shown in Figure 6, the serine at position 40 in the E. coli TrpE protein sequence is conserved in Streptomyces coelicolor and Bacillus subtilis. Furthermore, a domain with the amino acid sequence TXLLES is located near the tryptophan binding site, where X is any amino acid. To demonstrate that replacing the last serine in this domain with methionine relieves feedback inhibition, three-dimensional structures of anthranilate synthases from Streptomyces coelicolor and Bacillus subtilis and TXLLES were constructed using Swiss-Model. Molecular docking of the four structures with tryptophan was performed using Autodock Vina. The docking box size was set to 40*40*40 angstroms, and the docking box was placed near the TXLLES(M) domain. All other parameters were set to default. The docking results were aligned with the crystal structure of a co-crystal of TrpE from Mycobacterium tuberculosis and tryptophan to obtain the optimal conformation. Comparison of the binding energies of the optimal conformations of each structure revealed that the binding energy of anthranilate synthase from Streptomyces coelicolor to tryptophan increased by 2.0 kJ / mol (-8.2 to -6.2 kJ / mol) after the serine at position 48 was mutated to methionine; the binding energy of anthranilate synthase from Bacillus subtilis to tryptophan increased by 2.3 kJ / mol (-8.3 to -6.0 kJ / mol) after the serine at position 50 was mutated to methionine. These results indicate that the TXLLEM mutation created by the present invention also has the effect of reducing tryptophan feedback inhibition in anthranilate synthases from prokaryotes other than Escherichia coli. The amino acid sequences of TrpE (S48M) from Streptomyces coelicolor and TrpE (S50M) from Bacillus subtilis are shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively.

[0046] Example 6 Iterative Directed Evolution Experiment

[0047] Using pET28a-TrpE (S40M) as a template, error-prone PCR was used to mutate the TrpE gene. The mutant gene was inserted into pET28a using the method in Example 1 to obtain a mutant library. The mutant library was chemically transformed into BL21 (DE3) competent cells that had knocked out TrpD and cultured overnight at 37°C. 500 clones were randomly picked on the plate and cultured and expressed in 96-well plates according to Example 2. After centrifugation, the cells were resuspended in a buffer solution containing 2g / L tryptophan and pH=9. 5 μM chorismate was added using a pistol to start the reaction. The fluorescence value at 400nm after 320nm excitation was continuously measured using a fluorescence microplate reader. The clones corresponding to the highest fluorescence value changes within 10 minutes were sequenced. The results showed that the mutant with the highest activity under 2g / L tryptophan inhibition was S40M / S94N (amino acid sequence as SEQ ID NO.15), and its DNA sequence was as shown in SEQ ID NO.16. S40M / S94N was characterized with reference to Examples 2 and 3. As shown in FIG4 , the feedback inhibition constant of tryptophan on TrpE (S40M / S94N) was 0.5872 g / L, which was 533 times higher than that of the wild type, and has good industrial application prospects.

Claims

1. An anthranilate synthase mutant, characterized in that, The last amino acid in the TXLLES domain of anthranilate synthase from Streptomyces, Enterobacter, and Bacillus is mutated to methionine; more specifically, it is derived from Streptomyces coelicolor, Escherichia coli, and Bacillus subtilis, where X is any amino acid; More specifically, the 40th position of anthranilate synthase from Escherichia coli, the 48th position of anthranilate synthase from Streptomyces coelicolor, and the 48th position of anthranilate synthase from Bacillus subtilis are mutated to methionine; Further specifically, the 40th position of anthranilate synthase encoded by nucleotides as shown in SEQ ID NO.1 from Escherichia coli is mutated to methionine; furthermore, the serine at the 94th position is mutated to asparagine.

2. The anthranilate synthase mutant according to claim 1, wherein The amino acid sequences of the anthranilate synthase mutants are as shown in SEQ ID NO.3, SEQ ID NO.13, SEQ ID NO.14, or SEQ ID NO.

15.

3. A gene encoding the anthranilate synthase mutant as claimed in claim 1 or 2, preferably the nucleotide sequence of the gene is as shown in SEQ ID NO.2 or SEQ ID NO.

16.

4. A recombinant vector, characterized in that, The recombinant vector contains the encoding nucleic acid as claimed in claim 3.

5. A recombinant genetic engineering bacterium, characterized in that, Containing the recombinant vector as claimed in claim 4.

6. A method for preparing an anthranilate synthase mutant, characterized in that, Comprising the following steps: S1: Insert the nucleotide sequence as claimed in claim 3 into a vector plasmid to obtain a recombinant plasmid; S2: Transform the recombinant plasmid obtained in S1 into competent cells; S3: Culture the competent cells obtained in S2 under resistance conditions to obtain monoclonal colonies, and expand the colonies; S4: Fermentatively culture the colonies expanded in S3. S5: A method for collecting, disrupting, and purifying proteins from the cells cultured in S4.

7. The preparation method according to claim 6, characterized in that, The vector plasmid in step S1 is pET28a.

8. The preparation method according to claim 6, characterized in that, The competent cells in step S2 are Escherichia coli cells.

9. Use of the anthranilate synthase mutant as claimed in claim 1 in the preparation of tryptophan.

10. Use of the anthranilate synthase mutant as claimed in claim 1 in the synthesis of food, drugs, fine chemicals, health products, and cosmetics.

Images