Tryptophan synthase mutant as well as coding gene, vector, recombinant bacterium and application thereof

Abstract

The invention discloses a tryptophan synthase mutant as well as a coding gene, a vector, recombinant bacteria and application thereof. Comprising the following steps: carrying out error-prone PCR (Polymerase Chain Reaction) on a coding gene trpBA of TrpS from EscherichiaoliK12MG1655, screening mutants by combining high-throughput screening, and finding that the affinity of the TrpS to a substrate indole can be effectively improved by mutating 114-site Gln of a beta subunit of the TrpS into Met and mutating 84-site Gly into Ser, so that the synthesis of L-tryptophan is promoted. The efficiency of the TrpS for converting indole and L-serine into L-tryptophan is higher due to the improvement of the affinity of indole. A new idea is provided for efficient production of L-tryptophan in the fermentation industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and specifically relates to a tryptophan synthase mutant, its encoding gene, vector, recombinant bacterium and application. Background Art

[0002] L-tryptophan is an aromatic amino acid essential for humans and animals in nature, and is widely used in fields such as pharmaceuticals and feeds. At present, the industrial production of L-tryptophan mainly relies on microbial fermentation. Escherichia coli has become one of the main strains for the industrial production of L-tryptophan due to its advantages such as fast growth rate, simple growth medium, good fermentation robustness, and perfect means and methods for genetic modification. In Escherichia coli, the production of L-tryptophan is strictly regulated by the tryptophan operon. When the intracellular L-tryptophan content in Escherichia coli is relatively high, the five genes trpE, trpD, trpC, trpB, and trpA downstream of the tryptophan operon show a silent state. When the intracellular L-tryptophan content is relatively low, the downstream genes start to be transcribed and translated and show an active state. These five genes constitute the terminal synthesis pathway of tryptophan. Tryptophan synthase (TrpS) is encoded by the two genes trpA and trpB in this terminal synthesis pathway. Tryptophan synthase is a dimeric complex composed of an α subunit encoded by the trpA gene and a β subunit encoded by the trpB gene, and its arrangement is αββα. TrpS catalyzes the last two steps of the L-tryptophan synthesis process. First, the α subunit of tryptophan synthase cleaves indole-3-glycerol phosphate (IGP) into glyceraldehyde-3-phosphate (G3P) and indole. The indole produced by the cleavage enters the β subunit of tryptophan synthase along the channel between the α subunit and the β subunit. Then, the β subunit catalyzes the synthesis of L-tryptophan from indole and L-serine. The β subunit needs to rely on the cofactor pyridoxal phosphate to exhibit enzymatic activity. Studies have shown that the relationship between the α subunit and the β subunit of tryptophan synthase is inseparable. Some researchers have tried to separate the α subunit and the β subunit separately to achieve separate catalysis, but found that when the two subunits act alone, their catalytic efficiency for the substrate is greatly reduced. This is because there is an allosteric regulation between the α subunit and the β subunit. Since a special structure is formed between the flexible loop of the α subunit and the COMM domain of the β subunit of tryptophan synthase, this is not only the channel for the active sites of the α and β subunits, but also related to the allosteric regulation between the two subunits. This allosteric regulation mechanism is very complex. Studies have shown that when the substrate IGP of the α subunit binds to the active site of the α subunit, the activity of the reaction stage 1 of the active site of the β subunit is increased by 3-10 times. This indicates that there is a close relationship between the α subunit and the β subunit of tryptophan synthase, and it is not just a simple two-step reaction.

[0003] Tryptophan synthase has become a hot topic in enzyme engineering research due to its special structure and function. Many researchers have attempted to modify the β subunit of tryptophan synthase to enable it to catalyze efficiently on its own, independent of the α subunit. Miguel et al. identified the distal amino acid residues of the β subunit of tryptophan synthase and changed the conformation of the isolated β subunit by altering these residues, converting it from an inefficient conformation to an efficient one, thus changing the phenomenon of poor activity of the β subunit after separation from the α subunit of tryptophan synthase. Frances H. Arnold et al. performed directed evolution on the β subunit of tryptophan synthase from Pyrococcus furiosus, enabling it to have good activity when catalyzing alone, independent of the α subunit. The T292S mutant obtained through directed evolution had a 3.5-fold increase in kcat compared to the wild-type β subunit. Shingo Ito et al. used a quantum mechanics / molecular mechanics (QM / MM) hybrid model to analyze the reason for the increased efficiency of the reaction in the active site of the β subunit after the ligand binds to the α subunit. Some researchers have also modified tryptophan synthase through structure analysis to endow it with other functions. For example, Patrick J. Almhjell et al. modified the β subunit of tryptophan synthase through directed evolution to make it a tyrosine synthase, capable of catalyzing the synthesis of tyrosine.

[0004] Indole is an important substance in bacteria. There are research reports indicating that indole, as an important signaling molecule inside bacteria, seems to be very closely related to the bacteria's resistance to antibiotics. However, indole also has a certain degree of cytotoxicity. If indole accumulates excessively inside bacteria, it will have a very serious inhibitory effect on bacterial growth and product synthesis. The synthesis of indole in E. coli mainly relies on the intermediate PEP from the glycolysis pathway and the intermediate E4P from the pentose phosphate pathway through the shikimate pathway and the terminal synthesis pathway of tryptophan. It can also rely on tryptophanase to break down L-tryptophan into indole. Metabolic engineering to modify E. coli to overproduce L-tryptophan is also achieved by strengthening the enzymes in the shikimate and chorismate pathways to supply a large amount of the precursors for L-tryptophan synthesis.

[0005] However, as mentioned above, indole needs to be at a relatively balanced concentration inside E. coli to be beneficial for bacterial growth. Therefore, modifying tryptophan synthase to enable rapid and efficient metabolism of excessive indole into L-tryptophan can result in higher yields of L-tryptophan in industrial production, bringing better social and economic benefits. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this section, the abstract, and the title, but such simplifications or omissions shall not be used to limit the scope of the present invention.

[0007] In view of the above and / or problems existing in the prior art, the present invention is proposed.

[0008] Therefore, an object of the present invention is to overcome the deficiencies in the prior art and provide a tryptophan synthase mutant, its encoding gene, vector, recombinant bacterium, and application.

[0009] To solve the above technical problems, the present invention provides the following technical solution: A tryptophan synthase mutant, characterized in that: the tryptophan synthase mutant is TrpS(B) Q114M,G84S , which is obtained by mutating Gln at position 114 of the β subunit of tryptophan synthase in the parental sequence shown in SEQ ID NO.2 to Met and mutating Gly at position 84 to Ser.

[0010] As a preferred embodiment of the tryptophan synthase mutant of the present invention, wherein: the amino acid sequence of the TrpS(B) Q114M,G84S is as shown in SEQ ID NOs.5 - 6, and the nucleotide sequence is as shown in SEQ ID NOs.7 - 8.

[0011] Another object of the present invention is to overcome the deficiencies in the prior art and provide a coding gene containing the tryptophan synthase mutant.

[0012] Another object of the present invention is to overcome the deficiencies in the prior art and provide an expression vector carrying the coding gene.

[0013] As a preferred embodiment of the expression vector of the present invention, wherein: the expression vector is a bacterial plasmid, phage, yeast plasmid, plant cell virus, or mammalian cell virus.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide a recombinant bacterium expressing the tryptophan synthase mutant.

[0015] As a preferred embodiment of the recombinant bacterium of the present invention, wherein: the recombinant bacterium uses bacteria, fungi, plants, insects, or animal cells as host cells.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of the tryptophan synthase mutant in catalytically synthesizing L - tryptophan using indole and L - serine as substrates and pyridoxal phosphate as a cofactor.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide an enzyme preparation of a tryptophan synthase mutant.

[0018] As a preferred embodiment of the enzyme preparation of the present invention, wherein: the enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

[0019] Beneficial effects of the present invention:

[0020] In the present invention, by mutating tryptophan synthase from Escherichia coli K12 MG1655, Gln at position 114 of the β subunit of tryptophan synthase is mutated to Met and Gly at position 84 is mutated to Ser, so that it can exhibit higher indole affinity. The improvement of indole affinity makes it more efficient in converting indole and L-serine into L-tryptophan. This provides effective guidance for the high-efficient production of L-tryptophan in the fermentation industry. Description of the drawings

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them:

[0022] Figure 1 Comparison of the specific fluorescence values and OD 600 of the K-E8 mutant of tryptophan synthase and the wild-type tryptophan synthase in the embodiments of the present invention.

[0023] Figure 2 Comparison of the flask fermentation yields of L-tryptophan between JW-5 and JW-5 carrying different tryptophan synthase mutants in the embodiments of the present invention.

[0024] Figure 3 For the JW-5 strain and JW-TrpS(B) in the embodiments of the present invention Q114M,G84S Growth and L-tryptophan production curves at the 5L fermentor level of the strain.

[0025] Figure 4 SDS-PAGE diagram of the expression of tryptophan synthase in Escherichia coli BL21(DE3) in the embodiments of the present invention. The left figure is the protein expression gel diagram of the crude enzyme solution, and the right figure is the protein expression gel diagram of the purified tryptophan synthase.

[0026] Figure 5 For the tryptophan synthase mutant W-TrpS(B) of the present invention Q114M,G84S Protein molecular structure diagram obtained based on AlphaFold3 calculation. Detailed implementation manners

[0027] In order to make the above - mentioned objects, features and advantages of the present invention more obvious and understandable, the following specifically describes the embodiments of the present invention in detail in conjunction with the examples of the specification.

[0028] In the following description, many specific details are set forth to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0029] Secondly, the so - called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that can be included in at least one implementation manner of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it an individual or selectively mutually exclusive embodiment with other embodiments.

[0030] Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available. Among them, the starting bacterium is Escherichia coli K12 MG1655. The amino acid sequences are shown in SEQ ID NO.1 - 2, and the nucleotide sequences are shown in SEQ ID NO.3 - 4.

[0031] The JW - 5 strain (GenBank accession number: PRJNA1208919) is obtained by mutagenizing the E. coli MG1655 strain with atmospheric and room temperature plasma (ARTP), and then screening for resistance using p - fluorophenylalanine (PFP), 5 - fluorotryptophan (5 - FT), azaserine (AzaSer) and sulfaguanidine (SG), thereby obtaining a resistant mutant strain that produces L - tryptophan, namely Escherichia coli JW - 5 (i.e., Escherichia coli PFPr5 - FTrAzaSerr SGr Tyr -).

[0032] E. coli K12 MG1655 - △trpBA (tryptophan synthase - deficient strain): Based on the E. coli K12 wild - type strain, the gene trpBA was knocked out using the Crispr - cas9 technology.

[0033] Qualitative and quantitative analysis of substrates and products and monitoring of the growth of bacteria: Determination of bacterial liquid concentration: Pipette the sample bacterial liquid, dilute it by a certain multiple with distilled water, use distilled water as a blank control, and measure the OD at 600 nm and 1 cm optical path using a spectrophotometer. 600。The L-tryptophan production was determined by high performance liquid chromatography (HPLC), and its content was determined with reference to the standard curve. The purified tryptophan synthase and mutant proteins were obtained by purifying the crude enzyme solution through an affinity chromatography column, because the pET28a plasmid enables the expressed tryptophan synthase and its mutants to carry a 6His tag, and carrying the 6His tag allows the protein to bind to the chromatography column.

[0034] Example 1

[0035] Error-prone PCR was performed on the trpBA gene encoding tryptophan synthase:

[0036] A biosensor was constructed according to a high-throughput screening method reported by Mingyue Fang et al. that can characterize the L-tryptophan production through fluorescence intensity (Intermediate-sensor assisted push–pull strategy and its application in heterologous deoxyviolace in production in Escherichia coli). The construction method of the present invention is as follows:

[0037] First, the T7 promoter and T7 terminator of the plasmid pAcyc-Duet-1 plasmid were replaced with the Trc promoter and T2 terminator to achieve its availability in the Escherichia coli K12 strain. Then, the 24 bp sequence upstream of the start codon of the tnaC gene, the tnaC gene sequence, the intermediate sequence between the tnaC gene and the tnaA gene, the start codon of the tnaA gene and the 30 bp sequence downstream, that is, the enhanced green fluorescent protein sequence (eGFP), were assembled in the above order and constructed into the plasmid vector with the replaced promoter and terminator. That is, the sensor construction was completed.

[0038] The constructed biosensor was introduced into E. coli K12 MG1655-ΔtrpBA (tryptophan synthase-deficient strain) by chemical transformation. Error-prone PCR was performed on trpBA using the 2×Error-Prone PCR Mix kit from Gene Ze Company.

[0039] The error-prone trpBA library was seamlessly cloned between the EcoRI and XbaI restriction sites of the pTRCmob plasmid using the ClonExpress II One Step Cloning Kit from Nanjing vazyme Company.

[0040] Example 2

[0041] Obtaining tryptophan synthase with high indole affinity by high-throughput screening:

[0042] The pTRCmob plasmid carrying the trpBA mutant library was transformed into the tryptophan synthase-deficient E. coli K12 with the biosensor responsive to L-tryptophan production mentioned in Example 1, and the bacterial solution was spread on the double-antibiotic LB plates of C 20 and K 50 .

[0043] After single colonies grew, single colonies were picked and cultured in a 96-deep well plate. The medium used in the 96-deep well plate was M9 glucose inorganic salt medium supplemented with 0.5 g / L of L-serine and 0.35 g / L of indole. The 96-well plate was cultured overnight at 37 °C and 200 rpm.

[0044] After the culture was completed, 200 μL of the bacterial solution was transferred to an ELISA plate using a multichannel pipette, and the fluorescence value (excitation 479 nm; emission 520 nm) and OD 600 were measured in a multimode microplate reader.

[0045] After multiple rounds of error-prone and high-throughput screening, we obtained a strain with a high fluorescence value and an increased OD 600 compared with the wild type, which was named K-E8. The specific fluorescence value of K-E8 and OD 600 are compared with the wild type as Figure 1 shown.

[0046] Example 3

[0047] Perform Sanger sequencing on the mutant gene trpBA in K-E8:

[0048] We commissioned Genewiz Suzhou to sequence the mutant trpBA in K-E8.

[0049] After aligning the mutant trpBA, we found that there were 23 missense mutations and 7 nonsense mutations, and the final amino acid sequence is shown in SEQ ID NO.9-10, and the nucleotide sequence is shown in SEQ ID NO.11-12.

[0050] Example 4

[0051] Screen 23 missense mutation sites to obtain the best tryptophan synthase mutants:

[0052] We performed site-directed mutagenesis on wild-type TrpS for the above 23 missense mutation sites. Subsequently, these mutant TrpS were integrated into the pseudogene locus yeeP of strain JW-5 by CRISPR-Cas9 technology.

[0053] Strain JW-5 is a strain derived from E. coli K12 MG1655 and has been developed into a high-yield L-tryptophan producing strain after mutagenesis treatment in our laboratory. After the shake flask fermentation experiment of the strain containing mutant TrpS and the control strain JW-5, the yield of L-tryptophan was analyzed.

[0054] The results showed that the L-tryptophan yields of strains JW-TrpS(B) K61A , JW-TrpS(B) G84S , JW-TrpS(B) Q114M and JW-TrpS(B) F385V were significantly higher than those of the control strain JW-5. Among them, the yield of strain JW-TrpS(B) Q114M was the highest, reaching 6.60 g / L, which was about 29.9% higher than 5.08 g / L of JW-5. In addition, its α increased from 12.7% to 16.5%. The yields of JW-TrpS(B) K61A , JW-TrpS(B) G84S and JW-TrpS(B) F385V were 6.17 g / L, 6.13 g / L and 6.32 g / L respectively ( Figure 2 ).

[0055] To further increase the yield of L-tryptophan, we constructed combinatorial mutant strains by introducing K61A, G84S and F385V mutations respectively based on JW-TrpS(B) Q114M . The results of the shake flask experiment showed that the L-tryptophan yield of JW-TrpS(B) Q114M,G84S was the highest, reaching 7.10 g / L, and α was 17.8%. However, the L-tryptophan yields of the combinatorial mutant strains JW-TrpS(B) Q114M,K61A and JW-TrpS(B) Q114M,F385V did not exceed that of the single mutant strain JW-TrpS(B) Q114M , which were 6.41 g / L and 6.77 g / L respectively ( Figure 2 ). It is worth noting that the yield of JW-TrpS(B) Q114M,K61A was slightly lower than that of the single mutant strain JW-TrpS(B) Q114M (6.60 g / L). These results indicate that JW-TrpS(B) Q114M,G84S has significant potential in increasing the yield of L-tryptophan.

[0056] To verify JW-TrpS(B) Q114M,G84SPerformance in the bioreactor: We conducted a fed-batch fermentation experiment for 40 hours in a 5 L fermenter, using strain JW-5 as a control. In the batch fermentation, glucose was exhausted within approximately 4 hours, and then the glucose concentration was maintained at 0 to 1 g / L by feeding. L-tryptophan began to accumulate rapidly from approximately 5 hours and continued to increase after 35 hours to a final concentration of 34.43 g / L. In contrast, the final concentration of L-tryptophan of the control strain JW-5 was only 21.38 g / L. JW-TrpS(B) Q114M,G84S had a 61.03% higher L-tryptophan yield than JW-5. In addition, JW-TrpS(B) Q114M,G84S had an α of 21.6%, which was significantly higher than 14.8% of JW-5 ( Figure 3 ). These results fully demonstrated the significant advantage of JW-TrpS(B) Q114M,G84S in improving L-tryptophan production and highlighted its potential application value in industrial fermentation processes. Its amino acid sequence is shown in SEQ ID NO.5 - 6, and the nucleotide sequence is shown in SEQ ID NO.7 - 8. Table 1 shows the primers used for strain and plasmid construction.

[0057] Table 1 Primers used for strain and plasmid construction

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Example 5

[0065] Determination of the enzymatic properties of mutants with high L-tryptophan yields:

[0066] First, the genes encoding tryptophan synthase and its mutants were respectively constructed on the pET28a Escherichia coli inducible expression vector, and the constructed plasmids were chemically transformed into Escherichia coli BL21(DE3).

[0067] The strain was inoculated into TB medium and cultured at 37 °C for 2 - 3 h until OD 600It is between 0.5 and 0.6. After standing at room temperature for 30 min, 0.1 mmol / L IPTG was added to the bacterial solution, and then cultured at 30 °C for 12 - 16 h. The cultured bacterial solution was first rinsed 3 times with PBS, and then broken using an ultrasonic crusher. After breaking, the supernatant was taken respectively for SDS-PAGE to verify the protein expression situation( Figure 4 ).

[0068] The supernatant after breaking was purified by affinity chromatography. First, the crude enzyme solution was passed through the column 2 times, and then the impurity proteins were eluted using a 20 mM - 200 mM imidazole solution, and the tryptophan synthase was eluted using 300 mM imidazole. Finally, the purified tryptophan synthase protein was obtained( Figure 4 ).

[0069] Add 0.3 ml of 80 mmol / L L-serine, 0.16 ml of 80 mmol / L potassium phosphate buffer solution (pH 8.5), and 0.1 ml of pyridoxal phosphate solution containing 10 mg into a test tube, and then add 0.04 ml of 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, and 5 mmol / L indole solutions to the mixed solution respectively. Finally, add 0.4 ml of the pure enzyme extract to the reaction system and gently vortex. React in a 37 °C water bath for 90 min, and terminate the reaction using 0.2 ml of 5% NaOH solution. According to the Lineweaver - Burk double reciprocal method for plotting, the Michaelis constant (Km), maximum reaction rate (Vmax), and catalytic constant (kcat) of tryptophan synthase and its mutants can be calculated when using excessive L-serine and different concentrations of indole as substrates. The results show that the combined mutant TrpS(B) Q114M,G84S showed better affinity for indole (Table 2). This indicates that mutating Gln at position 114 of the β subunit of tryptophan synthase to Met and Gly at position 84 to Ser can significantly improve the affinity of tryptophan synthase for indole. This may be related to the change in the indole-binding pocket of tryptophan synthase, thus showing stronger affinity for indole, and thereby improving the conversion efficiency of L-tryptophan.

[0070] Figure 5 is the tryptophan synthase mutant W-TrpS(B) described in the present invention Q114M,G84S Protein molecular structure diagram obtained based on AlphaFold3 calculation.

[0071] Table 2 Kinetic parameters of tryptophan synthase and its mutants for the substrate indole

[0072]

[0073] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered within the scope of the present invention.

[0074] Escherichia coli K12 MG1655

[0075] SEQ ID NO.1:

[0076] α subunit

[0077] MERYENLFAQLNDRREGAFVPFVTLGDPGIEQSLKIIDTLIDAGADALELGVPFSDPLADGPTIQNANLRAFAAGVTPAQCFEMLALIREKHPTIPIGLLMYANLVFNNGIDAFYARCEQVGVDSVLVADVPVEESAPFRQAALRHNIAPIFICPPNADDDLLRQVASYGRGYTYLLSRSGVTGAENRGALPLHHLIEKLKEYHAAPALQGFGISSPEQVSAAVRAGAAGAISGSAIVKIIEKNLASPKQMLAELRSFVSAMKAASRA

[0078] SEQ ID NO.2:

[0079] β subunit

[0080] MTTLLNPYFGEFGGMYVPQILMPALNQLEEAFVSAQKDPEFQAQFADLLKNYAGRPTALTKCQNITAGTRTTLYLKREDLLHGGAHKTNQVLGQALLAKRMGKSEIIAETGAGQHGVASALASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWSGSYETAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILDKEGRLPDAVIACVGGGSNAIGMFADFINDTSVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPMMQTADGQIEESYSISAGLDFPSVGPQHAYLNSIGRADYVSITDDEALEAFKTLCRHEGIIPALESSHALAHALKMMREQPEKEQLLVVNLSGRGDKDIFTVHDILKARGEI

[0081] Escherichia coli K12 MG1655

[0082] SEQ ID NO.3:

[0083] trpA

[0084] atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcacgctcggtgatccgggcattgagcagtcattgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagttaggtatccccttctccgacccactggcggatggcccgacgattcaaaacgccactctgcgcgcctttgcggcaggtgtgactccggcacaatgttttgaaatgctggcactgattcgccagaaacacccgaccattcccattggcctgttgatgtatgccaatctggtgtttaacaaaggcattgatgagttttatgcccagtgcgaaaaagtcggcgtcgattcggtgctggttgccgatgtgccagttgaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcgcacctatcttcatctgcccgccaaatgccgatgacgacctgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcacgagcaggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctgaaagagtacaacgctgcacctccattgcagggatttggtatttccgccccggatcaggtaaaagcagcgattgatgcaggagctgcgggcgcgatttctggttcggccattgttaaaatcatcgagcaacatattaatgagccagagaaaatgctggcggcactgaaagtttttgtacaaccgatgaaagcggcgacgcgcagttaa

[0085] SEQ ID NO.4:

[0086] trpB

[0087]

[0088] TrpS(B) Q114M,G84S

[0089] SEQ ID NO.5:

[0090] α subunit

[0091] MERYENLFAQLNDRREGAFVPFVTLGDPGIEQSLKIIDTLIDAGADALELGVPFSDPLADGPTIQNANLRAFAAGVTPAQCFEMLALIREKHPTIPIGLLMYANLVFNNGIDAFYARCEQVGVDSVLVADVPVEESAPFRQAALRHNIAPIFICPPNADDDLLRQVASYGRGYTYLLSRSGVTGAENRGALPLHHLIEKLKEYHAAPALQGFGISSPEQVSAAVRAGAAGAISGSAIVKIIEKNLASPKQMLAELRSFVSAMKAASRA

[0092] SEQ ID NO.6:

[0093] β subunit

[0094] MTTLLNPYFGEFGGMYVPQILMPALNQLEEAFVSAQKDPEFQAQFADLLKNYAGRPTALTKCQNITAGTRTTLYLKREDLLHGSAHKTNQVLGQALLAKRMGKSEIIAETGAGMHGVASALASALLGLKCRIYMGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACNEALRDWSGSYETAHYMLGTAAGPHPYPTIVREFQRMIGEETKAQILDKEGRLPDAVIACVGGGSNAIGMFADFINDTSVGLIGVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPMMQTADGQIEESYSISAGLDFPSVGPQHAYLNSIGRADYVSITDDEALEAFKTLCRHEGIIPALESSHALAHALKMMREQPEKEQLLVVNLSGRGDKDIFTVHDILKARGEI

[0095] TrpS(B) Q114M,G84S

[0096] SEQ ID NO.7:

[0097] trpA

[0098] atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcacgctcggtgatccgggcattgagcagtcattgaaaattatcgatacgctaattgaagccggtgctgacgcgctggagttaggtatccccttctccgacccactggcggatggcccgacgattcaaaacgccactctgcgcgcctttgcggcaggtgtgactccggcacaatgttttgaaatgctggcactgattcgccagaaacacccgaccattcccattggcctgttgatgtatgccaatctggtgtttaacaaaggcattgatgagttttatgcccagtgcgaaaaagtcggcgtcgattcggtgctggttgccgatgtgccagttgaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcgcacctatcttcatctgcccgccaaatgccgatgacgacctgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcacgagcaggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctgaaagagtacaacgctgcacctccattgcagggatttggtatttccgccccggatcaggtaaaagcagcgattgatgcaggagctgcgggcgcgatttctggttcggccattgttaaaatcatcgagcaacatattaatgagccagagaaaatgctggcggcactgaaagtttttgtacaaccgatgaaagcggcgacgcgcagttaa

[0099] SEQ ID NO.8:

[0100] trpB

[0101]

[0102] K-E8

[0103] SEQ ID NO.9:

[0104] α subunit

[0105] MERYENLFAQLNDRREGAFVPFVTLGDPGTEQSLKIIDTPIDAGADALELGVPFSDPLADGPTIQNTNLRAFAAGVTPAQCFEMQALIREKHPTISIGLLMYANLVFNNGIDAFYARCEQVGVDSVLVADVPVEESAPFRQAALRHNIAPIFICPPNADDDLLRQVASYGRGYTYLLSRSGVTGAENRGALPLHHLIEKLKEYHAAPALQGFGISSPEQVSAVVRAGAAGAISGSAIVKIIEKNLASPKQMLAELRSFVSAMKAASRA

[0106] SEQ ID NO.10:

[0107] β subunit

[0108] MTTLLNPYFGEFGGMYVPQILMPALNQLEEAFVSAQKDPEFQAQLADLLKNYAGRPTALTACQNITAGTRTTLYLKREDLLHGSAHKTNQVLGQALLAKRMGKSEIIAETGAGMHGVASALASALLGLKCRIYVGAKDVERQSPNVFRMRLMGAEVIPVHSGSATLKDACSEALRDWSGSYETAQYMLGTAAGPHPYPTIVSEFQRMIGEETKAQIQDKEGRLPDAVNACVGGGSNSIGMFADFINDTSVCLIGVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPTMQTADGQIEESYSISAGLDFPSVGPQHANLNRIGRADYVSITDDEALEAFKTLCRHEGIIPALESSHTLAHALKMMREQPEKEQLLVVNLSGRGDKDIVTVHDILKARGEI

[0109] K-E8

[0110] SEQ ID NO.11:

[0111] trpA

[0112] atggaacgctacgaatctctgtttgcccagttgaaggagcgcaaagaaggcgcattcgttcctttcgtcacgctcggtgatccgggcactgagcagtcattgaaaattatcgatacgccaattgaagccggtgctgacgcgctggagttaggtatccccttctccgacccactggcggatggcccgacgattcaaaacaccactctgcgcgcctttgcggcaggtgtgactccggcacaatgttttgaaatgcaggcactgattcgccagaaacacccgaccatttccattggcctgttgatgtatgccaatctggtgtttaacaaaggcattgatgagttttatgcccagtgcgaaaaagtcggcgtcgattcggtgctggttgccgatgtgccagttgaagagtccgcgcccttccgccaggccgcgttgcgtcataatgtcgcacctatcttcatctgcccgccaaatgccgatgacgacctgctgcgccagatagcctcttacggtcgtggttacacctatttgctgtcacgagcaggcgtgaccggcgcagaaaaccgcgccgcgttacccctcaatcatctggttgcgaagctgaaagagtacaacgctgcgcctccattgcagggatttggtatttccgccccggatcaggtaaaagcagtgattgatgcaggagctgcgggcgcgatttctggttcggccattgttaaaatcatcgagcaacatattaatgagccagagaaaatgctggcggctctgaaagtttttgtacaaccgatgaaagcggcgacgcgcagttaa

[0113] SEQ ID NO.12:

[0114] trpB

[0115]

Claims

1. A tryptophan synthase mutant, characterized in that: The tryptophan synthase mutant described above is TrpS(B). Q114M,G84S It is derived from the parental sequence shown in SEQ ID NO.2 by mutating Gln at position 114 of the tryptophan synthase β subunit to Met and Gly at position 84 to Ser.

2. The tryptophan synthase mutant according to claim 1, wherein: The TrpS(B) Q114M,G84S has the amino acid sequences shown in SEQ ID NO.5-6 and the nucleotide sequences shown in SEQ ID NO.7-8.

3. A coding gene containing the tryptophan synthase mutant as described in claim 1 or 2.

4. An expression vector carrying the coding gene as described in claim 3.

5. The expression vector according to claim 4, characterized in that, The expression vector is a bacterial plasmid, phage, yeast plasmid, plant cell virus or mammalian cell virus.

6. A recombinant bacterium expressing the tryptophan synthase mutant as described in claim 1.

7. The recombinant bacterium according to claim 6, characterized in that, The recombinant bacterium uses a bacterium, fungus, plant, insect or animal cell as the host cell.

8. Use of the tryptophan synthase mutant as described in claim 1 in the catalytic synthesis of L-tryptophan using indole and L-serine as substrates and pyridoxal phosphate as a cofactor.

9. An enzyme preparation containing the tryptophan synthase mutant as described in claim 1.

10. The enzyme preparation according to claim 9, characterized in that, The enzyme preparation is a solid enzyme preparation or a liquid enzyme preparation.

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