Tryptophan synthetase mutant and application thereof in synthesis of L-serine derivative

Abstract

The invention relates to a tryptophan synthetase mutant and application of the tryptophan synthetase mutant in synthesis of an L-serine derivative, and belongs to the technical field of enzyme engineering. The tryptophan synthetase mutant provided by the invention takes an amino acid sequence as shown in SEQ ID NO.1 as a starting sequence, and is obtained by mutating 160th histidine into threonine, when L-tryptophan is produced, the enzyme activity is increased by 19.2% and the yield is increased by 16.25% compared with that of a wild type, and when L-cysteine is produced, the enzyme activity is increased by 17.62% and the yield is increased by 19.39% compared with that of the wild type. Serine hydroxymethyltransferase is introduced into an expression system, a reaction system with glycine as a substrate is constructed, the yield of the mutant H160T during production of L-tryptophan is increased by 19.86% compared with that of a wild type enzyme, the yield of the mutant H160T during production of L-cysteine is increased by 16.67% compared with that of the wild type enzyme, multiple purposes of one enzyme and multiple purposes of one bacterium are achieved, and the yield of the mutant H160T during production of L-tryptophan is increased by 19.86% compared with that of the wild type enzyme. A new thought and a new direction are provided for industrial production of the L-serine derivative.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a tryptophan synthase mutant and its application in the synthesis of L-serine derivatives. Background Art

[0002] L-Serine is a precursor of many important substances. It is an important precursor involved in the synthesis of intracellular biomolecules such as purines, pyrimidines, and phospholipids. It is also an essential precursor substance for the synthesis of two amino acids, L-tryptophan and L-cysteine.

[0003] L-Tryptophan is an important aromatic amino acid with two isomers, L-type and D-type. It has a wide range of applications in the fields of medicine, food, feed, etc., and is also a direct precursor for the synthesis of many high-value-added compounds. Tryptophan is considered an over-the-counter drug in many countries. It can effectively cross the blood-brain barrier and can be used as a nutritional supplement, sleep aid, appetite suppressant, and antidepressant. Tryptophan plays an important role in the treatment of various diseases, such as depression, obesity, cerebellar ataxia, persistent headache, fibromyalgia, and insomnia.

[0004] As an essential amino acid for the human body, L-cysteine can combine with other amino acids to form proteins and play physiological functions. L-Cysteine forms disulfide bonds in vivo, which plays an important role in stabilizing the protein structure. And L-cysteine has a wide range of applications in the fields of medicine, food, and cosmetics.

[0005] In traditional industries, L-tryptophan and L-cysteine are mainly produced by chemical synthesis, biotransformation, and enzymatic transformation methods. Nowadays, the production of amino acids by direct fermentation has become the main method of industrial production due to its green, more efficient, low-cost, and environmentally friendly advantages. Strains that can be used for the fermentation production of L-tryptophan and L-cysteine include Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. Among them, Escherichia coli has the advantages of fast growth rate, easy cultivation, and rich modification means, and has become the preferred strain for producing amino acids. In the tryptophan synthesis pathway, the tryptophan synthase encoded by trpBA is a key enzyme in the tryptophan synthesis pathway. Increasing its activity is beneficial to increasing the yield of tryptophan. And tryptophan synthase is a multifunctional enzyme. By adding different substrates, corresponding L-serine derivatives can be generated. Some studies have shown that it has the catalytic ability to synthesize L-cysteine. Therefore, increasing its activity is also beneficial to the synthesis of L-cysteine, which also provides a basis for "using one strain for multiple purposes".

[0006] However, there are few reports on how to improve the activities of producing L-tryptophan and L-cysteine by directed modification of tryptophan synthase. SUMMARY OF THE INVENTION

[0007] To solve the above technical problems, the present invention analyzed the sequence of wild-type tryptophan synthase with the amino acid sequence shown in SEQ ID NO.1, determined multiple mutation sites, and screened out a tryptophan synthase mutant H160T with improved tryptophan synthase activity and yield, which can be simultaneously applied to the production of L-serine derivative L-tryptophan and L-cysteine.

[0008] The first object of the present invention is to provide a tryptophan synthase mutant, which is obtained by mutating histidine at position 160 of the starting sequence with the amino acid sequence shown in SEQ ID NO.1 to threonine.

[0009] Further, the nucleotide sequence of the tryptophan synthase is shown in SEQ ID NO.2.

[0010] Further, the tryptophan synthase is composed of TrpB and TrpA. The amino acid sequence of TrpB is shown in SEQ ID NO.3, and the amino acid sequence of TrpA is shown in SEQ ID NO.4.

[0011] The second object of the present invention is to provide a gene encoding the above tryptophan synthase mutant.

[0012] The third object of the present invention is to provide an expression vector containing the above gene.

[0013] In one embodiment of the present invention, the expression vector is based on pET22b.

[0014] The fourth object of the present invention is to provide a host cell containing the above tryptophan synthase mutant, the above gene or the above expression vector.

[0015] In one embodiment of the present invention, the host cell is Escherichia coli BL21(DE3).

[0016] The fifth object of the present invention is to provide the application of the above tryptophan synthase mutant, the above gene, the above expression vector or the above host cell in the production of L-serine derivatives.

[0017] Further, the L-serine derivative is L-tryptophan and / or L-cysteine.

[0018] The sixth object of the present invention is to provide a method for synthesizing L-tryptophan, which comprises adding the above-mentioned tryptophan synthase mutant or an expression system containing the mutant to a reaction system containing a substrate for reaction.

[0019] Further, the substrate is L-serine or glycine.

[0020] Further, indole is added to the reaction system.

[0021] Further, when the substrate is glycine, the reaction system further contains serine hydroxymethyltransferase and formaldehyde.

[0022] In one embodiment of the present invention, the nucleotide sequence of the serine hydroxymethyltransferase is as shown in SEQ ID NO.5.

[0023] The seventh object of the present invention is to provide a method for synthesizing L-cysteine, which comprises adding the above-mentioned tryptophan synthase mutant or an expression system containing the mutant to a reaction system containing a substrate for reaction.

[0024] Further, the substrate is L-serine or glycine.

[0025] Further, sodium hydrosulfide is added to the reaction system.

[0026] Further, when the substrate is glycine, the reaction system further contains serine hydroxymethyltransferase and formaldehyde.

[0027] In one embodiment of the present invention, the nucleotide sequence of the serine hydroxymethyltransferase is as shown in SEQ ID NO.5. The beneficial effects of the present invention:

[0028] The tryptophan synthase mutant H160T provided by the present invention has a 19.2% increase in enzyme activity and a 16.25% increase in production when producing L-tryptophan compared to the wild-type enzyme. When producing L-cysteine, it has a 17.62% increase in enzyme activity and a 19.39% increase in production compared to the wild-type enzyme. By introducing serine hydroxymethyltransferase into the expression system and constructing a reaction system with glycine as the substrate, the production of mutant H160T is increased by 19.86% compared to the wild-type enzyme when producing L-tryptophan, and the production is increased by 16.67% compared to the wild-type enzyme when producing L-cysteine, significantly improving the catalytic characteristics of the wild enzyme. This mutation not only enhances the catalytic efficiency of the enzyme towards L-serine, but the same mutant can efficiently catalyze the synthesis of two high-value-added amino acids, L-tryptophan and L-cysteine, achieving "one enzyme, dual use". Through the development of the glycine substrate route, it provides a new way to simplify the production process and reduce raw material costs. From genetic engineering to the systematic design of host cells, the high-efficiency expression and large-scale preparation of the mutant enzyme are realized. This system exhibits high conversion rate and stability in biosynthesis, providing a reliable technical platform for the continuous production of L-serine derivatives and having significant industrial application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the accompanying drawings, where:

[0030] Figure 1 It is a diagram showing the change results of the enzyme activity of tryptophan synthase and its mutant in producing L-tryptophan in Example 3 of the present invention;

[0031] Figure 2 It is a diagram showing the change results of the enzyme activity of tryptophan synthase and its mutant in producing L-cysteine in Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The following further describes the present invention in combination with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0033] The culture media and culture methods involved in the following examples are as follows:

[0034] LB medium (g / L): NaCl 10, tryptone 10, yeast extract 5;

[0035] 0.5% Tween 80 1M Tris-HCl buffer: Tris(hydroxymethyl)aminomethane 121.1, Tween 80 5, and the pH is adjusted to 8 with concentrated hydrochloric acid.

[0036] Escherichia coli competent cell transformation method:

[0037] First, place the fresh Escherichia coli competent cells on ice to thaw naturally. Add 10 μL of the recombinant product to each tube of competent cells, mix well, and immediately place on ice for 30 min. Then, heat-shock the competent cells in a 42 °C water bath for 90 s, and let them stand on ice for another 3 - 5 min. Subsequently, add 900 μL of sterilized LB medium without resistance. Incubate the above mixture at 220 rpm and 37 °C for about 45 min. Centrifuge the revived competent cells for 1 min, use a sterile pipette tip to aspirate 900 μL of the culture medium supernatant, and gently pipette to resuspend the cell pellet. After mixing well, spread the cells on a plate containing Amp resistance, and then place it in a 37 °C incubator for 12 - 16 h.

[0038] The detection methods involved in the following examples are as follows:

[0039] Detection of L-tryptophan and L-cysteine concentrations: Detected using a high-performance liquid chromatograph (HPLC). After the whole-cell catalysis is processed and the supernatant is filtered through a 0.22 μm microporous filter membrane, the content of tryptophan is detected using a VWD (ultraviolet detector). The liquid chromatography method is as follows: The high-performance liquid chromatograph is a product of Waters, USA, model 1515, and the chromatographic column is a Venusil AA (4.6×250 nm) liquid chromatography column. Column temperature: 30 °C; mobile phase: 100% methanol, flow rate: 1 mL / min; injection volume: 10 μL.

[0040] Calculate the enzyme activity based on the number of millimoles of L-tryptophan / L-cysteine produced per gram of wet cells per hour.

[0041] Determination of biomass: Take an appropriate amount of the catalytic solution and dilute it by an appropriate multiple, and then measure the OD at 600 nm using an ultraviolet spectrophotometer 600 as the amount of bacterial cells.

[0042] Example 1: Point mutation of tryptophan synthase

[0043] Use a website to predict and perform multiple sequence alignment analysis on the wild-type tryptophan synthase (the amino acid sequence of TrpBA is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2, where the amino acid sequence of TrpB is shown in SEQ ID NO.3, the amino acid sequence of TrpA is shown in SEQ ID NO.4, and the stop codon of TrpB and the start codon of Trp share one nucleotide). Use a hotspot prediction website (https: / / espript.ibcp.fr / ESPrist / cgi-bin / ESPript.cgi) to screen out sites with low homology (D47, N51, L121, H160, R219, N246, G265, and A284).

[0044] Site-directed mutagenesis was carried out by the PCR method on the tryptophan synthase gene. Using plasmid pET22b-TrpBA as a template, inverse PCR was performed, and the homologous arm gene fragment containing the point mutation was amplified using primers trpBA-F and trpBA-R. The amplified gene fragment was transformed into Escherichia coli for circularization and cultured at 37 °C for 12 - 16 h. The single colonies that grew were recombinant colonies. Single colonies were picked and sent to Wuxi Tianlin Co., Ltd. for sequencing. The strains with correct results were the successfully mutated strains, and the recombinant strains BL21(DE3)-pET22b-TrpBA and tryptophan synthase mutant strains BL21(DE3)-pET22b-TrpBA* (* represents different mutants) containing different mutants were obtained.

[0045] Among them, the sequence of primer trpBA-F is 5’-ATCCCGGTGACTAGCGGTTCCGCG ACG-3’; the sequence of primer trpBA-R is 5’-ACCGCTAGTCACCGGGATCACTTCCG C-3’.

[0046] Example 2: Construction of recombinant strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA

[0047] The recombinant strain prepared in Example 1 was introduced with the pET28a-SHMT plasmid carrying serine hydroxymethyltransferase (SHMT, nucleotide sequence as shown in SEQ ID NO.5) to construct a dual plasmid system, and the recombinant strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA and the tryptophan synthase mutant strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA * (* represents different mutants).

[0048] Example 3: Production of L-tryptophan by tryptophan synthase mutants using L-serine as a substrate

[0049] The enzyme activities of the recombinant strains and tryptophan synthase mutant strains prepared in Example 1 were measured to analyze the effect of point mutation on the activity of tryptophan synthase. The enzyme activities of tryptophan synthase of the starting strain BL21(DE3)-pET22b-TrpBA and the tryptophan synthase mutant strain BL21(DE3)-pET22b-TrpBA * (* represents different mutants) were measured by whole-cell catalysis for 6 h.

[0050] The specific steps of whole-cell catalysis are as follows:

[0051] Take 10 mL of the bacteria cultured in an LB liquid vial and inoculate it into a 50 mL LB large flask at an inoculation amount of 1%. Incubate it in a rotary shaker at 37 °C and 220 rpm until the OD 600 reaches 0.6 - 0.9. Then add IPTG with a final concentration of 0.04 mM. After culturing for 4 h, pour the cultured bacterial solution into a 50 mL centrifuge tube for centrifugation. The centrifugation conditions are 7000 rpm for 7 min, and discard the supernatant.

[0052] Subsequently, wash the bacteria with 1 M Tris-HCl buffer (pH = 8) twice and discard the supernatant. Add 9.9 mL of 1 M Tris-HCl buffer containing 200 mM L-serine (about 20 g / L L-serine) and 0.5% Tween 80. Add 42.7 mM indole (about 5 g / L indole, dissolved in 100 μL of ethyl acetate) for catalysis, and then place it in a rotary shaker at 30 °C and 220 rpm for culturing. After 6 h of catalysis, L-tryptophan is obtained.

[0053] (1) Changes in the enzyme activity of tryptophan synthase in producing L-tryptophan

[0054] The results of the changes in the enzyme activity of tryptophan synthase and its mutants are shown in Table 1. Using the unmutated tryptophan synthase starting strain BL21(DE3)-pET22b-TrpBA as the control group, and the tryptophan synthase mutant strain obtained in Example 2 as the experimental group, compare the enzyme activity of tryptophan synthase after culturing each strain. It is found that when histidine at position 160 is mutated to threonine, the activity of tryptophan synthase increases. The H160T point mutation causes the activity of tryptophan synthase to increase by 19.2% compared to the control, while other site-directed mutations result in no significant change in enzyme activity compared to the control group (only the control and the changed data are listed in the table).

[0055] Table 1 Enzyme activities of tryptophan synthase and mutants in producing L-tryptophan

[0056] Strain Average enzyme activity (U) Relative enzyme activity Control 33.87 100.00％ H160T 40.38 119.20％

[0057] (2) Changes in the yield of tryptophan synthase in producing L-tryptophan

[0058] The yield results of wild-type tryptophan synthase and mutants using L-serine as a substrate to produce L-tryptophan are as follows Figure 1As shown, the L-tryptophan yield of the original control group strain, i.e., the starting strain BL21(DE3)-pET22b-TrpBA, was 4.9 g / L, and the L-tryptophan yield of the final mutant strain H160T was 5.7 g / L, which was 16.25% higher than that of the control starting strain BL21(DE3)-pET22b-TrpBA. However, the mutants H160Q, H160Y, H160R, R219Q, N51E, G265H, N246E, L121T, D47Y, and A284S led to a decrease in L-tryptophan yield, and other point mutations did not cause changes in tryptophan yield. The relevant mutants are shown in Table 2. Combining the above results, it can be seen that compared with the control starting strain BL21(DE3), the enzyme activity of tryptophan synthase in the mutant strain H160T increased by 19.2%, and the corresponding L-tryptophan yield increased by 16.25% compared with the control starting strain BL21(DE3)-pET22b-TrpBA.

[0059] From the control results, it can be seen that not all point mutations have a beneficial promoting effect, and some point mutations may even have an inhibitory effect; the H160T mutant strain is the only one among all mutant strains that has obtained a beneficial promoting effect, which also indicates that it is of great significance to predict through the website and perform multiple sequence alignments, and then further guide the rational design and construction of high-yield strains. At the same time, it also shows that tryptophan synthase (encoding gene trpBA) is indeed related to the synthesis of L-tryptophan. Tryptophan synthase can promote the synthesis of L-tryptophan. Correspondingly, increasing its activity may increase the tryptophan yield.

[0060] Table 2 Tryptophan synthase mutants that change the L-tryptophan yield

[0061] Name Mutation D47Y Aspartic acid at position 47 is mutated to tyrosine N51E Asparagine at position 51 is mutated to glutamic acid L121T Leucine at position 121 is mutated to threonine H160T Histidine at position 160 is mutated to threonine H160Q Histidine at position 160 is mutated to glutamine H160Y Histidine at position 160 is mutated to tyrosine H160R Histidine at position 160 is mutated to arginine R219Q Arginine at position 219 is mutated to glutamine N246E Asparagine at position 246 is mutated to glutamic acid G265H Glycine at position 265 is mutated to histidine A284S Alanine at position 284 is mutated to serine

[0062] Example 4: Tryptophan synthase mutants produce L-cysteine using L-serine as a substrate

[0063] The enzyme activity of the recombinant strain prepared in Example 1 was measured to analyze the effect of point mutations on the enzyme activity of tryptophan synthase. The enzyme activity of tryptophan synthase in the starting strain BL21(DE3)-pET22b-TrpBA and the mutant strain was measured by high performance liquid chromatography (HPLC) for 6 h of whole-cell catalysis.

[0064] The specific steps of whole-cell catalysis are as follows:

[0065] Take the bacteria cultured in a 10 mL LB liquid vial and inoculate them into a 50 mL LB large vial at an inoculation amount of 1%, and culture them in a rotary shaker at 37 °C and 220 rpm until OD 600When = 0.6 - 0.9, IPTG with a final concentration of 0.04 mM was added. After culturing for 4 h, the cultured bacterial solution was poured into a 50 mL centrifuge tube for centrifugation. The centrifugation conditions were 7000 rpm for 7 min, and the supernatant was discarded.

[0066] Subsequently, the cells were washed with 1 M Tris-HCl buffer (pH = 8) twice, and the supernatant was discarded. 9.9 mL of 1 M Tris-HCl buffer containing 200 mM L-serine (about 20 g / L L-serine) and 0.5% Tween 80 was added, and 5 g / L sodium hydrosulfide was added. Then it was placed in a rotary shaker at 30 °C and 220 rpm for cultivation, and L-cysteine was obtained by catalysis for 12 h.

[0067] (1) Changes in the enzyme activity of tryptophan synthase in producing L-cysteine

[0068] The results are shown in Table 3. Using the unmutated tryptophan synthase starting strain as the control group, and the mutant strains with the tryptophan synthase point-mutated to H160T in Example 1 and multiple-site point mutations of the tryptophan synthase obtained in Example 2 as the experimental group, the enzyme activities of tryptophan synthase after culturing each strain were compared. It was found that when histidine at position 160 was mutated to threonine, the activity of tryptophan synthase increased. The H160T point mutation led to a 17.62% increase in the activity of tryptophan synthase compared to the control, while the other 11 site-directed mutations did not result in significant changes in enzyme activity compared to the control group (only the control and the changed data are listed in the table).

[0069] Table 3 Enzyme activities of tryptophan synthase and mutants in producing L-cysteine

[0070] Strain Average enzyme activity (U) Relative enzyme activity Control 8.13 100.00％ H160T 9.56 117.62％

[0071] (2) Changes in the yield of tryptophan synthase in producing L-cysteine

[0072] The yield results of wild-type tryptophan synthase and mutants in producing L-cysteine using L-serine as the substrate are as Figure 2As shown, the L-cysteine production of the original control group strain, i.e., the starting strain BL21(DE3)-pET22b-TrpBA, was 0.985 g / L, and the L-cysteine production of the mutant strain H160T was 1.176 g / L, which was 19.39% higher than that of the control starting strain BL21(DE3)-pET22b-TrpBA. However, the mutants H160Q, H160Y, H160R, R219Q, N51E, G265H, N246E, L121T, D47Y, and A284S (consistent with Table 2) led to a decrease in L-cysteine production, and other point mutations did not change the cysteine production. Combining the above results, it can be seen that compared with the control starting strain BL21(DE3), the enzyme activity of tryptophan synthase in the mutant strain H160T increased by 17.62% compared with the control, and the corresponding L-cysteine production increased by 19.39% compared with the control starting strain BL21(DE3)-pET22b-TrpBA.

[0073] From the control results, it can be seen that not all point mutations have a beneficial promoting effect, and some point mutations will instead have an inhibitory effect; in this example, H160T was selected as the mutant strain, which was the only one to obtain a beneficial promoting effect among all mutant strains. This also shows that it is of great significance to further guide the rational design and construction of high-yield strains through website prediction and multiple sequence alignment. At the same time, it also shows that tryptophan synthase (encoding gene trpBA) is indeed related to L-cysteine synthesis, and tryptophan synthase can promote L-cysteine synthesis. Correspondingly, increasing its activity may increase cysteine production.

[0074] Example 5: Production of L-tryptophan and L-cysteine using glycine as a substrate by tryptophan synthase mutants

[0075] (1) Changes in L-tryptophan production

[0076] The obtained recombinant strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA carrying wild-type tryptophan synthase and the tryptophan synthase mutant strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA carrying the H160T mutant H160T were subjected to a multi-enzyme cascade whole-cell catalytic reaction. The specific method was as follows:

[0077] 10 μL of the preserved recombinant strain was taken for three-zone streaking and cultured for 8 - 12 h. After clear single colonies grew, a single colony was picked and inoculated into a 10 mL LB liquid vial, cultured at 37°C and 220 rpm on a rotary shaker for 8 - 12 h, and then inoculated into a 50 mL LB flask at an inoculation amount of 1%, and cultured at 37°C and 220 rpm on a rotary shaker until OD 600When = 0.6 - 0.9, add IPTG with a final concentration of 0.04 mM and culture for 4 h. Then pour the cultured bacterial solution into a 50 mL centrifuge tube and centrifuge at 7000 rpm for 7 min. After completion, discard the supernatant.

[0078] Subsequently, wash the bacterial cells with 0.1 M Tris-HCl buffer (pH = 7.8) twice, discarding the supernatant each time. Add glycine at a concentration of 5 g / L, 37% formaldehyde solution at a concentration of 3 g / L, and 10 mL of 0.25 M Tris-HCl buffer (containing 0.5% Tween 80) for catalysis. After 12 h of catalysis, add 5 g / L indole dissolved in 100 μL of ethyl acetate. Then place the buffer system in a rotary shaker at 30 °C and 220 rpm for culture and catalysis for 12 h to obtain the catalytic product L-tryptophan.

[0079] The L-tryptophan production of the original control group strain, i.e., the starting strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA, was 0.584 g / L. The L-tryptophan production of the final recombinant strain H160T with the H160T mutation was 0.7 g / L, which was 19.86% higher than that of the control starting strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA.

[0080] From the control results, it can be seen that the H160T mutant has a beneficial promoting effect on the production of L-tryptophan using glycine as a substrate. This also indicates that the recombinant strain can complete the multi-enzyme cascade reaction to produce tryptophan from glycine. Constructing a strain from glycine to L-tryptophan is of great significance and provides an idea for constructing a metabolic pathway for producing tryptophan from glycine.

[0081] (2) Changes in L-cysteine production

[0082] The obtained recombinant strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA with wild-type tryptophan synthase and the tryptophan synthase mutant strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA with the H160T mutation H160T Carry out the whole-cell catalytic reaction of multi-enzyme cascade. The specific method is as follows:

[0083] Aspirate 10 μL of the preserved recombinant strain for three-zone streaking and culture for 8 - 12 h. Wait until clear single colonies grow, pick a single colony and inoculate it into a 10 mL LB liquid vial, culture in a rotary shaker at 37 °C and 220 rpm for 8 - 12 h, and inoculate it into a 50 mL LB flask at an inoculation amount of 1%, and culture in a rotary shaker at 37 °C and 220 rpm until OD 600When = 0.6 - 0.9, after adding IPTG with a final concentration of 0.04 mM and culturing for 4 h, the cultured bacterial solution was poured into a 50 mL centrifuge tube and centrifuged at 7000 rpm for 7 min, and the supernatant was discarded.

[0084] Subsequently, the cells were washed with 0.1 M Tris-HCl buffer (pH = 7.8) twice, and the supernatant was discarded. 5 g / L glycine, 2 g / L pyridoxal phosphate, 3 g / L 37% formaldehyde solution, and 10 mL 0.25 M Tris-HCl buffer (containing 0.5% Tween 80) were added for catalysis. After 12 h of catalysis, 5 g / L sodium hydrosulfide was added, and then the buffer system was placed in a gyratory shaker at 30 °C and 220 rpm for culture and catalysis for 12 h to obtain the catalytic product L-cysteine.

[0085] The L-cysteine yield of the original control group strain, the starting strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA, was 0.138 g / L, and the recombinant strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA H160T with the H160T mutant had an L-cysteine yield of 0.161 g / L, which was 16.67% higher than that of the control starting strain BL21(DE3)-pET28a-SHMT-pET22b-TrpBA.

[0086] From the control results, it can be seen that the H160T mutant has a beneficial promoting effect on the production of L-cysteine using glycine as a substrate, which also indicates that the recombinant strain can complete the multi-enzyme cascade reaction to produce cysteine from glycine, providing a new idea for constructing a metabolic pathway for producing L-cysteine from glycine.

[0087] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A tryptophan synthase mutant, characterized in that: The mutant is obtained by mutating the histidine at position 160 of the starting sequence with the amino acid sequence shown in SEQ ID NO.1 to threonine.

2. A gene encoding the tryptophan synthase mutant according to claim 1.

3. An expression vector containing the gene according to claim 2.

4. A host cell containing the tryptophan synthase mutant according to claim 1, the gene according to claim 2, or the expression vector according to claim 3.

5. Use of the tryptophan synthase mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3, or the host cell according to claim 4 in the production of L-serine derivatives.

6. The application according to claim 5, characterized in that: The L-serine derivative is L-tryptophan and / or L-cysteine.

7. A method for synthesizing L-tryptophan, characterized in that: The tryptophan synthase mutant according to claim 1 or an expression system containing the mutant is added to a reaction system containing a substrate for reaction.

8. The method according to claim 7, wherein: The substrate is L-serine or glycine.

9. A method for synthesizing L-cysteine, characterized in that: The tryptophan synthase mutant according to claim 1 or an expression system containing the mutant is added to a reaction system containing a substrate for reaction.

10. The method according to claim 9, characterized in that: The substrate is L-serine or glycine.

Images