A tryptophanase mutant and its application in indole production

Abstract

The present invention discloses a tryptophanase mutant and its application in the production of indole, which relates to the technical field of microbial engineering. Through random mutation and site screening, the present invention performs an amino acid substitution at the 389th position of SEQ ID NO: 1, obtaining a new tryptophanase. Compared with the existing biosynthesis methods, the recombinant bacterium constructed using the gene fragment of the modified tryptophanase mutant can efficiently produce indole; at the same time, the indole production method provided by the present invention has low costs, easily available raw materials, and greatly reduces production costs; moreover, the fermentation cycle of the production method provided by the present invention is short, the process is simple, the efficiency is high and it is not restricted by regions. Therefore, the tryptophan mutant and the indole production method provided by the present invention have good industrial practicability.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial engineering, and in particular, to a tryptophanase mutant and its application in the production of indole. Background Art

[0002] Indigo is an ancient and classic natural dye, which is derived from indigo plants and has a history of thousands of years. Indigo has excellent color stability and glossiness and is widely used in textile dyeing, art painting, printing and other fields. Indigo can also be used as a raw material for drugs and has biological activities such as antioxidant and antibacterial properties. There are mainly two traditional methods for the production of indigo. One is to extract indigo from indigo plants and then obtain indigo through chemical reactions. This method has a high production cost and a large environmental load. The other is to directly obtain indigo by chemical synthesis. This method has high production efficiency and low cost, but requires the use of chemical reagents harmful to the human body and will produce toxic wastewater during the production process. Compared with traditional chemical synthesis and plant extraction, the production of indigo by microbial fermentation has the advantages of environmental friendliness, low cost and high product purity. With the further development of technology in the future, microbial fermentation is expected to become the dominant method for indigo production.

[0003] Existing studies have shown that a variety of microorganisms such as Pseudomonas ( Pseudomonas sp .), Acinetobacter ( Acinetobacter sp .) and Bacillus megaterium ( Bacillus megatreium ) etc. have the ability to produce indigo. These microorganisms can catalyze the production of indigo from indole through a series of key oxidases such as naphthalene dioxygenase, styrene dioxygenase, flavin oxygenase and cytochrome P450 monooxygenase. Indole and its homologues and derivatives are widely present in nature, such as auxin, tryptophan, etc. Indole is mainly used as a raw material for spices, dyes, amino acids, pesticides. However, at present, the production of indole is mainly carried out by chemical methods such as one-step synthesis of aniline and ethylene glycol or separation from coal tar. Due to the cytotoxicity of high-concentration indole, it is difficult to produce indole at a high level at present, so there is no large-scale biosynthesis method.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a tryptophanase mutant and its application in the production of indole. The recombinant bacterium constructed by using the gene fragment of the tryptophanase mutant can produce indole using tryptophan as a substrate, thereby overcoming the problem in the prior art that high-concentration indole has cytotoxicity and cannot produce indole at a high level.

[0006] The present invention is implemented as follows:

[0007] In a first aspect, the present invention provides a tryptophanase TnaA mutant, whose amino acid sequence is obtained by mutating the amino acid sequence shown in SEQ ID NO: 1, and the mutation sites of this mutant include: valine at position 389 is mutated to aspartic acid.

[0008] In a second aspect, the present invention provides a nucleic acid molecule that encodes the above-mentioned tryptophanase TnaA mutant.

[0009] In a third aspect, the present invention provides a recombinant vector that contains the above-mentioned nucleic acid molecule.

[0010] In a fourth aspect, the present invention provides a recombinant bacterium that contains the above-mentioned recombinant vector.

[0011] In a fifth aspect, the present invention also provides a method for constructing the above-mentioned recombinant bacterium, which includes: inserting the gene of the tryptophanase TnaA mutant into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into a starting strain to obtain the above-mentioned recombinant bacterium.

[0012] In a sixth aspect, the present invention also provides the use of the above-mentioned tryptophanase TnaA mutant, nucleic acid molecule, recombinant vector or recombinant bacterium in the production of indole and its downstream products.

[0013] In a seventh aspect, the present invention also provides a method for producing indole, which includes: adding the above-mentioned recombinant bacterium into a reaction system containing tryptophan for whole-cell catalysis to obtain indole.

[0014] The present invention has the following beneficial effects:

[0015] Based on the tryptophanase shown in SEQ ID NO: 1, through random mutation and site screening, amino acid substitution was carried out at position 389 of SEQ ID NO: 1 to obtain a new tryptophanase. Compared with the existing biosynthesis methods, the recombinant bacterium constructed with the gene fragment of the modified tryptophanase mutant can efficiently produce indole; at the same time, the indole production method provided by the present invention has low cost, easily available raw materials, and greatly reduces the production cost; moreover, the fermentation period of the production method provided by the present invention is short, the process is simple, the efficiency is high and it is not restricted by regions. Therefore, the tryptophan mutant and indole production method provided by the present invention have good industrial practicability. Detailed Embodiments

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0017] Tryptophanase (TnaA) can reversibly degrade tryptophan into indole, pyruvate, and ammonia. This invention utilizes this synthetic route to produce indole. However, during the microbial fermentation process, high concentrations of indole are cytotoxic, thus inhibiting microbial fermentation. To overcome this problem, this invention screened tryptophanase from specific sources and mutated it to obtain a tryptophanase mutant. After constructing a recombinant bacterium with the gene of this mutant, through biocatalytic reaction, using tryptophan as the raw material, indole can be efficiently produced, which also provides a new strategy for supplying the precursor substrate for subsequent indigo production.

[0018] In this invention, tryptophanase TnaA is derived from Providencia rettgeri and its amino acid sequence is shown as SEQ ID NO: 1. The amino acid sequence of the tryptophanase TnaA mutant obtained based on it is shown as SEQ ID NO: 3.

[0019] Among them, the mutation site of the tryptophanase TnaA mutant in this invention is: the 389th position; the mutation mode is: valine is mutated to aspartic acid.

[0020] Specifically, the preparation method of the tryptophanase TnaA mutant gene in this invention is as follows: Codon optimization is performed on the tryptophanase TnaA gene derived from Providencia rettgeri and its amino acid sequence is shown as SEQ ID NO: 1. The optimized tryptophanase TnaA gene is amplified by PCR, and the amplified tryptophanase TnaA gene is diluted as a template for error-prone PCR amplification to obtain the tryptophanase TnaA mutant gene.

[0021] The recombinant vector provided by this invention is a DNA molecule containing a nucleotide molecule encoding the above-mentioned tryptophanase TnaA mutant or its complementary sequence.

[0022] Among them, the vector can select conventional expression vectors in the art, such as pCDFDuet, pET-28a(+), etc.

[0023] The above-mentioned recombinant vector can be obtained by existing preparation methods. For example: The gene fragment of the tryptophanase TnaA mutant is obtained by PCR amplification. Using the expression vector as a template, primers are designed to obtain a linearized vector by inverse PCR amplification, and then the linearized vector and the mutant gene fragment are ligated by a recombinase to obtain a recombinant vector inserted with the tryptophanase TnaA mutant gene fragment.

[0024] The recombinant bacterium provided by this invention contains the above-mentioned recombinant vector.

[0025] In some embodiments, the host cell of the above-mentioned recombinant bacterium can be a prokaryotic cell or a eukaryotic cell; the prokaryotic cell can be Escherichia coli, preferably, Escherichia coli can be Escherichia coli BL21(DE3), Escherichia coli DH5α and Escherichia coli XL-Blue.

[0026] Furthermore, based on this, the present invention can also provide a whole-cell catalyst containing the above-mentioned recombinant bacterium.

[0027] Using the above-mentioned recombinant bacterium as a whole-cell catalyst, the expressed tryptophanase TnaA mutant can efficiently convert tryptophan into indole.

[0028] The present invention also provides a method for constructing the above-mentioned recombinant bacterium, which includes: inserting the gene of the above-mentioned tryptophanase TnaA mutant into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into a starting strain to obtain the recombinant bacterium.

[0029] Based on the constructed recombinant bacterium, the present invention also provides a screening method for the above-mentioned recombinant bacterium, which includes: picking the recombinant bacterium into a 96-well plate containing LB medium, transferring the cultured bacterial liquid to a 96-well plate containing fermentation medium, culturing until the OD 600nm of the bacterial cells reaches 0.6 - 0.8, adding an inducer, taking the fermentation broth after fermentation, diluting it, adding a chromogenic agent, measuring its absorbance, and selecting the strain with a high absorbance for preservation.

[0030] In some embodiments, the culture conditions of the above-mentioned recombinant bacterium in LB medium are: culturing at 37 °C and 1000 rpm for 6 h.

[0031] In some embodiments, the culture conditions of the above-mentioned recombinant bacterium in fermentation medium are: culturing at 30 °C and 1000 rpm for 24 h, and the inoculation amount of the well plate is 5%.

[0032] In some embodiments, the components of the fermentation medium in the screening process include: yeast powder 23.6 g / L, peptone 11.8 g / L, K2HPO4 2.2 g / L, KH2PO4 9.4 g / L, MgSO4 0.241 g / L, CaCl2 0.011 g / L, tryptophan 1 g / L, glucose 4 g / L.

[0033] In some embodiments, the induction expression conditions of the above-mentioned recombinant bacterium are: the added concentration of the inducer IPTG is 0.1 mM, and the induction temperature is 30 °C.

[0034] In some embodiments, the dilution ratio of the fermentation broth after fermentation is 10 times, the added volume of the chromogenic agent is 10%, and the chromogenic reaction time is 10 min.

[0035] In some embodiments, the preparation method of the above-mentioned developer is as follows: Weigh 5 g of p-dimethylaminobenzaldehyde and dissolve it in 5 mL of ethanol, and then add 5 mL of concentrated sulfuric acid.

[0036] In some embodiments, the absorbance wavelength for detection is 568 nm.

[0037] The present invention obtains the above-mentioned tryptophanase TnaA mutant through random mutation and screening. Using this mutant and its related biological materials, indole and its downstream products can be efficiently produced. Therefore, based on the above-mentioned mutant, the present invention can also provide a method for producing indole, which can be applied to different production scenarios according to production conditions and production demands, such as shake flask fermentation and fermenter fermentation.

[0038] Among them, the method of shake flask fermentation includes the following steps:

[0039] (1) Pick the recombinant bacterial liquid and streak it onto the culture medium plate for cultivation;

[0040] (2) Pick the fresh colonies in step (1) into a shake flask for cultivation;

[0041] (3) Transfer the bacterial liquid after cultivation in step (2) to the fermentation medium for shake flask cultivation;

[0042] (4) Add an inducer at the initial stage of inoculation for induced expression;

[0043] (5) After fermentation, take samples to detect the indole concentration.

[0044] In some embodiments, the cultivation conditions in step (1) are: cultivate at 37 °C and 220 rpm for 16 h, and the culture medium used is LB medium.

[0045] In some embodiments, the cultivation conditions in step (2) are: cultivate at 37 °C and 220 rpm until the logarithmic growth phase, OD 600nm = 5, cultivate for 6 h, the culture medium used is LB medium, and the liquid loading volume is 10%.

[0046] In some embodiments, the cultivation conditions in step (3) are: when the OD of the cells pre-cultivated in the shake flask 600nm reaches about 5, transfer them to a triangular flask containing the fermentation medium (10% liquid loading volume) according to an inoculation amount of 10%, ferment at 30 °C and 220 rpm, and stop fermentation after 24 h.

[0047] Among them, the components of the fermentation medium include: 23.6 g / L of yeast powder, 11.8 g / L of peptone, 2.2 g / L of K2HPO4, 9.4 g / L of KH2PO4, 0.241 g / L of MgSO4, 0.011 g / L of CaCl2, 3 g / L of tryptophan, 40 g / L of glucose, and 20% dibutyl sebacate.

[0048] In some embodiments, in step (4), when the culture reaches OD 600nm = 0.6 - 0.8, an inducer is added. The inducer is 0.1 mM IPTG, the induction conditions are 30 °C, and after induction for 2 h, 3 g / L of tryptophan is added as a substrate.

[0049] The method for fermentation in a fermenter includes the following steps:

[0050] (1) Pick the recombinant bacterial solution and streak it onto a culture medium plate for cultivation;

[0051] (2) Pick the fresh recombinant colonies in step (1) into a shake flask for cultivation;

[0052] (3) Transfer the bacterial solution after the cultivation in step (2) to a fermenter for fermentation;

[0053] (4) Add an inducer for induced expression;

[0054] (5) Start to supplement tryptophan as a substrate after induction.

[0055] In some embodiments, the cultivation conditions in step (2) are: cultivation at 37 °C and 220 rpm, the culture medium used is LB medium, and the liquid loading amount is 10%.

[0056] In some embodiments, the cultivation conditions in step (3) are: fermentation temperature 30 °C, pH = 7.5, dissolved oxygen content 25% - 35%, stir to relate to dissolved oxygen, and stop fermentation after 48 h.

[0057] Specific fermenter parameters are: control pH = 7.5 with ammonia water during the fermentation process, fermentation temperature is 30 °C, DO dissolved oxygen content is 25% - 35%, feed according to the residual amount of glucose, add tryptophan as a substrate 4 h after induction, and control the tryptophan concentration above 2 g / L.

[0058] Among them, the components of the fermentation medium include: 23.6 g / L of yeast powder, 11.8 g / L of peptone, 2.2 g / L of K2HPO4, 9.4 g / L of KH2PO4, 0.241 g / L of MgSO4, 0.011 g / L of CaCl2, 40 g / L of glucose, 0.002 g / L of pyridoxal phosphate, 20% dibutyl sebacate, 0.5 mL / L of antifoaming agent, and the initial liquid loading amount is 2 L.

[0059] In some embodiments, the induction conditions in step (4) are: culturing at 37 °C until OD 600nm = 20, adjusting the temperature to 30 °C, and adding 0.2 mM IPTG for induction.

[0060] Using the above production method, indole with high yield can be obtained by microbial fermentation. Moreover, the process is simple, the production cost is low, and the raw materials are easily available, providing a new idea for the biosynthesis of indole.

[0061] The present invention will be further described below in conjunction with embodiments. However, it should be understood that the protection scope of the present invention is not limited by the embodiments.

[0062] Example 1

[0063] This example is for the construction of the expression strain pCDFDuet-TnaA-BL21, and the specific steps are as follows:

[0064] S1. Codon optimization is performed on the tryptophanase gene TnaA derived from Providencia rettgeri , and the sequence of the tryptophanase gene TnaA is shown in SEQ ID NO:2;

[0065] S2. The optimized TnaA gene is amplified by PCR. The nucleotide sequences of the required primers are shown in SEQ ID NO:4 and SEQ ID NO:5, and the TnaA gene of the target fragment is obtained;

[0066] Among them, the PCR amplification conditions are denaturation at 98 °C, annealing at 55 / 59 / 61 °C, extension at 72 °C, and cycling 30 times;

[0067] S3. Using the plasmid pCDFDuet-1 as a template, linearized amplification is performed by inverse PCR to obtain the linearized expression vector pCDFDuet-1. The nucleotide sequences of the required primers are shown in SEQ ID NO:6 and SEQ ID NO:7;

[0068] Among them, the PCR amplification conditions are denaturation at 98 °C, annealing at 55 °C, extension at 72 °C, and cycling 30 times;

[0069] S4. Through Novizan recombinase C112, the target fragment TnaA and the linearized vector pCDFDuet-1 are ligated. The enzyme reaction conditions are 37 °C for 30 min; the expression vector pCDFDuet-TnaA is obtained and transferred into Escherichia coli BL21 competent cells by chemical transformation method.

[0070] The primers and gene sequences involved in the PCR amplification of the present invention in this example are shown in Table 1:

[0071] Table 1 Sequence Information

[0072]

[0073] Example 2

[0074] This example is for the construction of the random mutation vector pCDF-TnaA*, and the specific steps are as follows:

[0075] S1. Dilute the TnaA gene amplified in step S2 of Example 1 by 100 times as a template for error-prone PCR amplification. The nucleotide sequences of the required primers are shown in SEQ ID NO:4 and SEQ ID NO:5.

[0076] The error-prone PCR system is a 100 μL system, and the components are as follows: 51 μL ddH2O, 10 μL 100 mM Tris-Cl, 2.5 μL 2 M KCl, 3.5 μL 200 mM MgCl2, 4 μL 25 mM dCTP, 4 μL 25 mM dTTP, 4 μL 5 mM dATP, 4 μL 5 mM dGTP, 4 μL primer, 2 μL 25 mM MnCl2, 1 μL taq DNA polymerase, 10 μL template.

[0077] The PCR amplification conditions are denaturation at 98℃, annealing at 65℃ - 55℃, extension at 72℃, and 40 cycles.

[0078] S2. After obtaining the mutant TnaA* gene, use the Tsingke Biological DNA Gel Extraction Kit for gel extraction and purification.

[0079] S3. Through Novizan recombinase C112, ligate the purified target fragment TnaA* in step a and the linearized vector pCDFduet-1 obtained in step C of Example 1. The enzyme reaction conditions are 37℃ for 30 min. Obtain the expression vector pCDF-TnaA*, and transfer it into Escherichia coli BL21 competent cells by the chemical transformation method.

[0080] Example 3

[0081] This example is a method for rapidly screening high-yield indole strains with pCDF-TnaA*, and the specific steps are as follows:

[0082] S1. Pick fresh pCDFDuet-TnaA and pCDF-TnaA*-BL21 colonies into a 96-well plate containing 400 μL of LB medium per well, and culture at 37 °C and 1000 rpm for 6 h. Among them, pick 2 pCDFDuet-TnaA colonies as controls, and pick pCDF-TnaA*-BL21 colonies for the remaining 90 wells;

[0083] S2. Transfer the cultured bacterial liquid in step S1 to a 96-well plate containing 760 μL of fermentation medium per well according to an inoculation amount of 5%, and culture. The culture conditions are 30 °C and 1000 rpm, and the total fermentation time is 24 h;

[0084] S3. Add IPTG with a final concentration of 0.1 mM for induction when the cell OD 600nm reaches 0.6 - 0.8 after inoculation, and the induction temperature is 30 °C;

[0085] S4. After 24 h of fermentation, take 10 μL of the fermentation broth sample from each well and dilute it in 900 μL of water, add 10% of the chromogenic agent, react at room temperature for 5 min, and measure the absorbance with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 568 nm after the reaction;

[0086] S5. Compare the absorbance values, and preserve the strain with a higher absorbance value with 40% glycerol at a ratio of 1:1.

[0087] The formula of the fermentation medium used is: yeast powder 23.6 g / L, peptone 11.8 g / L, K2HPO4 2.2 g / L, KH2PO4 9.4 g / L, MgSO4 0.241 g / L, CaCl2 0.011 g / L, tryptophan 1 g / L, glucose 4 g / L.

[0088] Example 4

[0089] This example is the shake flask fermentation verification of pCDF-TnaA*-BL21, and the specific steps are as follows:

[0090] 1) Streak the preserved pCDF-TnaA*-BL21 bacterial liquid in Example 3 onto an LB medium plate, and culture at 37 °C and 220 rpm for 16 h;

[0091] 2) Pick the cultured pCDF-TnaA*-BL21 colonies in step 1) into a 500 mL shake flask with an LB medium loading volume of 10%, and place it at 37 °C and 220 rpm for shake flask culture until the OD 600nm reaches about 5, which takes about 6 hours;

[0092] 3) Transfer the cultured pCDF-TnaA*-BL21 bacterial liquid at the end of step 2) to a 5 L shake flask with a filling volume of 10% of the fermentation medium according to an inoculation amount of 10%, and place it at 30 °C and 220 rpm for shake flask culture until the fermentation ends. The total fermentation time is 24 h;

[0093] 4) Add IPTG with a final concentration of 0.1 mM when the culture reaches OD 600nm = 0.6 - 0.8 after inoculation, and perform induction expression at 30 °C;

[0094] 5) After 2 h of induction, supplement 3 g / L of tryptophan as a substrate.

[0095] The fermentation medium formula is: yeast powder 23.6 g / L, peptone 11.8 g / L, K2HPO4 2.2 g / L, KH2PO4 9.4 g / L, MgSO4 0.241 g / L, CaCl2 0.011 g / L, glucose 40 g / L, 20% dibutyl sebacate.

[0096] Perform shake flask fermentation on the strain screened in Example 4 to determine the indole yield, extract the plasmid of the strain with high yield in shake flask fermentation for sequencing identification. The protein sequence is shown in SEQ ID NO:3, where the mutation site is V389D, and the highest yield of this strain can reach 1.6 g / L.

[0097] Comparative Example 1

[0098] 1) Using BL21 as the chassis strain, construct the pCDF-PreTnaA plasmid and introduce it into the BL21 strain by chemical transformation method; PreTnaA is derived from Providencia rettgeri , and its protein sequence is shown in SEQ ID NO:1;

[0099] 2) Perform shake flask fermentation according to the steps in Example 4;

[0100] 3) Detect the indole yield by liquid phase after fermentation ends.

[0101] Comparative Example 2

[0102] 1) Using BL21 as the chassis strain, construct the pCDF-PvTnaA plasmid and introduce it into the BL21 strain by chemical transformation method. PvTnaA is derived from Proteus vulgaris , and its protein sequence is shown in SEQ ID NO:8;

[0103] 2) Perform shake flask fermentation according to the steps in Example 4;

[0104] 3) Detect the indole yield by liquid phase after fermentation ends.

[0105] Experimental Example 1

[0106] The fermentation products after the end of fermentation in Example 4 and Comparative Examples 1-2 were detected for indole production using HPLC (high performance liquid chromatography), and the detection conditions were as follows:

[0107] High performance liquid chromatograph: Shimadzu LC-2030Plus;

[0108] Chromatographic column: ShimNex HE C8 4.6×150mm 3μm;

[0109] Mobile phase A: 0.1% aqueous acetic acid solution;

[0110] Mobile phase B: methanol;

[0111] Elution ratio Mobile phase A: Mobile phase B = 3:7;

[0112] Flow rate: 1 ml / min;

[0113] Injection volume: 10 μL;

[0114] Column temperature: 35°C;

[0115] Detection wavelength: 269 nm.

[0116] The data of indole content detected in each group are shown in Table 2:

[0117] Table 2 Indole production data of each group

[0118]

[0119] Example 5

[0120] This example was a verification of the fermentation effect of pCDF-TnaA*-BL21 in a fermenter, and the specific steps were as follows:

[0121] A single colony of the high-yield strain pCDF-TnaA*-BL21 verified in Example 4 was picked into 100 mL of LB liquid, and cultured at 37°C and 220 rpm until OD 600nm = 2-3 to obtain a seed solution;

[0122] The seed solution was inoculated into a 5 L fermenter containing 2 L of fermentation medium, and cultured at 37°C until OD 600nm = 20, then the temperature was adjusted to 30°C, and 0.2 mM IPTG was added for induction;

[0123] The components of the fermentation medium are as follows: yeast powder 23.6 g / L, peptone 11.8 g / L, K2HPO4 2.2 g / L, KH2PO4 9.4 g / L, MgSO4 0.241 g / L, CaCl2 0.011 g / L, glucose 40 g / L, pyridoxal phosphate 0.002 g / L, 20% dibutyl sebacate, 0.5 mL / L antifoaming agent;

[0124] Fermenter parameters: During the fermentation process, ammonia water is used to control the pH to 7.5, the fermentation temperature is 30 °C, the DO dissolved oxygen is 25% - 35%, feeding is carried out according to the residual amount of glucose, and tryptophan is added as a substrate 4 h after induction, controlling the tryptophan concentration above 2 g / L.

[0125] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A tryptophanase TnaA mutant, characterized in that, The amino acid sequence of the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO: 1, and the mutation site of the mutant is the substitution of valine at position 389 with aspartic acid.

2. A nucleic acid molecule, characterized in that, Encoding the tryptophanase TnaA mutant as claimed in claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as claimed in claim 2.

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

5. The method for constructing the recombinant bacterium according to claim 4, wherein Including: Inserting the gene of the tryptophanase TnaA mutant into an expression vector to obtain a recombinant vector, and then introducing the recombinant vector into a starting strain to obtain the recombinant bacterium.

6. The construction method according to claim 5, characterized in that, The starting strain includes Escherichia coli.

7. Use of the tryptophanase TnaA mutant as claimed in claim 1, the nucleic acid molecule as claimed in claim 2, the recombinant vector as claimed in claim 3 or the recombinant bacterium as claimed in claim 4 in the production of indole.

8. A method for producing indole, characterized in that, Including: Adding the recombinant bacterium as claimed in claim 4 into a reaction system containing tryptophan for whole-cell catalysis to obtain indole.

9. The method according to claim 8, characterized in that, The culture method of the recombinant bacterium includes: streaking the recombinant bacterium onto a plate for culture, and then picking the colonies obtained by culture into a shake flask for scale-up culture; Among them, the conditions for plate culture are: overnight culture at 37 °C and 220 rpm, and the medium is LB medium; The conditions for scale-up culture are: culture at 37 °C and 220 rpm, the medium is LB medium, and the liquid loading is 10%.

10. The method according to claim 9, wherein The method further includes fermentation after scale-up culture; the fermentation includes shake flask fermentation and fermenter fermentation; The shake flask fermentation includes: inoculating the bacterial liquid obtained after scale-up culture into a fermentation medium for shake flask culture, and adding an inducer at the initial stage of inoculation for induced expression; The fermenter fermentation includes: inoculating the bacterial liquid obtained after scale-up culture into a fermentation medium for fermenter culture, adding an inducer for induced expression, and after induction, supplementing tryptophan as a substrate.

11. The method according to claim 10, wherein The conditions for shake flask fermentation are: fermentation at 30 °C and 220 rpm, and stop fermentation after 24 h; the conditions for induction expression are: add inducer when cultured to OD 600nm = 0.6 - 0.

8. The inducer is 0.1 mM IPTG, the induction temperature is 30 °C, and add 3 g / L tryptophan as substrate after 2 h of induction.

12. The method according to claim 10, wherein, The components of the fermentation medium in the shake flask fermentation include: 23.6 g / L of yeast powder, 11.8 g / L of peptone, 2.2 g / L of K2HPO4, 9.4 g / L of KH2PO4, 0.241 g / L of MgSO4, 0.011 g / L of CaCl2, 3 g / L of tryptophan, 40 g / L of glucose, 20% dibutyl sebacate.

13. The method according to claim 10, wherein The fermentation conditions of the fermenter are as follows: temperature is 30°C, pH = 7.5, dissolved oxygen is 25% - 35%, stirring is related to dissolved oxygen, and the fermentation time is 48 h; the conditions of the induction table are as follows: culture at 37°C until OD 600nm = 20, adjust the temperature to 30°C, add 0.2 mM IPTG for induction, and supplement tryptophan at a concentration of more than 2 g / L as a substrate after 4 h of induction.

14. The method according to claim 10, characterized in that, The components of the fermentation medium in the fermenter fermentation include: 23.6 g / L of yeast powder, 11.8 g / L of peptone, 2.2 g / L of K2HPO4, 9.4 g / L of KH2PO4, 0.241 g / L of MgSO4, 0.011 g / L of CaCl2, 40 g / L of glucose, 0.002 g / L of pyridoxal phosphate, 20% dibutyl sebacate, 0.5 mL / L of antifoaming agent.

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