Tryptophan enzyme mutant and application thereof in production of indole

Abstract

The invention discloses a tryptophanase mutant and application thereof in production of indole, and relates to the technical field of microbial engineering. Through random mutation and site screening, amino acid replacement is carried out at the 389th site of SEQ ID NO: 1 to obtain a new tryptophan enzyme, and compared with the existing biosynthesis method, the recombinant bacteria constructed by using the gene segment of the modified tryptophan enzyme mutant can efficiently produce indole; meanwhile, the indole production method provided by the invention is low in cost, raw materials are easy to obtain, and the production cost is greatly reduced; the production method provided by the invention is short in fermentation period, simple in process, high in efficiency and not limited by regions. Therefore, the tryptophan mutant and the indole production method provided by the invention have good industrial practicability.

Description

Technical Field

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

[0002] Indigo is an ancient and classic natural dye, which comes 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 medicine, with antioxidant, antibacterial and other biological activities. There are two main methods for the production of traditional indigo. One is to extract indigo from indigo plants, and then produce indigo through chemical reactions. This method has high production costs and a large environmental load; the other is to directly produce indigo through chemical synthesis. This method has high production efficiency and low cost, but it requires the use of chemical reagents that are harmful to the human body, and toxic wastewater is generated during the production process. Compared with traditional chemical synthesis and plant extraction, microbial fermentation to produce indigo has the advantages of environmental friendliness, low cost, and high product purity. In the future, with the further development of technology, microbial fermentation is expected to become the dominant method of indigo production.

[0003] Studies have shown that a variety of microorganisms such as Pseudomonas sp., Acinetobacter sp. and Bacillus megatreium have the ability to produce indigo. These microorganisms can catalyze indole to produce indigo 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 and tryptophan. Indole is mainly used as a raw material for spices, dyes, amino acids and pesticides. However, indole is currently produced mainly through 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 currently difficult to produce indole by high-level fermentation, so there is no large-scale biosynthesis method.

[0004] In view of this, the present invention is 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 bacteria constructed 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 concentrations of indole are cytotoxic and cannot produce indole at a high level.

[0006] The present invention is achieved in that:

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

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

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

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

[0011] In a fifth aspect, the present invention also provides a method for constructing the above-mentioned recombinant bacteria, which comprises: 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 bacteria.

[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 bacteria in the production of indole and its downstream products.

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

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

[0015] The present invention is based on the tryptophanase shown in SEQ ID NO: 1, and through random mutation and site screening, an amino acid substitution is performed at the 389th position of SEQ ID NO: 1 to obtain a new tryptophanase. Compared with the existing biosynthesis method, the recombinant bacteria constructed by 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 is low in cost, the raw materials are easy to obtain, and the production cost is greatly reduced; and the production method provided by the present invention has a short fermentation cycle, a simple process, high efficiency and is not restricted by region. Therefore, the tryptophan mutant and the indole production method provided by the present invention have good industrial applicability. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0017] Tryptophanase (TnaA) can reversibly degrade tryptophan into indole, pyruvic acid and ammonia. The present invention utilizes this synthetic route to produce indole, but high concentrations of indole are cytotoxic during microbial fermentation, thereby inhibiting microbial fermentation. In order to overcome this problem, the present invention screens tryptophanase from a specific source and mutates it to obtain a tryptophanase mutant. After the gene of the mutant is constructed into a recombinant bacterium, indole can be efficiently produced through a biocatalytic reaction using tryptophan as a raw material, and a new strategy is provided for the subsequent production of indigo to supply precursor substrates.

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

[0019] The mutation site of the tryptophanase TnaA mutant of the present invention is position 389, and the mutation mode is: valine is mutated into aspartic acid.

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

[0021] The recombinant vector provided by the present invention is a DNA molecule containing a nucleotide molecule encoding the tryptophanase TnaA mutant or a complementary sequence thereof.

[0022] The vector may be a conventional expression vector in the art, such as pCDFDuet, pET-28a(+), etc.

[0023] The above-mentioned recombinant vector can be obtained by existing preparation methods, for example: PCR amplification obtains the gene fragment of the tryptophanase TnaA mutant, using the expression vector as a template, designing primers to obtain a linearized vector through reverse PCR amplification, and then connecting the linearized vector with the mutant gene fragment through a recombinase to obtain a recombinant vector with the tryptophanase TnaA mutant gene fragment inserted.

[0024] The recombinant bacteria provided by the present invention contain the above-mentioned recombinant vector.

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

[0026] Furthermore, on this basis, the present invention can also provide a whole-cell catalyst, which contains the above-mentioned recombinant bacteria.

[0027] The above recombinant bacteria are used as whole-cell catalysts, and the tryptophanase TnaA mutant expressed by the recombinant bacteria can efficiently convert tryptophan into indole.

[0028] The present invention also provides a method for constructing the above-mentioned recombinant bacteria, which comprises: 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 bacteria.

[0029] Based on the constructed recombinant bacteria, the present invention also provides a screening method for the above recombinant bacteria, which comprises: picking the recombinant bacteria into a 96-well plate containing LB medium, transferring the cultured bacterial solution into a 96-well plate containing fermentation medium, and culturing until the bacterial OD reaches 0. 600nm =0.6-0.8, add the inducer, dilute the fermentation liquid after fermentation, add the colorimetric agent, measure the absorbance, and select the strain with high absorbance for preservation.

[0030] In some embodiments, the culture conditions of the above-mentioned recombinant bacteria in LB medium are: 37° C., 1000 rpm, 6 hours.

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

[0032] In some embodiments, the components of the fermentation medium during 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, and glucose 4 g / L.

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

[0034] In some embodiments, the dilution ratio of the fermentation liquid after the fermentation is 10 times, the volume of the color developer added is 10%, and the color development reaction time is 10 minutes.

[0035] In some embodiments, the developer is prepared by weighing 5 g of p-dimethylaminobenzaldehyde, dissolving it in 5 mL of ethanol, and adding 5 mL of concentrated sulfuric acid.

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

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

[0038] The shake flask fermentation method comprises the following steps:

[0039] (1) streaking the recombinant bacterial solution onto a culture medium plate for cultivation;

[0040] (2) picking up the fresh bacterial colonies in step (1) and culturing them in a shake flask;

[0041] (3) transferring the bacterial solution after culturing in step (2) to a fermentation medium for shake flask culture;

[0042] (4) adding an inducer at the initial stage of inoculation to induce expression;

[0043] (5) After the fermentation is completed, samples are taken to detect the indole concentration.

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

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

[0046] In some embodiments, the culture conditions in step (3) are: the OD of cells pre-cultured in a shake flask is 600nm When the temperature reached about 5, the mixture was transferred to a conical flask containing fermentation medium (10% liquid volume) according to 10% inoculation amount, and fermented at 30°C and 220 rpm. The fermentation was stopped after 24 hours.

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

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

[0049] The method of fermentation in a fermenter comprises the following steps:

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

[0051] (2) picking up the fresh recombinant colonies from step (1) and culturing them in a shake flask;

[0052] (3) transferring the bacterial liquid cultured in step (2) to a fermentation tank for fermentation;

[0053] (4) adding an inducer to induce expression;

[0054] (5) After induction, tryptophan is supplemented as a substrate.

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

[0056] In some embodiments, the culture conditions in step (3) are: fermentation temperature 30°C, pH=7.5, dissolved oxygen content 25%-35%, stirring associated dissolved oxygen, and stopping fermentation after 48 hours.

[0057] The specific fermentation tank parameters are: during the fermentation process, ammonia water is used to control the pH = 7.5, the fermentation temperature is 30°C, the DO dissolved oxygen content is 25%-35%, feeding is carried out according to the residual amount of glucose, tryptophan is added as a substrate after 4 hours of induction, and the tryptophan concentration is controlled above 2g / L.

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

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

[0060] The above production method can obtain a higher yield of indole through microbial fermentation, and the process is simple, the production cost is low, and the raw materials are easy to obtain, which provides a new idea for the biosynthesis of indole.

[0061] The present invention will be further described below in conjunction with embodiments, but 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 the construction of the expression strain pCDFDuet-TnaA-BL21, and the specific steps are as follows:

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

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

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

[0067] S3. Using plasmid pCDFDuet-1 as a template, linear expression vector pCDFDuet-1 was obtained by linear amplification through inverse PCR, and the required primer nucleotide sequences are shown in SEQ ID NO: 6 and SEQ ID NO: 7;

[0068] Among them, the PCR amplification conditions were denaturation at 98°C, annealing at 55°C, and extension at 72°C for 30 cycles;

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

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

[0071] Table 1 Sequence information

[0072]

[0073] Example 2

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

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

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

[0077] The PCR amplification conditions were denaturation at 98°C, annealing at 65°C-55°C, and extension at 72°C for 40 cycles.

[0078] S2. After obtaining the mutant TnaA* gene, use the Qingke Biotechnology DNA Gel Recovery Kit to perform gel recovery and purification.

[0079] S3. The purified target fragment TnaA* in step a and the linearized vector pCDFduet-1 obtained in step C of Example 1 were connected by Novizan recombinase C112, and the enzyme reaction conditions were 37°C for 30 min. The expression vector pCDF-TnaA* was obtained and transferred into E. coli BL21 competent cells by chemical transformation.

[0080] Example 3

[0081] This example is a method for quickly screening high-yielding indole strains using pCDF-TnaA*. 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 LB medium in each well, and culture at 37°C, 1000 rpm for 6 h. Pick 2 pCDFDuet-TnaA colonies as controls, and pick pCDF-TnaA*-BL21 colonies in all the remaining 90 wells.

[0083] S2. The bacterial solution cultured in step S1 was transferred to a 96-well plate containing 760 μL of fermentation medium in each well at a 5% inoculation rate for culture at 30°C and 1000 rpm for a total fermentation time of 24 h;

[0084] S3. After inoculation, culture until the bacterial OD 600nm =0.6-0.8, add IPTG with a final concentration of 0.1mM for induction, and the induction temperature is 30°C;

[0085] S4. After 24 hours of fermentation, take 10 μL of fermentation broth sample from each well and dilute it in 900 μL of water, add 10% of color developer, and react at room temperature for 5 minutes. After the reaction, measure the absorbance at a wavelength of 568 nm using an enzyme reader;

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

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

[0088] Example 4

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

[0090] 1) The pCDF-TnaA*-BL21 bacterial solution preserved in Example 3 was streaked onto an LB medium plate and cultured at 37°C and 220 rpm for 16 h;

[0091] 2) Pick the pCDF-TnaA*-BL21 colony cultured in step 1) and place it in a 500 mL shake flask filled with 10% LB medium, and culture it at 37°C and 220 rpm until the OD 600nm About 5, it takes about 6 hours;

[0092] 3) The pCDF-TnaA*-BL21 bacterial solution cultured in step 2) was transferred to a 5L shake flask filled with 10% fermentation medium at a 10% inoculation rate, and the shake flask was cultured at 30°C and 220 rpm until the fermentation was completed. The total fermentation time was 24 hours;

[0093] 4) After inoculation, culture until OD 600nm =0.6-0.8, add IPTG with a final concentration of 0.1 mM, and induce expression at 30°C;

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

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

[0096] The strain screened in Example 4 was subjected to shake flask fermentation to determine indole production, and the plasmid of the strain with high indole production during shake flask fermentation was extracted for sequencing and identification. The protein sequence was shown in SEQ ID NO: 3, wherein the mutation site was V389D, and the maximum production of the strain could 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; PreTnaA is derived from Providencia rettgeri, and its protein sequence is shown in SEQ ID NO: 1;

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

[0100] 3) After the fermentation is completed, the indole production is detected in the liquid phase.

[0101] Comparative Example 2

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

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

[0104] 3) After the fermentation is completed, the indole production is detected in the liquid phase.

[0105] Experimental Example 1

[0106] The fermentation products of Example 4 and Comparative Example 1-2 after fermentation were tested for indole yield using HPLC (high performance liquid chromatography), and the test 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% acetic acid in water;

[0110] Mobile phase B: methanol;

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

[0112] Flow rate: 1ml / min;

[0113] Injection volume: 10 μL;

[0114] Column temperature: 35 °C;

[0115] Detection wavelength: 269nm.

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

[0117] Table 2 Indole production data of each group

[0118] Expression vector Yield g / L Comparative Example 1 pCDF-PreTnaA 1.04 Comparative Example 2 pCDF-PvTnaA 0.89 Example 4 <![CDATA[pCDF-TnaA V389D ]]> 1.62

[0119] Example 4

[0120] This example is to verify the effect of pCDF-TnaA*-BL21 fermentation tank fermentation, and the specific steps are as follows:

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

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

[0123] The composition of the fermentation medium 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, pyridoxal phosphate 0.002 g / L, 20% dibutyl sebacate, 0.5 mL / L defoamer;

[0124] Fermentation tank parameters: During the fermentation process, ammonia water was used to control pH = 7.5, the fermentation temperature was 30°C, the DO dissolved oxygen content was 25%-35%, and feeding was performed according to the residual amount of glucose. Tryptophan was added as a substrate 4 hours after induction to control the tryptophan concentration to be above 2 g / L.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in 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 mutation of the amino acid sequence shown in SEQ ID NO: 1, and the mutation sites of the mutant include: the 389th valine is mutated to aspartic acid.

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

3. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule according to claim 2.

4. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the recombinant vector according to claim 3.

5. The method for constructing a recombinant bacterium according to claim 4, characterized in that: include: The gene of the tryptophanase TnaA mutant is inserted into an expression vector to obtain a recombinant vector, and then the recombinant vector is introduced 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 according to claim 1, the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3 or the recombinant bacterium according to claim 4 in the production of indole and its downstream products.

8. A method for producing indole, characterized in that: include: Indole can be obtained by adding the recombinant bacteria described in claim 4 to a reaction system containing tryptophan for whole-cell catalysis.

9. The method according to claim 8, characterized in that The method for culturing the recombinant bacteria comprises: streaking the recombinant bacteria onto a plate for culturing, and then picking the colonies obtained by the culture to a shake bottle for expansion culture; The conditions for plate culture are: overnight culture at 37°C and 220 rpm, and the culture medium is LB medium; The conditions for the expanded culture are: culture at 37°C and 220 rpm, the culture medium is LB culture medium, and the liquid volume is 10%.

10. The method according to claim 9, characterized in that The method further comprises fermenting after the expanded culture; the fermentation comprises shake flask fermentation and fermentation tank fermentation; The shake flask fermentation comprises: inoculating the bacterial liquid obtained after the expanded culture into the fermentation medium for shake flask culture, and adding an inducer at the initial stage of inoculation to induce expression; The fermentation in a fermenter comprises: inoculating the bacterial liquid obtained after the expanded culture into a fermentation medium for fermentation culture, adding an inducer to induce expression, and after the induction, supplementing tryptophan as a substrate; Preferably, the conditions for the shake flask fermentation are: fermentation at 30°C, 220rpm, and stopping the fermentation after 24h; the conditions for inducing expression are: culturing until OD 600nm =0.6-0.8, the inducer was added, the inducer was 0.1mM IPTG, the induction temperature was 30°C, and 3g / L tryptophan was added as a substrate after 2h of induction; Preferably, the components of the fermentation medium in the shake flask fermentation include: 23.6 g / L yeast powder, 11.8 g / L peptone, 2.2 g / L K2HPO4, 9.4 g / L KH2PO4, 0.241 g / L MgSO4, 0.011 g / L CaCl2, 3 g / L tryptophan, 40 g / L glucose, and 20% dibutyl sebacate; Preferably, the fermentation conditions of the fermentation tank are: temperature of 30°C, pH=7.5, dissolved oxygen 25%-35%, stirring and dissolved oxygen, and fermentation time of 48h; the conditions of the induction table are: culturing at 37°C until OD 600nm =20, adjust the temperature to 30°C, add 0.2 mM IPTG for induction, and add more than 2 g / L tryptophan as substrate after 4 h of induction; Preferably, the components of the fermentation medium in the fermentation tank fermentation include: yeast powder 23.6g / L, peptone 11.8g / L, K2HPO4 2.2g / L, KH2PO4 9.4g / L, MgSO4 0.241g / L, CaCl2 0.011g / L, glucose 40g / L, pyridoxal phosphate 0.002g / L, 20% dibutyl sebacate, and 0.5mL / L defoamer.

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