Tryptophan producing strain and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly discloses a tryptophan production strain and application thereof. The L-tryptophan high-yield strain T29 is developed and obtained through methods of combining gene modification with mutagenesis screening and the like. The strain T29 is fermented in a 5L tank, the highest yield of L-tryptophan can reach 60.7 g / L, and the conversion rate reaches 18.6%. Therefore, the strain provided by the invention has important industrial application value for industrial production of L-tryptophan and development of an L-tryptophan production strain with higher fermentation performance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a tryptophan-producing bacterium and its applications. Background Technology

[0003] Currently, the industrial production of L-tryptophan mainly relies on microbial fermentation. This method uses inexpensive carbon sources such as glucose and molasses as substrates, and achieves the large-scale accumulation of L-tryptophan through high-density cultivation of engineered strains. To improve the fermentation performance of strains, reduce production costs, and increase production efficiency, recent research has focused on two main directions: strain modification and fermentation process optimization. Among these, strain modification has become a research focus because it can systematically solve key technical problems in the L-tryptophan fermentation process from the source. Its core objective is to increase L-tryptophan yield and substrate conversion rate, while reducing the accumulation of byproducts.

[0004] However, L-tryptophan, as an aromatic amino acid, undergoes a complex and lengthy metabolic pathway for synthesis from glucose in microorganisms, encompassing the central carbon metabolism pathway, shikimic acid synthesis pathway, and branched acid synthesis pathway, involving approximately 26 biochemical reactions. This synthesis pathway has two major regulatory nodes: first, the expression products of key genes such as aroG / aroF / aroH (encoding 3-deoxy-D-arabinohepenolate-7-phosphate synthase) and trpE (encoding anthranilic acid synthase) are subject to multiple feedback regulation, limiting the activity of key enzymes; second, there is significant metabolic flux competition between the central carbon metabolism pathway and the aromatic amino acid synthesis pathway, resulting in insufficient carbon source allocation to L-tryptophan. These bottlenecks make it difficult to achieve breakthroughs in L-tryptophan yield and substrate conversion rate in existing fermentation processes, severely restricting its large-scale, industrialized production.

[0005] Therefore, in order to obtain superior strains that can meet the needs of large-scale industrialization and alleviate the growing contradiction between market supply and demand for L-tryptophan, it is urgent to strengthen the research on the construction and breeding technology of high-yield L-tryptophan strains. Summary of the Invention

[0006] This invention develops a high-yield strain of L-tryptophan through gene modification and mutagenesis screening, providing support for the industrial production of L-tryptophan or the development of strains with higher fermentation performance.

[0007] This invention provides a tryptophan-producing strain, Escherichia coli T29, which was deposited at the China Center for Type Culture Collection on June 17, 2024, with accession number CCTCC NO: M 20241245.

[0008] Genome sequencing revealed that strain T29, compared to the originating strain *E. coli* W3110, lacked several genes, including: pspBCDE (phage shock protein), ycjW (DNA-binding transcriptional repressor), tyrR (DNA-binding transcriptional dual regulator), tpx (lipid hydroperoxide peroxidase), ycjG (L-Ala-D / L-Glu epimerase), and mppA (murein tripeptide ABC transporter periplasmic binding protein).

[0009] The present invention further provides the use of the aforementioned tryptophan-producing strain in the preparation of tryptophan or tryptophan derivatives, or tryptophan-based downstream metabolites.

[0010] Specifically, the tryptophan derivative is a product obtained by metabolic derivatization of L-tryptophan as a direct synthetic precursor or intermediate.

[0011] More specifically, the tryptophan derivatives are 7-chlorotryptophan, 5-hydroxytryptophan, α-methyltryptophan, β-thiotryptophan, N-acetyltryptophan, and tryptophan-oligopeptide conjugates; the downstream metabolites based on tryptophan are kynurenine, 5-hydroxytryptamine, melatonin, indole-3-acetic acid, indirubin, nicotinic acid mononucleotide (NaMN), and β-carboline alkaloids.

[0012] Specifically, the application of the tryptophan-producing strain in the preparation of downstream metabolites of tryptophan includes: further modifying the metabolic pathways of the tryptophan-producing strain to endow it with the ability to continue metabolizing tryptophan into downstream metabolites. Taking the production of 5-hydroxytryptamine (5-HT) as an example: L-tryptophan is converted to 5-HT through a two-step reaction of hydroxylation and decarboxylation by tryptophan hydroxylase (TPH) and aromatic amino acid decarboxylase (AADC). Therefore, by endowing or enhancing the activity of tryptophan hydroxylase (TPH) and aromatic amino acid decarboxylase (AADC) in L-tryptophan-producing strains to obtain 5-hydroxytryptamine-producing strains, they can be used to produce 5-HT.

[0013] The present invention also provides a method for preparing tryptophan, which includes the step of fermenting the tryptophan-producing strain to produce tryptophan;

[0014] Preferably, fermentation is carried out in a culture medium with glucose as the carbon source.

[0015] Specifically, the fermentation is shake-flask fermentation or fermenter fermentation.

[0016] More specifically, the shake flask fermentation conditions are: 33-39℃, 150-250rpm; during shake flask fermentation, the pH is adjusted to 7.0±0.2 every 4-8 hours with 10-20% ammonia solution.

[0017] In a specific example, the shake-flask fermentation medium consisted of: glucose 20 g / L, magnesium sulfate heptahydrate 1 g / L, potassium dihydrogen phosphate 2 g / L, ammonium sulfate 4 g / L, yeast extract 1 g / L, ferrous sulfate heptahydrate 100 mg / L, citric acid monohydrate 2 g / L, and calcium carbonate 30 g / L.

[0018] Specifically, the fermentation temperature in the fermenter is 34-39℃, and the pH of the fermentation broth is maintained at 7.0±0.2 by automatically adding ammonia solution through a bioreactor, with dissolved oxygen controlled at 30-50%.

[0019] More specifically, the fermenter culture medium consists of: 20g glucose, 2g yeast extract, 3g ammonium sulfate, 7g dipotassium hydrogen phosphate, 1g methionine, 2g citric acid monohydrate, 1g magnesium sulfate heptahydrate, 1mL trace element stock solution (0.04g ferrous sulfate heptahydrate, 0.02g manganese sulfate monohydrate, 0.005g calcium chloride, 0.005g zinc sulfate heptahydrate, 0.0002g cobalt chloride hexahydrate, 0.0002g cobalt sulfate pentahydrate), and 1mL vitamin stock solution (0.001g VB1, 0.001g VB3, 0.001g VB5, 0.001g VB7, 0.001g VB12), diluted with distilled water to a final volume of 1L.

[0020] Furthermore, samples were taken and tested during fermentation to control the residual sugar content below 5 g / L. Fermentation was stopped when a decrease in cell activity or a cessation of L-tryptophan production was detected.

[0021] Optionally, the process also includes a step of separating the resulting tryptophan.

[0022] This invention developed a high-yield L-tryptophan strain, T29, through genetic modification and mutagenesis screening. Fermentation of strain T29 in a 5L tank showed that the highest L-tryptophan yield reached 60.7 g / L, with a conversion rate of 18.6%. Therefore, this strain has significant industrial value for the industrial production of L-tryptophan and the development of L-tryptophan-producing strains with higher fermentation performance. Attached Figure Description

[0023] Figure 1The results are from the 5L fermentation of strain T29.

[0024] Information on the preservation of biological materials:

[0025] Strain T29 was deposited on June 17, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO: M 20241245, and classified as Escherichia coli. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments. In the embodiments, unless otherwise specified, the percentage sign "%" refers to the mass percentage; the percentage of a solution refers to the number of grams of solute contained in 100 mL; the percentage between liquids refers to the volume ratio of the solution at 25°C.

[0027] 1. Information on the strains, plasmids, kits, solutions, and culture media used in the following examples is as follows:

[0028] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2.

[0029] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder, pH 7.2.

[0030] Shake-flask fermentation medium: glucose 20g / L, magnesium sulfate heptahydrate 1g / L, potassium dihydrogen phosphate 2g / L, ammonium sulfate 4g / L, yeast extract 1g / L, ferrous sulfate heptahydrate 100mg / L, citric acid monohydrate 2g / L, calcium carbonate 30g / L, pH 7.0.

[0031] Seed culture medium: peptone 12 g / L, yeast extract 24 g / L, glycerol 4 mM / L, potassium dihydrogen phosphate 2.313 g / L, dipotassium hydrogen phosphate 12.54 g / L, pH 7.0.

[0032] Fermentation medium: 20g glucose, 2g yeast extract, 3g ammonium sulfate, 7g dipotassium hydrogen phosphate, 1g methionine, 2g citric acid monohydrate, 1g magnesium sulfate heptahydrate, 1mL trace element stock solution (0.04g ferrous sulfate heptahydrate, 0.02g manganese sulfate monohydrate, 0.005g calcium chloride, 0.005g zinc sulfate heptahydrate, 0.0002g cobalt chloride hexahydrate, 0.0002g cobalt sulfate pentahydrate), 1mL vitamin stock solution (0.001g VB1, 0.001g VB3, 0.001g VB5, 0.001g VB7, 0.001g VB12), add distilled water to a final volume of 1L.

[0033] Strains, vectors, and related gene / promoter / primer sequences: The starting strain used in the examples was wild-type Escherichia coli W3110 (accession number ATCC 27325); the pGRB vector was purchased from Miaoling Company; the primer information used to construct the plasmid and the strain is shown in Table 1; the corresponding promoters and genes are shown in the sequence listing.

[0034] Table 1

[0035]

[0036]

[0037]

[0038] 2. The methods involved in the following embodiments are as follows:

[0039] 2.1 PCR reaction method:

[0040] PCR reaction system (50 μL): ddH2O 20 μL, primer F (10 mM) 2 μL, primer R (10 mM) 2 μL, template 1 μL, 2×Phanta Mix high-fidelity enzyme 25 μL;

[0041] The PCR reaction program is as follows: ① 95℃, 3 min; ② 95℃, 30 s; Tm℃, 30 s; 72℃, 30 s / kb; repeat 30-32 cycles; ③ 72℃, 5 min; store the product at 4℃.

[0042] 2.2 Overlap PCR method:

[0043] PCR reaction system (50 μL): primer F (10 mM) 2 μL, primer R (10 mM) 2 μL, template fragment 1 μL each, 2×Phanta Mix high-fidelity enzyme 25 μL, ddH2O added to 50 μL;

[0044] The PCR reaction program is as follows: ① 95℃, 3 min; ② 95℃, 30 s; Tm℃, 30 s; 72℃, 30 s / kb; repeat 30-32 cycles; ③ 72℃, 5 min; store the product at 4℃.

[0045] 2.3 Performance verification of L-tryptophan production by the strain:

[0046] 2.3.1 Shake-flask fermentation:

[0047] ① Activation culture: Streak the glycerol tube-preserved strain onto LB agar plates and incubate at 36-37℃ for 10-12 hours; pick a single colony and inoculate it into LB liquid medium and incubate overnight at 36-37℃ and 200 rpm.

[0048] ② Shake flask fermentation culture: Inoculate the culture medium from step 1 into the shake flask fermentation medium at an inoculation rate of 0.1% (v / v). The shake flask has a baffle on the side. Ferment at 35℃ and 200 rpm. During fermentation, adjust the pH to 7.0±0.2 with ammonia water (15%) every 6 hours. Take samples at about 22 hours to determine the OD value and tryptophan yield.

[0049] 2.3.2 Fermentation in a 5L tank:

[0050] ① Activation culture: Inoculate the strain preserved in glycerol tubes onto LB plates and incubate at 36-37℃ for 10-12 hours; pick a single colony and inoculate it into LB liquid medium and incubate overnight at 36-37℃ and 200 rpm.

[0051] ② Seed culture: Inoculate the culture solution from step 1 into the seed culture medium at an inoculation rate of 1% (v / v), and incubate at 36-37℃ for 8-10 hours until the OD is 8-10;

[0052] ③ Fermentation in a 5L tank: Inoculate the seed culture into the fermentation medium at a rate of 10% (v / v), and incubate at 37℃. Add ammonia water through the fermenter's automatic pump to maintain the pH at 7.0±0.2, dissolved oxygen at 30%, and residual sugar content below 5g / L. Take samples regularly to determine the L-tryptophan content and conversion rate. Terminate fermentation when a decrease in cell activity or a cessation of L-tryptophan production is detected.

[0053] 2.4 Calculation of sugar-acid conversion rate (%): The ratio of the total mass (g) of L-tryptophan at the bottom of the tank to the total mass (g) of glucose consumed. The calculation formula is: Conversion rate (%) = Tank volume (L) x L-tryptophan concentration (g / L) ÷ Total glucose consumed (g) × 100%.

[0054] 2.5 L-Tryptophan Concentration Detection: Take 1 ml of fermentation broth, dilute with dilute sulfuric acid aqueous solution (pH 3.0), and make up to 100 ml. Take 0.5 ml of the diluted solution, filter it through a 0.22 μm aqueous filter membrane, and perform high performance liquid chromatography (HPLC) for determination. The chromatographic column is HILIC Amphion II, 5 μm, 4.6 × 150 mm.

[0055] Mobile phase preparation: Solution A is 50mM dipotassium hydrogen phosphate, pH adjusted to 3.0 with phosphoric acid, and then filtered through a 0.22μm aqueous filter membrane; Solution B is chromatographic grade acetonitrile. Mix A and B at a volume ratio of 1:3, degas by sonication, and the mobile phase is obtained.

[0056] The HPLC detection conditions were as follows: detection time: 7 min; detection wavelength: 250 nm; column oven temperature: 35 ℃; flow rate: 1 mL / min; injection volume: 10 μL.

[0057] 2.6 Glucose concentration detection: Glucose concentration was determined using the SBA-40D-100 biosensor analyzer from the Institute of Biology, Shandong Academy of Sciences.

[0058] Example 1: Construction and mutagenesis screening of L-tryptophan strains

[0059] This embodiment aims to illustrate the specific construction steps of the strain. The gene editing method is based on the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21). Unless otherwise specified, all technical terms involved in this invention are explained in this article. In particular, if the same type of gene manipulation method is used in the embodiment, only one annotation is provided.

[0060] 1. Construction of the recombinant L-tryptophan-producing strain T9

[0061] The genotype of recombinant strain T9 is W3110,ΔtnaAB,yjiV::Ptrc-trpBA,ΔlacI,yeeP::Pj23106-ywkB(Bs),ΔpykF,yncI::Ptrc-trpE S40F ,ylbE::Ppck-Pck,mbha::Ptrc-pyc P458S ,trpE::Ptrc-trpE S40FStarting with *Escherichia coli* W3110, the following gene editing was performed: tnaA and tanB genes (tanA and tanB are adjacent, abbreviated as tanAB; tnaA encodes tryptophanase, tnaB encodes tryptophan:H cotransporter), lacI gene (encoding lactose operon repressor protein), pykF gene (encoding pyruvate kinase), yjiV, yeeP, pykF, yncI, ylbE, and mbha genes were knocked out; the expression cassette Ptrc-trpBA (encoding tryptophan synthase) controlled by the Ptrc promoter was integrated into the yjiV pseudogene site; the ywkB gene (encoding AEC family transporter protein) from *Bacillus subtilis* was integrated into the yeeP site under the control of the Pj23106 promoter; and trpE was... S40F The gene (encoding anthranilate synthase, with an S40F mutation in its amino acid sequence compared to the wild type) is integrated into the yncI site and replaces the wild-type trpE gene under the control of the Ptrc promoter, respectively; the pck gene (encoding phosphoenolpyruvate carboxykinase) is integrated into the ylbE site and pyc site under the control of the Ppck promoter itself. P458S The gene (encoding pyruvate carboxylase, with a P458S mutation in its amino acid sequence compared to the wild type) was integrated into the mbha site under the control of the Ptrc promoter.

[0062] The sources of the edited genes are as follows: trpBA and pck are from *Escherichia coli* W3110 (gene sequences SEQ ID NO.1 and SEQ ID NO.2, respectively); ywkB(Bs) is a gene for a transport protein of the *Bacillus subtilis* AEC family (gene sequence shown in SEQ ID NO.3); trpE S40F It is a mutant obtained by mutating trpE from E. coli W3110 to S40F (gene sequence shown in SEQ ID NO.4); pyc P458S This is a mutant of pyc derived from Corynebacterium glutamicum ATCC 13032, obtained by undergoing a P458S mutation (gene sequence shown in SEQ ID NO. 5). All of the above genes are expressed by the Ptrc promoter (Ptrc sequence shown in SEQ ID NO. 6); ywkB is expressed by the Pj23106 promoter (Pj23106 sequence shown in SEQ ID NO. 7); and pck is expressed by its own promoter Ppck (Ppck sequence shown in SEQ ID NO. 8). The specific construction steps are as follows:

[0063] (1) Knockout of the tnaAB gene: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using tnaAB-up-F / tnaAB-up-R and tnaAB-down-F / tnaAB-down-R primers, respectively. Using the upstream and downstream homologous arm fragments as templates, overlapping fragments were obtained by overlapping PCR amplification using tnaAB-up-F / tnaAB-down-R primers. Using the pGRB vector as a template, plasmid tnaAB-pGRB was obtained by PCR amplification using pamF-tnaAB / pamR-tnaAB primers. Following the method in Molecular Cloning (3rd Edition), E. coli W3110 electroporation competent cells were prepared. The overlapping fragment and plasmid tnaAB-pGRB were electroporated into E. coli W3110 competent cells. Positive transformants were obtained by verification and screening using primers VF-tnaAB and VR-tnaAB, and strain T1 was obtained.

[0064] (2) Knockout of the yjiV gene and integration of Ptrc-trpBA at this site: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms and the target fragment were obtained by PCR amplification using primers trpBA-up-F / trpBA-up-R, ttrpBA-down-F / trpBA-down-R, and trpBA-F / trpBA-R, respectively. Using the upstream and downstream homologous arms and the target gene fragment as templates, overlapping fragments were obtained by overlapping PCR amplification using primers trpBA-up-F / trpBA-down-R. Using the pGRB vector as a template, plasmid trpBA-pGRB was obtained by PCR amplification using primers pamF-trpBA / pamR-trpBA. The overlapping fragment and plasmid trpBA-pGRB were electroporated together into T1 competent cells. Positive transformants were obtained by verification and screening using primers VF-trpBA and VR-trpBA, and strain T2 was obtained.

[0065] (3) Knock out lacI: Using the same operation method as in step (1), the difference is that the primers used are lacI-up-F / lacI-up-R, lacI-down-F / lacI-down-R, and pamF-lacI / pamR-lacI, the competent cells are T2, the verification primers are VF-lacI and VR-lacI, and strain T3 is obtained.

[0066] (4) Knockout of yeeP and integration of Pj23106-ywkB at this site: Using the E. coli W3110 genome as a template, and ywkB-up-F / ywkB-up-R and ywkB-down-F / ywkB-down-R as primers, upstream and downstream homologous arms were obtained by PCR amplification. Using the Bacillus subtilis genome (Gene ID: 936875) as a template, and ywkB-F / ywkB-R as primers, the target fragment was obtained by PCR amplification. Using upstream and downstream homologous arms and the target gene fragment as templates, and with ywkB-up-F / ywkB-down-R as primers, overlapping fragments were amplified by overlapping PCR to obtain overlapping fragments. Using the pGRB vector as a template and with pamF-ywkB / pamR-ywkB as primers, plasmid ywkB-pGRB was amplified by PCR to obtain plasmid ywkB-pGRB. The overlapping fragments and plasmid ywkB-pGRB were electroporated into T3 competent cells, and strain T4 was obtained by verification using primers VF-ywkB and VR-ywkB.

[0067] (5) Knock out pykF: Using the same operation method as in step (1), the difference is that the primers used are pykF-up-F / pykF-up-R, pykF-down-F / pykF-down-R, and pamF-pykF / pamR-pykF, the competent cells are T4, the verification primers are VF-pykF and VR-pykF, and strain T5 is obtained.

[0068] (6) Knock out yncI and integrate Ptrc-trpE at this site S40F Using the same operating method as in step (2), the difference lies in the primers yncI-trpE*-up-F / yncI-trpE*-up-R, yncI-trpE*-down-F / yncI-trpE*-down-R, and yncI-trpE*-F / yncI-trpE*-R, upstream and downstream homologous arms and the target fragment are obtained by PCR amplification. Then, using the target fragment as a template, primers are used...

[0069] The mutant fragment of the target fragment was prepared using S40F-yncI-trpE*-F / S40F-yncI-trpE*-R. Using the pGRB vector as a template and pamF-yncI-trpE* / pamR-yncI-trpE* as primers, PCR amplification yielded the plasmid yncI-trpE*-pGRB. The competent cells were T5. The strain T6 was obtained through verification using primers VF-yncI-trpE* and VR-yncI-trpE*.

[0070] (7) Knock out ylbE and integrate Ppck-Pck at this site: Using the same operation method as in step (2), the difference is that the primers used are pck-up-F / pck-up-R, pck-down-F / pck-down-R, pck-F / pck-R, pamF-pck / pamR-pck, the competent cells are T6, the primers VF-pck and VR-pck are verified, and strain T7 is obtained.

[0071] (8) Knock out mbha and integrate Ptrc-pyc at that site P458S Using the same operation method as in step (4), the difference is that, using the E. coli W3110 genome as a template, and pyc*-up-F / pyc*-up-R and pyc*-down-F / pyc*-down-R as primers, the upstream and downstream homologous arms are amplified by PCR. P458S The gene was obtained by Qingke Biotechnology Co., Ltd. through codon preference optimization of the gene encoding pyruvate carboxylase (protein sequence number NCBI Reference Sequence: WP_040967057.1) from Corynebacterium glutamicum, and by designing a mutation at position 458 of the protein sequence, replacing proline (P) with serine (S). P458S The sequence is shown in SEQ ID NO.5. The primers used for amplification were pyc*-F / pyc*-R. The plasmid pyc*-pGRB was constructed using the primers pamF-pyc* / pamR-pyc*. The competent cells were T7 cells. The verification primers were VF-pyc* and VR-pyc*, and strain T8 was obtained.

[0072] (9) Introduction of mutations into the trpE gene (trpE) S40F The Ptrc promoter was replaced, and the same operation method as in step (6) was used. The primers used were Ptrc-trpE*-up-F / Ptrc-trpE*-up-R, Ptrc-trpE*-down-F / Ptrc-trpE*-down-R, Ptrc-trpE*-F / Ptrc-trpE*-R and pamF-Ptrc-trpE* / pamR-Ptrc-trpE*. The competent cells were T8. The verification primers were VF-Ptrc-trpE* and VR-Ptrc-trpE*. The strain T9 was obtained.

[0073] 2. Mutagenesis screening of L-tryptophan-producing mutant bacteria T9x

[0074] Shake-flask fermentation was performed on strain T9, and the results are shown in Table 2. When editing a single gene or a few genes was completed, the L-tryptophan yield was low. Then, the researchers attempted to perform ARTP (atmospheric pressure room temperature plasma) mutagenesis and screening on strain T9 in order to screen for mutant strains with significantly improved L-tryptophan yield and conversion rate. The specific steps are as follows.

[0075] (1) Activation of strain: The T9 strain preserved in glycerol tubes was inoculated into a test tube containing 10 mL of LB liquid medium and cultured at 37℃ and 200 rpm for 12-16 h.

[0076] (2) Strain Mutagenesis: This involves three steps: ① The T9-activated culture is transferred to 50 mL of fresh LB liquid medium and cultured at 37°C and 200 rpm until the logarithmic growth phase is reached. 600 Approximately 0.6. ② Beforehand, autoclave and dry the slides. Take 10 μL of logarithmic-phase bacterial suspension and evenly spread it on an ARTP-specific slide. Turn on the instrument switch, condensate valve, and helium valve sequentially, and irradiate with UV light for 30 min. Place the ARTP-specific slide in the slide for mutagenesis. The mutagenesis conditions are: operating power 120 W, gas flow rate 10 L / min, and mutagenesis time gradients of 40 s, 50 s, 60 s, 70 s, 80 s, and 90 s. ③ After mutagenesis, place all samples in 2 mL EP tubes containing 800 μL / L liquid medium, mix on a shaker for 1 min, centrifuge, remove approximately 600 μL of supernatant, resuspend the bacteria, plate on LB agar plates, and incubate overnight at 37°C. Calculate the lethality of ARTP mutagenesis at different treatment times, and repeat the above operations at appropriate mutagenesis times to obtain a sufficient number of mutant libraries.

[0077] (3) Screening of dominant bacteria by well plate: This involves three steps: ① Pick a single colony from the mutant library plate and inoculate it into a 96-well plate containing 800 μL of seed culture medium (approximately 2.2 mL). Incubate at 37℃ and 800 rpm for approximately 16-18 h. ② Transfer 10 μL of the seed culture from the 96-well plate to a 48-well plate containing 1 mL of fermentation medium (approximately 4.6 mL). Incubate at 35℃ and 700 rpm for 16-20 h. ③ Take 200 μL of bacterial culture from the 48-well plate (mix well before sampling) for L-tryptophan concentration detection. The sample preparation steps are: add 200 μL of supernatant to 800 μL of pure water (diluted 5 times), filter through a 0.22 μm aqueous filter membrane, and perform liquid chromatography.

[0078] Dominant strain rescreening: After screening 18 well plates (approximately 1584 single colonies) through the above screening process, a rescreening was performed. The top 5 dominant single colonies from each plate in the initial screening were pooled into a new 96-well plate for rescreening. Using strain T9 as a control, 10 high-yielding single colonies were selected from the rescreening process for shake-flask verification. Combining the results of well plate screening and shake-flask verification, the mutant strain with improved yield and conversion rate was identified and numbered T9x (shake-flask fermentation results are shown in Table 2). Glycerol tubes were prepared and stored at -80℃.

[0079] 3. Construction of strain T29, which produces high levels of L-tryptophan

[0080] After gene editing and traditional mutagenesis breeding, strain T9x was obtained from *E. coli*, but its fermentation performance still failed to break through the bottleneck. Therefore, researchers continued to perform metabolic engineering on strain T9x, including enhancing the shikimic acid synthesis pathway, the branched acid synthesis pathway, and tryptophan efflux. Specifically, the ilvG and mtr genes were knocked out, and the aroE gene (encoding shikimic acid dehydrogenase) and aroK (encoding shikimic acid kinase) were placed under the control of the Ptrc promoter and integrated into the ilvG site. The yddG gene (edited tryptophan efflux protein) was placed under the control of the Ptrc promoter and integrated into the mtr site. Among them, aroEK and yddG are from *E. coli* W3110 (gene sequences are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively). The specific modification steps are as follows:

[0081] (1) Knock out ilvG and integrate aroEK at the site: Using the same operation method as in step (2), except that the primers used are aroEK-up-F / aroEK-up-R, aroEK-down-F / aroEK-down-R, aroE-F / aroE-R, aroK-F / aroK-R, pamF-Ptrc-trpE* / pamR-Ptrc-trpE*, the competent cells are T9x, the verification primers are VF-aroEK and VR-aroEK, and strain T20 is obtained.

[0082] (2) Knock out mtr and integrate Yddg at the site: Using the same operation method as in step (2), the difference is that the primers used are Yddg-up-F / Yddg-up-R, Yddg-down-F / Yddg-down-R, Yddg-F / Yddg-R, pamF-Yddg / pamR-Yddg, the competent cells are T20, the verification primers are VF-Yddg and VR-Yddg, and strain T29 is obtained.

[0083] Shake-flask fermentation was performed on strains T20 and T29, and the results are shown in Table 2. The yield of the mutagenized strain T9x increased from 1.79 g / L to 2.03 g / L (an increase of about 13% compared to T9), and the conversion rate increased from 9.97% to 11.13%. There was no significant difference in biomass. This indicates that the mutagenized strain T9x was successfully obtained, which further improved the yield and conversion rate of tryptophan, laying the foundation for subsequent pathway optimization.

[0084] T20 was derived from the overexpression of aroE and aroK in T9x, which significantly increased the supply of tryptophan precursors, resulting in an increase of approximately 13% in L-tryptophan production and approximately 12% in conversion rate. Furthermore, the overexpression of the tryptophan efflux protein Yddg in T20 relieved the inhibition of intracellular products, and the resulting strain T29 further increased the yield to 2.52 g / L (an increase of approximately 10% compared to T20), and the conversion rate to 13.96% (an increase of approximately 12% compared to T20), without extending the fermentation period.

[0085] In summary, the T29 strain, obtained through three rounds of iterations of "mutation screening - pathway enhancement - efflux upgrade", achieved a stepwise leap in synthesis efficiency by approximately 2.2 times and 1.9 times in tryptophan production and conversion rate, respectively, while maintaining a basically unchanged biomass.

[0086] Table 2

[0087] strain <![CDATA[OD 600 ]]> L-Tryptophan content (g / L) Conversion rate (%) T7 20.0 0 0 T9 16.2 1.79 9.97 T9x 14.6 2.03 11.13 T20 15.6 2.30 12.46 T29 16.0 2.52 13.96

[0088] The strain T29 was validated by fermentation in a 5L tank, and the results are as follows: Figure 1 As shown, the highest yield of L-tryptophan reached 60.7 g / L, with a conversion rate of 18.6%, which has significant industrial value for the industrial production of L-tryptophan and the development of L-tryptophan-producing strains with higher fermentation performance. Strain T29 was deposited on June 17, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO: M20241245, and classified as *Escherichia coli*.

Claims

1. A tryptophan-producing strain, characterized in that, It is Escherichia coli, with accession number CCTCC NO:M 20241245.

2. The use of the tryptophan-producing strain as described in claim 1 in the preparation of tryptophan or tryptophan derivatives, or tryptophan-based downstream metabolites.

3. The application as described in claim 2, characterized in that, The tryptophan derivative is a product derived from L-tryptophan as a direct synthetic precursor or intermediate metabolite.

4. The application as described in claim 2, characterized in that, The tryptophan derivatives are 7-chlorotryptophan, 5-hydroxytryptophan, α-methyltryptophan, β-thiotryptophan, N-acetyltryptophan, and tryptophan-oligopeptide conjugates; the downstream metabolites based on tryptophan are kynurenine, 5-hydroxytryptamine, melatonin, indole-3-acetic acid, indirubin, nicotinic acid mononucleotide (NaMN), and β-carboline alkaloids.

5. A method for preparing tryptophan, characterized in that, Includes the step of producing tryptophan by fermentation culture of the tryptophan-producing strain as described in claim 1; Preferably, fermentation is carried out in a culture medium with glucose as the carbon source.

6. The method of claim 5, characterized in that, The fermentation is either shake-flask fermentation or fermenter fermentation.

7. The method as described in claim 6, characterized in that, The shake flask fermentation conditions are: 33-39℃, 150-250 rpm; during shake flask fermentation, the pH is adjusted to 7.0±0.2 every 4-8 hours with 10-20% ammonia solution. Specifically, the shake-flask fermentation medium consisted of: glucose 20 g / L, magnesium sulfate heptahydrate 1 g / L, potassium dihydrogen phosphate 2 g / L, ammonium sulfate 4 g / L, yeast extract 1 g / L, ferrous sulfate heptahydrate 100 mg / L, citric acid monohydrate 2 g / L, and calcium carbonate 30 g / L.

8. The method as described in claim 6, characterized in that, The fermentation in the fermenter was carried out at a temperature of 34-39℃, and the pH of the fermentation broth was maintained at 7.0±0.2, with dissolved oxygen controlled at 30-50% during the fermentation process. Specifically, the fermenter culture medium consists of: glucose 20 g / L, yeast extract 2 g / L, ammonium sulfate 3 g / L, dipotassium hydrogen phosphate 7 g / L, methionine 1 g / L, citric acid monohydrate 2 g / L, magnesium sulfate heptahydrate 1 g / L, trace element stock solution 1 mL / L, and vitamin stock solution 1 mL / L. The 1 mL trace element stock solution comprises: ferrous sulfate heptahydrate 0.04 g, manganese sulfate monohydrate 0.02 g, calcium chloride 0.005 g, zinc sulfate heptahydrate 0.005 g, cobalt chloride hexahydrate 0.0002 g, and cobalt sulfate pentahydrate 0.0002 g. The 1 mL vitamin stock solution comprises: VB1 0.001 g, VB3 0.001 g, VB5 0.001 g, VB7 0.001 g, and VB12 0.001 g.

9. The method as described in claim 8, characterized in that, During fermentation, the residual sugar content should be controlled to be below 5g / L.

10. The method according to any one of claims 6 to 9, characterized in that, It also includes the step of separating the obtained tryptophan.

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