Genetic engineering strain with high yield of L-tryptophan as well as construction method and application of genetic engineering strain

Abstract

The invention discloses a genetic engineering strain with high yield of L-tryptophan as well as a construction method and application of the genetic engineering strain. The genetic engineering strain expresses a mutant FlhD protein and / or a mutant GabD protein; the 84th amino acid of the mutant FlhD protein is phenylalanine; the 130th amino acid of the mutant GabD protein is proline. According to the invention, the flhD mutant gene is introduced, and the gabD mutant gene is combined to modify escherichia coli, so that the content of glutamic acid which is a precursor substance in cells is jointly increased, the yield of L-tryptophan is remarkably increased, and a new thought is provided for industrial production of L-tryptophan.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a genetically engineered strain that produces high levels of L-tryptophan, its construction method, and its applications. Background Technology

[0002] L-Tryptophan, an essential amino acid, is in high demand in the pharmaceutical, food, and feed industries. Microbial fermentation is the mainstream method for producing L-Tryptophan, with Escherichia coli becoming an important engineered host bacterium due to its clear genetic background and ease of operation.

[0003] To increase yield, traditional metabolic engineering strategies mainly focus on directly enhancing the L-tryptophan synthesis pathway itself, such as overexpressing the trp operon gene and removing feedback inhibition. However, L-tryptophan synthesis is highly dependent on the supply of precursors, especially branched acids and glutamate. Among them, glutamate, as a core molecule linking carbon and nitrogen metabolism, is the direct donor of indole ring nitrogen atoms in tryptophan synthesis, and its intracellular concentration often becomes a bottleneck limiting yield.

[0004] In recent years, research has gradually shifted from direct pathway modification to the discovery of global regulatory factors. For example, the flagellar synthesis master controller, FlhD, has been found to have global regulatory effects beyond its original function. Deletion of the flhD gene leads to a decrease in tryptophan production in *E. coli*, the mechanism of which may be related to the FlhDC complex inhibiting glutamate efflux, thereby affecting the intracellular glutamate pool. On the other hand, succinate semialdehyde dehydrogenase (GabD) catalyzes the conversion of succinate semialdehyde to succinate, simultaneously producing NADPH. This reaction not only replenishes the reducing power required for synthesis, but its product, succinate, entering the TCA cycle can also increase the production of α-ketoglutarate and downstream glutamate.

[0005] Although the flhD and gabD genes are recognized as potentially indirectly linked to tryptophan synthesis by influencing glutamate metabolism, there are still significant gaps in current technology: First, no studies have reported on the precise regulation of this metabolic network through specific gain-of-function point mutations (rather than knockout or overexpression) of the flhD or gabD genes themselves; second, no studies have revealed whether there is a synergistic effect between these two genes belonging to different regulatory levels, or whether a synergistic breakthrough in L-tryptophan production bottlenecks can be achieved through combined mutations.

[0006] Therefore, developing a strain construction strategy based on the synergistic modification of key regulatory nodes to more efficiently improve precursor supply and metabolic flux is of great value for overcoming the performance bottleneck of existing L-tryptophan-producing strains. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a genetically engineered strain that produces high levels of L-tryptophan, along with its construction method and applications. By introducing a mutant flhD gene and simultaneously modifying Escherichia coli with a gabD mutant gene, the content of glutamate, a precursor substance, within the cells is increased, significantly enhancing the yield of L-tryptophan and providing a new approach for the industrial production of L-tryptophan.

[0008] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a genetically engineered strain that produces high levels of L-tryptophan, wherein the genetically engineered strain expresses mutant FlhD protein and / or mutant GabD protein; The 84th amino acid of the mutant FlhD protein is phenylalanine. The mutant GabD protein has proline at position 130.

[0009] This invention enhances L-tryptophan production in microorganisms by introducing mutant flhD and / or mutant gabD genes, wherein the mutant flhD gene changes the 84th amino acid of its encoded protein to phenylalanine (F), and the mutant gabD gene changes the 130th amino acid of its encoded protein to proline (P). Simultaneous introduction of a double mutation—the mutant flhD gene encoding protein V84F and the mutant gabD gene encoding protein T130P—in Escherichia coli increases L-tryptophan production by approximately 21.9% compared to the original strain.

[0010] Preferably, the amino acid sequence of the mutant FlhD protein includes the sequence shown in SEQ ID NO.3.

[0011] Preferably, the amino acid sequence of the mutant GabD protein includes the sequence shown in SEQ ID NO.4.

[0012] The amino acid mutations in this invention include substitution, deletion, insertion, addition, or inversion of one or more amino acid residues, including at least one of the methods such as mutagenesis, site-directed mutagenesis by PCR, and / or homologous recombination, and the final modified sequence has at least 90% homology with the amino acid sequences of the flhD gene-encoded protein and the gabD gene-encoded protein, all of which are within the protection scope of this invention.

[0013] Preferably, the genetically engineered strain includes a recombinant Escherichia coli engineered strain.

[0014] In a second aspect, the present invention provides a method for constructing a genetically engineered strain that produces high levels of L-tryptophan as described in the first aspect. The method includes: using homologous recombination technology, integrating a mutant flhD gene and / or a mutant gabD gene into the genome of a host microorganism to replace its corresponding wild-type gene, thereby obtaining the genetically engineered strain that produces high levels of L-tryptophan.

[0015] Preferably, the homologous recombination technology includes: expressing λ-Red recombinase in a host microorganism, electroporating a linear DNA fragment with homologous arms at both ends to the target site in the genome into the host microorganism, wherein the linear DNA fragment contains a mutant flhD gene and / or a mutant gabD gene, and obtaining the correct recombinant genetically engineered strain through resistance screening and genotype verification.

[0016] Preferably, the method for preparing the linear DNA fragment includes: using a plasmid containing the mutant flhD gene and / or the mutant gabD gene as a template, and performing PCR amplification using primers shown in SEQ ID NO.5-SEQ ID NO.8 and / or SEQ ID NO.9-SEQ ID NO.12; the nucleic acid sequence of the mutant flhD gene includes the sequence shown in SEQ ID NO.1, and the nucleic acid sequence of the mutant gabD gene includes the sequence shown in SEQ ID NO.2.

[0017] SEQ ID NO.1 (nucleotide sequence of the flhD mutant gene): atgcatacctccgagttgctgaaacacatttatgacatcaacttgtcatatttactacttgcacagcgtttgattgttcaggacaaagcgtccgctatgtttcgtctcggcataaatgaagaaatggcgacaacgttagcggcactgactcttccgcaaatggttaagctggcaga aaccaatcaactggtttgtcacttccgttttgacagccaccagacgattactcagttgacgcaagattcccgttttgacgatctccagcaaattcataccggcatgctctcaacacgcttgctgaatgatgttaatcagcctgaagaagcgctgcgcaagaaaagggcctga.

[0018] SEQ ID NO.2 (nucleotide sequence of the gabD mutant gene):

[0019] SEQ ID NO.3 (FlhD mutant protein sequence): MHTSELLKHIYDINLSYLLLAQRLIVQDKASAMFRLGINEEMATTLAALTLPQMVKLAETNQLVCHFRFDSHQTITQLTQDSRFDDLQQIHTGIMLSTRLLNDVNQPEEALRKKRA。

[0020] SEQ ID NO.4 (GabD mutant protein sequence): MKLNDSNLFRQQALINGEWLDANNGEAIDVTNPANGDKLGSVPKMGADETRAAIDAANRALPAWRALTAKERATILRNWFNLMMEHQDDLARLMTLEQGKPLAEAKGEISYAASFIEWFAEEGKRIYGDPIPGHQADKRLIVIKQPIGVTAAITPWNFPAAMITRKAGPALAAGCTMVLKPASQTPFSALALAELAIRAGVPAGVFNVVTGSAGAVGNELTSNPLVRKLSFTGSTEIGRQLMEQCAKDIKKVSLELGGNAPFIVFDDADLDKAVEGALASKFRNAGQTCVCANRLYVQDGVYDRFAEKLQQAVSKLHIGDGLDNGVTIGPLIDEKAVAKVEEHIADALEKGARVVCGGKAHERGGNFFQPTILVDVPANAKVSKEETFGPLAPLFRFKDEADVIAQANDTEFGLAAYFYARDLSRVFRVGEALEYGIVGINTGIISNEVAPFGGIKASGLGREGSKYGIEDYLEIKYMCIGL。

[0021] SEQ ID NO.5 (PflhD-F-1): GACATCACGGGGTGCGGTGAAAC。

[0022] SEQ ID NO.6 (PflhD-R-1): GGGTGATCAATTCCATTGCCAGCTGA。

[0023] SEQ ID NO.7 (PflhD-F-2): AACCGCATAAAAATAAAGTTGGTTATTCTGGGTGGGAATAATGCATACCTCCGAGTTGCTGAAACA。

[0024] SEQ ID NO. 8 (PflhD-R-2): ATATCCCGCGCTTCCTGAACAATGCTTTTTTCACTCATGATCAGGCCCTTTTTCTTGCGCAG.

[0025] SEQ ID NO. 9 (PgabD-F-1): GGCACGTAGTGTGGATGCCTTACAC.

[0026] SEQ ID NO. 10 (PgabD-R-1): CCACGGGGAATCGCCTGACTG.

[0027] SEQ ID NO. 11 (PgabD-F-2): GCCGCATTTAATCAATAACCTTTGAAAACAGGATGTAGCGATGAAACTTAACGACAGTAACTTATTCC.

[0028] SEQ ID NO. 12 (PgabD-R-2): GCTGCATTAACTCTTTATTGCTGTTCATTCGCATTCTCCAGTTAAAGACCGATGCACATATATTTGATTTCTA.

[0029] Preferably, the host microorganism includes Escherichia coli.

[0030] Thirdly, the present invention provides the application of the genetically engineered strain of high L-tryptophan production described in the first aspect in the production of L-tryptophan.

[0031] Fourthly, the present invention provides a method for producing L-tryptophan, the method comprising: inoculating the genetically engineered strain of high L-tryptophan production described in the first aspect into a seed culture medium for cultivation to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium for fermentation culture; and recovering L-tryptophan from the fermentation broth after the fermentation culture is completed.

[0032] Preferably, the fermentation culture temperature is 35-38℃ (e.g., 35℃, 36℃ or 38℃), the stirring speed is 150-250 rpm (e.g., 150 rpm, 200 rpm, 250 rpm), and the culture time is 20-28 hours (e.g., 20 hours, 24 hours, 28 hours).

[0033] Compared with the prior art, the present invention has the following beneficial effects: (1) Significant increase in yield: By introducing the V84F mutation of the mutant flhD gene and the T130P point mutation of the mutant gabD gene, the L-tryptophan synthesis pathway was synergistically enhanced. The L-tryptophan yield of the double mutant strain was increased by 21.9% compared with the original strain, which is far more effective than the single gene mutation. (2) Novel mechanism: This invention is the first to discover and verify that the V84F point mutation of the mutant flhD gene encoded protein and the T130P point mutation of the mutant gabD gene encoded protein can work synergistically. By optimizing the supply of precursors and the allocation of metabolic flux, the production bottleneck can be broken. (3) Strong universality: This mutation strategy is based on the regulation of core metabolic nodes. It is not only applicable to Escherichia coli, but also provides new targets and clear technical inspiration for metabolic modification in other L-tryptophan producing bacteria (such as Corynebacterium glutamicum). (4) Good prospects for industrial application: The recombinant strain described in this invention has a fast growth rate, a clear genetic background, and is easy to carry out industrial scale-up culture. Attached Figure Description

[0034] Figure 1 XJFFtrp-2-flhD V84F Graph showing strain construction and validation; Figure 2 XJFFtrp-2-gabD T130P Graph showing strain construction and validation; Figure 3 XJFFtrp-2-gabD T130P -flhD V48F Graph showing strain construction and validation. Detailed Implementation

[0035] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0036] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0037] According to existing literature, knocking out the ptsI and ptsG genes encoding the glucose-specific phosphotransferase system (PTS) in *E. coli*, introducing the glucose-promoted diffusion transporter gene glf and the glucokinase gene glk from *Fermentomonas motilityis*, and overexpressing the tryptophan operon gene trpEDCBA (with trpGD and trpCF fused) can enable *E. coli* to produce a certain amount of tryptophan. In this invention, strains XJFFtrp-2 and XJFFtrp-3 are strains with a certain tryptophan production capacity. These two tryptophan-producing strains were derived from *E. coli* MG1655. Strain XJFFtrp-2 was obtained by knocking out the ptsI and ptsG genes in *E. coli* MG1655 and introducing the glf and glk genes from *Fermentomonas motilityis*; strain XJFFtrp-3 was obtained by overexpressing the tryptophan operon gene trpEDCBA in *E. coli* MG1655.

[0038] The specific sequences of the primers in the following examples are as follows: SEQ ID NO. 13 (Pkan-F-1): CCGGGTTCTTTTAAAAATCAGTCACAAGTAAGGTAGGGTTAGGCTGGAGCTGCTTC.

[0039] SEQ ID NO. 14 (Pkan-R-1):GATCTTCTCCTAAGCAGTAAATTGGGCCGCATCTCGTGGATCCGGGGATCCGTCGACC.

[0040] SEQ ID NO. 15 (Pglf-F-1): CCGGGTTCTTTTAAAAATCAGTCACAAGTAAGGTAGGGTTATGCGATTTTGTGTTGTTGGGG.

[0041] SEQ ID NO. 16 (Pglf-R-1): GATCTTCTCCTAAGCAGTAAATTGGGCCGCATCTCGTGGACTATAATGGATCAACAAAAAATGATGGC.

[0042] SEQ ID NO. 17 (Pkan-F-2): GAACGTAAAAAAAGCACCCATACTCAGGAGCACTCTCAATTAGGCTGGAGCTGCTTC.

[0043] SEQ ID NO. 18 (Pkan-R-2): GCAGCCATCTGGCTGCCTTAGTCTCCCCAACGTCTTACGGATCCGGGGATCCGTCGACC.

[0044] SEQ ID NO. 19 (Pglk-F-1): GAACGTAAAAAAAGCACCCATACTCAGGAGCACTCTCAATTATGGAAATTGTTGCGATTGACATC.

[0045] SEQ ID NO. 20 (Pglk-R-1): GCAGCCATCTGGCTGCCTTAGTCTCCCCAACGTCTTACGGATTATTCAACTTCAGAATATTTGTTGGCAT.

[0046] SEQ ID NO. 21 (Pkan-F-3): AACCGCATAAAAATAAAGTTGGTTATTCTGGGTGGGAATAAGGCTGGAGCTGCTTC.

[0047] SEQ ID NO. 22 (Pkan-R-3): ATATCCCGCGCTTCCTGAACAATGCTTTTTTCACTCATGATCCGGGGATCCGTCGACC.

[0048] SEQ ID NO. 23 (Pkan-F-4): GCCGCATTTAATCAATAACCTTTGAAAACAGGATGTAGCGAGGCTGGAGCTGCTTC.

[0049] SEQ ID NO. 24 (Pkan-R-4): GCTGCATTAACTCTTTATTGCTGTTCATTCGCATTCTCCAGTCCGGGGATCCGTCGACC.

[0050] SEQ ID NO. 25 (PpCP20-F): ATGTCTGAATTAGTTGTTTTCAAAGCAAATGAAC.

[0051] SEQ ID NO. 26 (PpCP20-R): GATCCTTCCGTATTTAGCCAGTATGTTCT.

[0052] Example 1 This embodiment describes the construction of L-tryptophan-containing chassis strain XJFFtrp-2 and its recombinant strain.

[0053] (1) Construction of L-tryptophan chassis strain XJFFtrp-2 In Escherichia coli, modifying the existing PTS system by introducing the exogenous glf and glk genes can enhance the ability of E. coli to produce tryptophan. In this example, a strain capable of producing L-tryptophan was first constructed. The ptsI gene in the E. coli MG1655 strain was knocked out using homologous recombination and replaced with the glf gene from *Fermentomonas motilityis*. The specific steps are as follows: Competent cells of *E. coli* MG1655 were prepared. The pKD46 plasmid was induced into competent *E. coli* MG1655 cells using arabinose stock solution, and the cells were cultured and screened in an ampicillin-resistant medium. Homologous arms were selected at both ends of the target gene. Primers Pkan-F-1 / Pkan-R-1, designed using plasmid PKD13 as a template, were used for PCR amplification of the homologous arm of the kanamycin resistance gene *kan*. The purified PCR product was electroporated into a strain containing the pKD46 plasmid to obtain the pKD46-kan strain. Homologous recombination occurred during culture at 30℃, yielding a homologous recombination strain. The pKD46 temperature-sensitive plasmid was removed by culture at 37℃, and the strain was then transformed with the pCP20 plasmid. PCR amplification using the 0-F / PpCP20-R primer pair was used to verify the expression of the invertase recombinase gene, promoting homologous recombination at the FRT site, ultimately achieving ptsI gene knockout. The strain was simultaneously cultured in LB medium and kanamycin-resistant medium. Strains that grew normally in LB medium but not in kanamycin-resistant medium were considered successfully knocked-out strains. Finally, the pCP20 temperature-sensitive plasmid was removed by incubation at 42°C. Sequencing confirmed the ptsI gene knockout, and the strain was named MG1655-△ptsI. Using the nucleotide sequence of the glf gene (Gene ID: 33073478) from *F. motile fermentum* as a template, homologous arm primers Pglf-F-1 / Pglf-R-1 were designed to construct the PCR amplification product of the exogenous glf gene. This PCR amplification product was transformed into *E. coli* MG1655-△ptsI, and homologous recombination replaced the target region, ultimately achieving glf gene knock-in. Sequencing confirmed the successful acquisition of strain MG1655-△ptsI-glf.

[0054] Following the above method, using Pkan-F-2 / Pkan-R-2 as primers, the ptsG gene in the MG1655-△ptsI strain was knocked out via homologous recombination. Final sequencing verification yielded the MG1655-△ptsI-△ptsG-glf strain. Using the glk nucleotide sequence (Gene ID: 33073808) from *Fermentomonas motilityis* as a template, homologous arm primers Pglk-F-1 / Pglk-R-1 were designed to introduce exogenous glk into the MG1655-△ptsI-△ptsG-glf strain, resulting in the MG1655-△ptsI-△ptsG-glf-glk strain, named XJFFtrp-2, which possesses a certain tryptophan production capacity.

[0055] (2)XJFFtrp-2-flhD V84F Construction of recombinant strains Referring to the method described in Example 1 (1), using Pkan-F-3 / Pkan-R-3 as primer pair, the flhD gene was knocked out by homologous recombination. PCR was performed using primer pair PflhD-F-1 / PflhD-R-1 to verify whether the target gene was successfully removed, resulting in the flhD knockout strain XJFFtrp-2-△flhD. Using PflhD-F-2 / flhD-R-2 as primer pair, the PCR amplification product was transformed into Escherichia coli XJFFtrp-2-△flhD, and the target region was replaced by homologous recombination. The specific implementation method was the same as above, ultimately achieving the knock-in of the flhD mutant gene. PCR verification of the above recombinant strain was performed using primer pair PflhD-F-1 / PflhD-R-1, and the verification results are as follows. Figure 1 As shown, Figure 1 In the diagram, M represents the DNA marker, lane E1 shows the amplification results of the XJFFtrp-2-△flhD strain, and lane E2 shows the validation results of the flhD mutant gene knock-in. Sequencing analysis was performed, and the sequencing results are shown in SEQ ID NO.1, confirming the successful acquisition of the V84F mutant strain encoding the flhD mutant gene protein, named XJFFtrp-2-flhD. V84F .

[0056] (3)XJFFtrp-2-gabD T130P Construction of recombinant strains Referring to the method described in Example 1 (1), using Pkan-F-4 / Pkan-R-4 as the primer pair, the gabD gene in Escherichia coli XJFFtrp-2 was replaced by homologous recombination to knock out the gabD gene. PCR was performed using primer pair PgabD-F-1 / PgabD-R-1 to verify whether the target gene was successfully removed, resulting in the gabD knockout strain XJFFtrp-2-△gabD. Using primer pair PgabD-F-2 / PgabD-R-2, the PCR amplification product was transformed into Escherichia coli XJFFtrp-2-△gabD, ultimately achieving the knock-in of the gabD mutant gene. PCR was performed using primer pair PgabD-F-1 / PgabD-R-1 for verification, and the verification results are as follows. Figure 2 As shown, Figure 2 In the diagram, M represents the DNA marker, lane E1 shows the amplification results of strain XJFFtrp-2-△gabD, and lane E2 shows the validation results of gabD mutant gene knock-in. Sequencing analysis was performed, and the results are shown in SEQ ID NO.2, confirming the successful acquisition of the gabD mutant gene-encoded protein T130P mutant strain XJFFtrp-2-gabD. T130P .

[0057] (4) XJFFtrp-2-gabD T130P -flhD V84F Construction of recombinant strains Referring to the method described in Example 1 (1), using Pkan-F-1 / Pkan-R-1 as the primer pair, homologous recombination was used to replace Escherichia coli XJFFtrp-2-gabD. T130P The flhD gene was knocked out. PCR was performed using primer pair PflhD-F-1 / PflhD-R-1 to verify successful gene removal. Homologous arm primers PflhD-F-2 / flhD-R-2 were designed using the flhD gene mutation sequence as a template to construct the PCR amplification product of the exogenous flhD mutant gene. This PCR amplification product was then transformed into *E. coli*, ultimately achieving the knock-in of the flhD mutant gene. PCR verification was performed using primer pair PflhD-F-1 / PflhD-R-1, and the verification results are as follows: Figure 3 As shown, Figure 3 In the diagram, M represents the DNA marker, lane E1 shows the validation results of the flhD gene knock-in, and lane E2 shows the flhD gene knockout results. The sequencing results are shown in SEQ ID NO.1, confirming the successful acquisition of the double-mutant strain XJFFtrp-2-gabD, which encodes the flhD gene protein V84F mutation and the gabD gene protein T130P mutation. T130P -flhD V84F .

[0058] Example 2 This embodiment describes the construction of the tryptophan-containing chassis strain XJFFtrp-3 and its recombinant strain.

[0059] An overexpression plasmid pTrp containing the trpEDCBA gene (trpE gene Gene ID: 945846, trpD gene Gene ID: 945109, trpC gene Gene ID: 945519, trpB gene Gene ID: 945768 and trpA gene Gene ID: 946204) was synthesized. The pTrp plasmid was electroporated into Escherichia coli MG1655 (electroplation parameters: 2.5 Kv, 5.8 ms). The successfully transformed strain was named XJFFtrp-3, which enabled it to produce tryptophan.

[0060] Based on the primers described in Examples 1(2), (3) and (4), single and double mutations of the flhD and gabD genes were respectively achieved to obtain XJFFtrp-3-flhD. V84F strain, XJFFtrp-3-gabD T130P strain and XJFFtrp-3-gabD T130P -flhD V84F strains.

[0061] Example 3 This embodiment utilizes the strains constructed in Examples 1 and 2 to produce L-tryptophan through fermentation.

[0062] The strains constructed in Examples 1 and 2 were inoculated onto seed culture medium and cultured to obtain seed solutions. The seed culture medium contained the following components at the following concentrations: glucose 60 g / L, yeast extract 2.5 g / L, ammonium sulfate [(NH4)2SO4·7H2O] 20 g / L, MgSO4 1 g / L, KH2PO4 2 g / L, sodium citrate 5 g / L, NaCl 1 g / L, L-tyrosine 0.1 g / L, L-phenylalanine 0.15 g / L, and CaCO3 40 g / L.

[0063] The successfully constructed strains were validated by shake-flask fermentation, with the starting strains XJFFtrp-2 and XJFFtrp-3 serving as controls. They were cultured overnight in LB solid medium. The growing strains were inoculated into 250 mL angle-baffle flasks containing 25 mL of fermentation medium and cultured at 37°C with shaking at 200 rpm for 22 hours. The fermentation medium consisted of 60 g / L glucose, 1 g / L yeast extract, 5 g / L KH₂PO₄, 2 g / L sodium citrate, 2 g / L MgSO₄·7H₂O, 5 g / L (NH₄)₂SO₄, 0.1 g / L MnSO₄·H₂O, 0.1 g / L FeSO₄·7H₂O, 0.1 g / L ZnSO₄·H₂O, 0.1 g / L CoCl₂·6H₂O, 0.03 g / L CuSO₄·5H₂O, and 20 g / L CaCO₃. After cultivation, the L-tryptophan content in the fermentation broth was determined using an amino acid analyzer (Hitachi, model LA8080).

[0064] Method for detecting glucose content in fermentation broth: Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 2 min, take 200 μL of supernatant, add 1800 μL of ddH2O, dilute to 10 times, shake to mix, and measure glucose content using SBA-90 biosensor analyzer.

[0065] The final L-tryptophan yield and conversion rate results are shown in Table 1 below (the yield and conversion rate results for each strain are the average of 3 results). The conversion rate is calculated as follows: Conversion rate = amino acid produced (g / L) / (amount of glucose input (g / L) - residual sugar (g / L)) × 100%.

[0066] Table 1 After valine was replaced by phenylalanine at position 84 of the flhD gene, the L-tryptophan production was significantly increased by 7.7% and 9.3% respectively compared to the original strains XJFFtrp-2 and XJFFtrp-3, and the OD... 600 The values ​​also showed a significant increase; after the threonine at position 130 of the gabD gene amino acid sequence was replaced by proline, the L-tryptophan production was significantly increased by 8.6% and 10.2% respectively compared to the original strains XJFFtrp-2 and XJFFtrp-3, and the OD value was also significantly increased. 600 The values ​​also showed a significant increase; when the flhD V84F mutant gene and the gabD T130P mutant gene were simultaneously introduced into the original strain, the L-tryptophan production was significantly increased by 17.7% and 21.9% respectively compared to the original strains XJFFtrp-2 and XJFFtrp-3, and the OD value was also significantly increased. 600The value is increased even more. This indicates that in the L-tryptophan production process, mutating either the flhD gene or the gabD gene can increase L-tryptophan production, but when both genes are mutated simultaneously, the bacterial cells grow faster, resulting in a greater increase in L-tryptophan production.

[0067] In summary, this invention modifies Escherichia coli by introducing mutation sites in the flhD gene and simultaneously combining them with mutation sites in the gabD gene, thereby increasing the content of glutamate, a precursor substance, within the cells and significantly improving the production of L-tryptophan, providing a new approach for the industrial production of L-tryptophan.

[0068] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A genetically engineered strain that produces high levels of L-tryptophan, characterized in that, The genetically engineered strain expresses mutant FlhD protein and / or mutant GabD protein; The 84th amino acid of the mutant FlhD protein is phenylalanine. The mutant GabD protein has proline at position 130.

2. The genetically engineered strain with high L-tryptophan production according to claim 1, characterized in that, The amino acid sequence of the mutant FlhD protein includes the sequence shown in SEQ ID NO.3; Preferably, the amino acid sequence of the mutant GabD protein includes the sequence shown in SEQ ID NO.

4.

3. The genetically engineered strain with high L-tryptophan production according to claim 1 or 2, characterized in that, The genetically engineered strains include recombinant Escherichia coli engineered strains.

4. A method for constructing a genetically engineered strain that produces high levels of L-tryptophan according to any one of claims 1-3, characterized in that, The construction method includes: using homologous recombination technology, integrating the mutant flhD gene and / or the mutant gabD gene into the genome of the host microorganism, replacing its corresponding wild-type gene, thereby obtaining the genetically engineered strain that produces high levels of L-tryptophan.

5. The construction method according to claim 4, characterized in that, The homologous recombination technology includes: expressing λ-Red recombinase in a host microorganism, electroporating a linear DNA fragment with homologous arms at both ends to the target site in the genome into the host microorganism, wherein the linear DNA fragment contains a mutant flhD gene and / or a mutant gabD gene, and obtaining the correct recombinant genetically engineered strain through resistance screening and genotype verification.

6. The construction method according to claim 4 or 5, characterized in that, The method for preparing the linear DNA fragment includes: using a plasmid containing the mutant flhD gene and / or the mutant gabD gene as a template, and performing PCR amplification using primers shown in SEQ ID NO.5-SEQ ID NO.8 and / or SEQ ID NO.9-SEQ ID NO.12; the nucleic acid sequence of the mutant flhD gene includes the sequence shown in SEQ ID NO.1, and the nucleic acid sequence of the mutant gabD gene includes the sequence shown in SEQ ID NO.

2.

7. The construction method according to any one of claims 4-6, characterized in that, The host microorganisms include Escherichia coli.

8. The use of the genetically engineered strain with high L-tryptophan production according to any one of claims 1-3 in the production of L-tryptophan.

9. A method for producing L-tryptophan, characterized in that, The method includes the following steps: inoculating the genetically engineered strain of high L-tryptophan production according to any one of claims 1-3 into a seed culture medium for cultivation to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium for fermentation culture; and recovering L-tryptophan from the fermentation broth after the fermentation culture is completed.

10. The method according to claim 9, characterized in that, The fermentation culture was carried out at a temperature of 35-38℃, a stirring speed of 150-250 rpm, and a culture time of 20-28 hours.

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