Genetically engineered bacteria with high yield of l-tryptophan, and construction method and application thereof
By inactivating or weakening the activity of cysteine synthase A in Escherichia coli and overexpressing the tryptophan operon gene, the serine-cysteine synthesis pathway is blocked, solving the problem of insufficient L-tryptophan production in traditional methods and achieving efficient tryptophan production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINJIANG FUFENG BIOTECH
- Filing Date
- 2026-05-08
- Publication Date
- 2026-07-03
AI Technical Summary
In existing technologies, traditional L-tryptophan production methods suffer from insufficient yield, especially in Escherichia coli, where the activity of cysteine synthase A affects the synthesis efficiency of tryptophan.
By inactivating or weakening the activity of cysteine synthase A in Escherichia coli, the metabolic pathway of serine synthesis into cysteine is blocked, and the tryptophan operon genes trpE, trpD, trpC, trpB, and trpA are overexpressed to optimize the tryptophan synthesis pathway and enhance tryptophan production.
It increased the yield of L-tryptophan in Escherichia coli by 8.7% compared to the original strain, achieving more efficient tryptophan production.
Smart Images
Figure CN122326499A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to a genetically engineered bacterium that produces high levels of L-tryptophan, its construction method, and its applications. Background Technology
[0002] L-Tryptophan is an important aromatic amino acid with diverse physiological functions in organisms and wide applications in food, medicine, and feed. Tryptophan is one of the essential amino acids that humans and animals cannot synthesize themselves and must obtain through diet or feed. With the development of the food, pharmaceutical, and feed industries, the market demand for tryptophan continues to grow. Traditional tryptophan production methods mainly rely on chemical synthesis and microbial fermentation, with microbial fermentation gaining significant attention due to its environmental friendliness and cost-effectiveness. Various bacteria can be used in L-Tryptophan production, such as wild-type mutant strains induced from Escherichia coli and Corynebacterium spp. as production strains. With the increasing global demand for L-Tryptophan, the construction and modification of high-yield L-Tryptophan strains are particularly important. Optimizing the tryptophan synthesis pathway, relieving feedback inhibition, increasing precursor supply, and enhancing product secretion through genetic engineering of production strains are currently effective methods for producing L-Tryptophan.
[0003] In E. coli cells, tryptophan synthesis begins with the central metabolite clade acid, and proceeds through five enzymatic reactions to produce tryptophan. First, clade acid is catalyzed by anthranilate synthase to produce anthranilic acid (ANTA), which is then catalyzed by anthranilate phosphoribosyltransferase to produce phosphoribosylanthranilic acid (PRAT). PRAT is then converted to carboxyphenylaminodeoxyribulose phosphate (CdRP) by PRAT isomerase. CdRP is then converted to indole-3-glycerophosphate (IGP) by indole-3-glycerophosphate synthase. Finally, IGP is cleaved to produce indole, which condenses with serine to produce tryptophan.
[0004] Cysteine synthase A (CysK) is a key enzyme in the cysteine synthesis pathway. In cysK gene knockout mutants, L-cysteine production is reduced to about 50% of that in wild-type strains. Overexpression of genes such as cysK in *E. coli* significantly increases L-cysteine production. Cysteine synthase A is primarily responsible for the binding of O-acetylserine with sulfides to generate cysteine, a reaction that is the final step in the serine-to-cysteine metabolic pathway. Simultaneously, serine is also a key substrate in the final step of the tryptophan synthesis pathway, participating in the final reaction where indole condenses with serine to form tryptophan. Therefore, the intracellular serine concentration in *E. coli* cells affects the final tryptophan production and is crucial for tryptophan synthesis. Summary of the Invention
[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a genetically engineered bacterium that produces high levels of L-tryptophan, along with its construction method and applications. This invention enhances tryptophan production by inactivating the cysK gene, blocking the serine-cysteine synthesis metabolic pathway, thereby allowing more serine to participate in the tryptophan synthesis metabolic pathway.
[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a recombinant strain that produces high levels of L-tryptophan, the recombinant strain being obtained by genetic modification of a starting strain, wherein the activity of cysteine synthase A in the recombinant strain is weakened or inactivated compared to the starting strain; the cysteine synthase A gene in the recombinant strain is partially or completely knocked out, and the amino acid sequence encoded by the cysteine synthase A gene includes the sequence shown in SEQ ID NO.1.
[0007] This invention enhances tryptophan production by inactivating the cysK gene, blocking the metabolic pathway of serine to cysteine, and allowing more serine to participate in the tryptophan synthesis pathway. The tryptophan production of the recombinant strain is 2.25 g / L, which is 8.7% higher than that of the original strain.
[0008] In this invention, the weakening or inactivation of cysteine synthase A activity can be achieved through any one or a combination of at least two of the following methods: partial or complete knockout of the cysK gene, frameshift mutation of the cysK gene, introduction of a stop codon in the cysK gene, or point mutation in the coding region of the cysK gene.
[0009] Preferably, the cysteine synthase A gene includes the cysK gene or its homolog, and the nucleic acid sequence of the cysK gene includes the sequence shown in SEQ ID NO.2.
[0010] SEQ ID NO.1: MSKIFEDNSLTIGHTPLVRLNRIGNGRILAKVESRNPSFSVKCRIGANMIWDAEKRGVLKPGVELVEPTSGNTGIALAYVAAARGYKLTLTMPETMSIERRKLLKALGANLVLTEGAKGMKGAIQKAEEIVASNPEKYLLLQQFSNPANPEIHEKTTGPEIWEDTDGQVDVFIAGVGTGGTLTGVSRYIKGTKGKTDLISVAVEPTDSPVIAQALAGEEIKPGPHKIQGIGAGFIPANLDLKLVDKVIGITNEAISTARRLMEEEGILAGISSGAAVAAALKLQEDESFTNKNIVVILPSSGERYLSTALFADLFTEKELQQ.
[0011] SEQ ID NO.2: ATGAGTAAGATTTTTGAAGATAACTCGCTGACTATCGGTCACACGCCGCTGGTTCGCCTGAATCGCATCGGTAACGGACGCATTCTGGCGAAGGTGGAATCTCGTAACCCCAGCTTCAGCGTTAAGTGCCGTATCGGTGCCAACATGATTTGGGATGCCGAAAAGCGCGGCGTGCTGAAACCAGGCGTTGAACTGGTTGAACCGACCAGCGGTAATACCGGGATTGCACTGGCCTATGTAGCTGCCGCTCGCGGTTACAAACTCACCCTGACCATGCCAGAAACCATGAGTATTGAACGCCGCAAGCTGCTGAAAGCGTTAGGTGCAAACCTGGTGCTGACGGAAGGTGCTAAAGGCATGAAAGGCGCAATCCAAAAAGCAGAAGAAATTGTCGCCAGCAATCCAGAGAAATACCTGCTGCTGCAACAATTCAGCAATCCGGCAAACCCTGAAATTCACGAAAAGACCACCGGTCCGGAGATATGGGAAGATACCGACGGTCAGGTTGATGTATTTATTGCTGGCGTTGGGACTGGCGGTACGCTGACTGGCGTCAGCCGCTACATTAAAGGCACCAAAGGCAAGACCGATCTTATCTCTGTCGCCGTTGAGCCAACCGATTCTCCAGTTATCGCCCAGGCGCTGGCAGGTGAAGAGATTAAACCTGGCCCGCATAAAATTCAGGGTATTGGCGCTGGTTTTATCCCGGCTAACCTCGATCTCAAGCTGGTCGATAAAGTCATTGGCATCACCAATGAAGAAGCGATTTCTACCGCGCGTCGTCTGATGGAAGAAGAAGGTATTCTTGCAGGTATCTCTTCTGGAGCAGCTGTTGCCGCGGCGTTGAAACTACAAGAAGATGAAAGCTTTACCAACAAGAATATTGTGGTTATTCTACCATCATCGGGTGAGCGTTATTTAAGCACCGCATTGTTTGCCGATCTCTTCACTGAGAAAGAATTGCAACAGTAA。
[0012] In one embodiment of the present invention, the recombinant strain overexpresses any one or a combination of at least two of the tryptophan operon genes trpE, trpD, trpC, trpB, or trpA.
[0013] In one embodiment of the present invention, the ptsI gene in the recombinant strain is knocked out and replaced with the glf gene from *Fermentomonas motilityis*, and / or the ptsG gene in the recombinant strain is knocked out and replaced with the glk gene from *Fermentomonas motilityis*.
[0014] In this invention, the NCBI Gene IDs corresponding to each target gene (glf gene, glk gene, trpE gene, trpD gene, trpC gene, trpB gene, trpA gene) are as follows: Gene ID: 33073478, Gene ID: 33073808, Gene ID: 945846, Gene ID: 945109, Gene ID: 945519, Gene ID: 945768, Gene ID: 946204.
[0015] Preferably, the starting strain includes Escherichia coli.
[0016] In a second aspect, the present invention provides a method for constructing the recombinant strain described in the first aspect, the method comprising: genetically modifying the starting strain to weaken or inactivate the cysteine synthase A activity in the starting strain; the method for weakening or inactivating the cysteine synthase A activity in the starting strain includes any one of homologous recombination, CRISPR / Cas9, transposon mutation or chemical mutagenesis, wherein the amino acid sequence encoded by the cysteine synthase A activity gene includes the sequence shown in SEQ ID NO.1.
[0017] Preferably, the cysteine synthase A gene includes the cysK gene or its homolog, and the nucleic acid sequence of the cysK gene includes the sequence shown in SEQ ID NO.2.
[0018] Thirdly, the present invention provides a method for producing L-tryptophan, the method comprising: culturing the recombinant strain described in the first aspect to obtain a fermentation broth; and recovering L-tryptophan from the fermentation broth.
[0019] Preferably, the culture is carried out in a fermentation medium containing a carbon source and a nitrogen source, wherein the carbon source includes glucose.
[0020] Preferably, the fermentation cycle of the culture is 20-25 hours (e.g., 20 hours, 22 hours or 25 hours).
[0021] Fourthly, the present invention provides the application of the recombinant strain described in the first aspect or the recombinant strain constructed by the method described in the second aspect in the production of L-tryptophan.
[0022] Fifthly, the present invention provides the use of the recombinant strain described in the first aspect or the recombinant strain constructed by the method described in the second aspect in the preparation of products containing L-tryptophan.
[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a recombinant strain with high tryptophan production, wherein the tryptophan production of the recombinant strain is 2.25 g / L, which is 8.7% higher than that of the original strain; (2) This invention confirms that the inactivation of the cysK gene in the recombinant strain will block the metabolic pathway of serine to cysteine, allowing more serine to participate in the tryptophan synthesis metabolic pathway, thereby enhancing the production of tryptophan. Attached Figure Description
[0024] Figure 1 This is a diagram illustrating the principle of homologous recombination gene knockout. Detailed Implementation
[0025] 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.
[0026] 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.
[0027] The specific sequences of the primers used in the following examples are as follows: SEQ ID NO. 3 (Pkan-F-1): ATCCCAATTTCATACAGTTAAGGACAGGCCTACCGTTCGTATAGCATACATTATACGAAGTTATAGGAAGCGGAACACGTAGAA.
[0028] SEQ ID NO. 4 (Pkan-R-1): GCTTTTTACGCATTTTTAACAAGCTGGCATACCGTTCGTATAATGTATGCTATACGAAGTTATTTAGAAGAACTCGTCAAGAAG.
[0029] SEQ ID NO.5(Pglf-F):ATGCGATTTTGTGTTGTTGGG。
[0030] SEQ ID NO.6(Pglf-R):CTCCTAAGCAGTAAATTGGGCCGCATCTCGTGGACTATAATGGATCAACAAAAAATGGC。
[0031] SEQ ID NO.7(Pkan-F-2):AAAATCAGTCAAGTAAGGTAGGGTTTACCGTTCGTATAGCATACATTATACGAAGTTATAGGAAGCGGAACACGTAGAA。
[0032] SEQ ID NO.8(Pkan-R-2):AAAGCCAGCCCCAACAACACAAATTCGCATTACCGTTCGTATAATGTATGCTATACGAAGTTATTTAGAAACTCGTCAAGAAGG。
[0033] SEQ ID NO.9(Pglk-F):ATGGAAATTGTTGCGATTGACATC。
[0034] SEQ ID NO.10(Pglk-R):GGCTGCCTTAGTCTCCCCAACGTCTTACGGATTATTCAACTTCAGAATATTTGTTGGCAT。
[0035] SEQ ID NO.11(Pkan-F-3):CACCCATACTCAGGAGCACTCTCAATTTACCGTTCGTATAGCATACATTATACGAAGTTATAGGAAGCGGAACACGTAGAA。
[0036] SEQ ID NO.12(Pkan-R-3):TCCACCGATGTCAATCGCAACAATTTCCATTACCGTTCGTATAATGTATGCTATACGAAGTTATTTAGAAACTCGTCAAGAAGG。
[0037] SEQ ID NO.13(PcysK):CCGTTTGTGTGAAACAGGG。
[0038] SEQ ID NO.14(PcysK):TTCCGACAGGCTGAATCAATG。
[0039] Example 1 Construction of XJFFtrp-3 and its recombinant strains.
[0040] (1) Construction of strain XJFFtrp-3 In this embodiment, a strain capable of producing L-tryptophan was first constructed. 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, thus enabling it to produce a certain amount of tryptophan.
[0041] (2) Construction of strain XJFFtrp-3-△cysK Homologous arms were selected at both ends of the target gene. Primers Pkan-F-1 / Pkan-R-1, containing homologous arms of the kan gene with lox71 / lox66 sites, were designed using plasmid PKD13 as a template. PCR amplification was performed to obtain the kan fragment containing homologous arms and lox71 / lox66 sites. *E. coli* XJFFtrp-3 competent cells were prepared, and the pKD46 plasmid was electroporated into *E. coli* XJFFtrp-3. The resulting XJFFtrp-3-pKD46 strain was cultured at 30℃. Competent cells of the XJFFtrp-3-pKD46 strain were prepared, and the PCR product was electroporated into the XJFFtrp-3-pKD46 strain. Homologous recombination was induced by adding 100 mg / mL arabinose inducer to the culture medium. The strain was cultured at 37℃ to remove the pKD46 temperature-sensitive plasmid, plated on kan resistance plates, and screened. Subsequently, the pSC101-cre plasmid was transformed into the correctly validated strain to express the flip recombinase gene, promoting site-specific recombination at the lox71 / lox66 loci, ultimately achieving cysK gene knockout. The strain was simultaneously cultured in LB medium and kanamycin-resistant medium. Strains that grew normally in LB medium but not in the resistant medium were considered to have successfully knocked out the cysK gene. Finally, the strain was cultured at 37°C to remove the temperature-sensitive pSC101-cre plasmid, thus achieving the knockout of the cysK gene (SEQ ID NO.2) and inactivating it. PCR was performed using primer pairs PcysK-F / PcysK-R to verify the successful removal of the target gene. The verification results are as follows: Figure 1As shown, M is the DNA marker, E1 is the cysK gene amplification result, and E2 is the cysK gene knockout verification result. The strain with partial cysK gene knockout was named XJFFtrp-3-△cysK.
[0042] Example 2 Construction of XJFFtrp-4 and its recombinant strains.
[0043] (1) Construction of strain XJFFtrp-4 Primers Pglf-F / Pglf-R and Pkan-F-2 / Pkan-R-2 were designed using the glf nucleotide sequence (Gene ID: 33073478) from *C. molybdenum* and the PKD13 plasmid as templates. The kan-glf fragment with lox71 / lox66 sites was amplified using the primers. Following the method described in Example 1, the target region was replaced by homologous recombination, and the exogenous glf gene was finally introduced to replace the ptsI gene in *E. coli*. Sequencing verification yielded the MG1655-△ptsI-glf strain.
[0044] Primers Pglk-F / Pglk-R and Pkan-F-3 / Pkan-R-3 were designed using the glk nucleotide sequence (Gene ID: 33073808) from *Bacillus muscularis* and the PKD13 plasmid as templates. The kan-glk fragment with lox71 / lox66 sites was amplified using the above primers. According to the method described in Example 1, the ptsG gene in the MG1655-△ptsI-glf strain was replaced with the glk gene by homologous recombination. After final sequencing verification, the MG1655-△ptsI-△ptsG-glf-glk strain was obtained and named XJFFtrp-4, which enabled it to have a certain tryptophan production capacity.
[0045] (2) Construction of strain XJFFtrp-4-△cysK Referring to the method and primers described in Example 1 (2), the cysK gene in strain XJFFtrp-4 was knocked out to inactivate the cysK gene. The target gene was verified by PCR using primer pair PcysK-F / PcysK-R to determine whether the removal of the target gene was successful. A strain with partial cysK gene sequence knockout was obtained and named FFtrp-4-△cysK.
[0046] Example 3 L-tryptophan was produced by fermentation using recombinant strains constructed in Examples 1 and 2.
[0047] The recombinant *E. coli* strains obtained in the above examples were inoculated onto seed culture medium and cultured to obtain seed culture. 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, magnesium sulfate (MgSO4) 1 g / L, potassium dihydrogen phosphate (KH2PO4) 2 g / L, sodium citrate 5 g / L, sodium chloride (NaCl) 1 g / L, L-tyrosine 0.1 g / L, L-phenylalanine 0.15 g / L, and calcium carbonate (CaCO3) 40 g / L.
[0048] The successfully constructed strains were validated by shake-flask fermentation, with the starting strains XJFFtrp-3 and XJFFtrp-4 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).
[0049] 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.
[0050] 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%.
[0051] Table 1 As shown in Table 1, knocking out the cysK gene in *E. coli* significantly increased L-tryptophan production compared to the original strains XJFFtrp-3 and XJFFtrp-4, by 8.5% and 8.7%, respectively, and the OD...600 The elevated value indicates that during the production of L-tryptophan in E. coli, the inactivation of the cysK gene blocks the metabolic pathway of serine to cysteine, allowing more serine to participate in the tryptophan synthesis pathway, thereby enhancing tryptophan production.
[0052] In summary, this invention enhances tryptophan production by inactivating the cysK gene, blocking the metabolic pathway of serine to cysteine, and allowing more serine to participate in the tryptophan synthesis metabolic pathway.
[0053] 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 recombinant strain producing L-tryptophan at a high yield, characterized in that, The recombinant strain was obtained by genetic modification of the starting strain. Compared with the starting strain, the recombinant strain has reduced or inactivated cysteine synthase A activity. The cysteine synthase A gene in the recombinant strain is partially or completely knocked out, and the amino acid sequence encoded by the cysteine synthase A gene includes the sequence shown in SEQ ID NO.
1.
2. The recombinant bacterial strain of claim 1, wherein, The cysteine synthase A gene includes the cysK gene or its homolog, and the nucleic acid sequence of the cysK gene includes the sequence shown in SEQ ID NO.
2.
3. The recombinant strain according to claim 1 or 2, characterized in that, The starting strain includes Escherichia coli.
4. A method for constructing the recombinant strain according to any one of claims 1-3, characterized in that, The method includes: genetically modifying the starting strain to weaken or inactivate the cysteine synthase A activity in the starting strain; the method for weakening or inactivating the cysteine synthase A activity in the starting strain includes any one of homologous recombination, CRISPR / Cas9, transposon mutation or chemical mutagenesis, and the amino acid sequence encoded by the cysteine synthase A activity gene includes the sequence shown in SEQ ID NO.
1.
5. The method according to claim 4, characterized in that, The cysteine synthase A gene includes the cysK gene or its homolog, and the nucleic acid sequence of the cysK gene includes the sequence shown in SEQ ID NO.
2.
6. A method for producing L-tryptophan, characterized in that, The method for producing L-tryptophan includes: culturing the recombinant strain according to any one of claims 1-3 to obtain a fermentation broth; and recovering L-tryptophan from the fermentation broth.
7. The method according to claim 6, characterized in that, The culture is carried out in a fermentation medium containing a carbon source and a nitrogen source, wherein the carbon source includes glucose.
8. The method for producing L-tryptophan according to claim 6 or 7, characterized in that, The fermentation cycle for the culture is 20-25 hours.
9. The use of the recombinant strain according to any one of claims 1-3 or the recombinant strain constructed by the method according to claim 4 or 5 in the production of L-tryptophan.
10. The use of the recombinant strain according to any one of claims 1-3 or the recombinant strain constructed by the method according to claim 4 or 5 in the preparation of products containing L-tryptophan.