L-tryptophan-producing recombinant bacterium as well as construction method and application thereof

Abstract

The invention relates to recombinant bacteria for producing L-tryptophan as well as a construction method and application of the recombinant bacteria, and belongs to the technical field of genetic engineering. In the recombinant bacterium, a functional amide phosphoribosyltransferase is deleted, or the functional amide phosphoribosyltransferase and 2-imino propionate deaminase are deleted. According to the method disclosed by the invention, a recombinant bacterium for high-yield tryptophan is constructed, and functional amide phosphoribosyltransferase is deleted in the recombinant bacterium, so that more PRPP and glutamine participate in an anabolic pathway of tryptophan, and a culture mode of supplementing threonine can be matched; according to the tryptophan phosphoribosyltransferase, the problem that a tryptophan synthesis route is lack of PRPP and glutamine and the problem that the cell activity is reduced due to the lack of functional amide phosphoribosyltransferase can be solved at the same time; the main amino supply in the purine synthesis original route is changed from glutamine to 2-aminosuccinic acid as a carrier, so that the tryptophan production capacity can be further improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and relates to an L-tryptophan-producing recombinant bacterium, its construction method, and its application. Background Technology

[0002] L-Tryptophan (hereinafter referred to as tryptophan) is an important aromatic amino acid with various 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, among which microbial fermentation has attracted much attention due to its environmental friendliness and cost-effectiveness. Various bacteria can be used in tryptophan production, such as wild-type mutant strains induced from Escherichia coli and Corynebacterium spp. as production strains. With the continuous increase in global demand for tryptophan, the construction and modification of high-yield tryptophan strains are particularly important.

[0003] Tryptophan is one of the amino acids with the longest metabolic pathway. Microbial tryptophan synthesis is a highly energy-intensive process, requiring large amounts of adenosine triphosphate (ATP), carbon, and nitrogen. Ribose-5-phosphate reacts with ATP under the action of phosphoribosyl pyrophosphate kinase to generate phosphoribosyl pyrophosphate (PRPP), which serves as an important precursor in the tryptophan synthesis pathway. Glutamine provides the amino group for the formation of anthranilic acid, directly participating in the catalytic reaction of key steps, and is also an important precursor in the tryptophan synthesis pathway. Amide phosphoribosyltransferase (PurF) catalyzes the synthesis of PRA, glutamate, and diphosphate from PRPP and glutamine, a process that consumes large amounts of PRPP and glutamine. One of the main modification strategies for high-tryptophan-producing strains is to enhance PRPP and glutamine metabolic fluxes. For example, CN118374425A significantly reduced the translation efficiency of the purF gene by mutating the RBS sequence, thereby reducing the participation of PRPP in purine pathway synthesis and increasing the supply of PRPP precursors for tryptophan biosynthesis, thus increasing tryptophan production. CN120249154A discloses a recombinant microorganism for L-tryptophan production, which weakens the purF gene and heterologously introduces glnA and prs genes at the ycaP gene locus, increasing the amount of tryptophan precursors PRPP and glnA and reducing the amount of byproduct glutamate. However, weakening purF gene expression reduces the intracellular purine synthesis efficiency of E. coli, which in turn affects the synthesis of cellular DNA, RNA, and GTP, resulting in lower cell activity during fermentation and limiting the improvement of tryptophan production.

[0004] In summary, the excessive consumption of PRPP and glutamine during the biosynthesis of tryptophan severely restricts the improvement of tryptophan yield and conversion rate, and is a core bottleneck problem that urgently needs to be solved. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a recombinant L-tryptophan-producing bacterium, its construction method, and its application, aiming to achieve efficient tryptophan production.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a recombinant L-tryptophan-producing bacterium, wherein the recombinant bacterium lacks a functional amide phosphoribosyltransferase, or lacks both a functional amide phosphoribosyltransferase and 2-iminopropionate deaminase.

[0008] This invention employs a novel modification strategy to construct a recombinant bacterium that produces high levels of tryptophan. This recombinant bacterium lacks a functional amide phosphoribosyltransferase, which weakens / blocks the consumption of PRPP and glutamine in the purine synthesis pathway. This allows more PRPP and glutamine to participate in the tryptophan synthesis pathway, complementing a culture method that supplements threonine in the culture medium to replenish the purine synthesis pathway. This not only effectively solves the problem of insufficient PRPP and glutamine in the tryptophan synthesis pathway but also addresses the decreased cell activity caused by the lack of functional amide phosphoribosyltransferase. Furthermore, the simultaneous absence of 2-iminopropionate deaminase prevents 2-aminosuccinate (2-aminocrotonate), generated from threonine metabolism, from participating in the isoleucine synthesis pathway. This changes the primary amino group supply in the original purine synthesis pathway from glutamine to 2-aminosuccinate, regulating metabolic flux and further improving production capacity.

[0009] Preferably, the missing functional amide phosphoribosyltransferase and 2-iminopropionate deaminase include: the gene encoding amide phosphoribosyltransferase (purF gene, SEQ ID NO.2) is missing (knocked out) or its expression is weakened, and the gene encoding 2-iminopropionate deaminase (ridA gene, SEQ ID NO.5) is missing or its expression is weakened.

[0010] In this invention, a strategy for enhancing the tryptophan production capacity of strains is designed. Theoretically, this strategy is applicable to currently known tryptophan-producing strains, such as strains that include the knockout of ptsI and ptsG genes, the exogenous introduction of glf and glk genes from motile fermentation monoclonal bacteria, and overexpression of the trpEDCBA operon.

[0011] Preferably, the starting strain of the L-tryptophan-producing recombinant bacteria includes Escherichia coli.

[0012] Preferably, the Escherichia coli includes Escherichia coli XJFF-151106S or Escherichia coli MG1655, etc.

[0013] Preferably, the L-tryptophan recombinant bacteria also lacks functional phosphoenolpyruvate-protein phosphotransferase and PTS system glucose-specific enzyme, introduces exogenous glucose-promoted diffusion transporter and glucokinase, and overexpresses the tryptophan operon.

[0014] In this invention, the glucose-specific phosphotransferase system (PTS) is weakened and glucose-promoted diffusion transporter and glucokinase are introduced exogenously, and the tryptophan operon is overexpressed to further promote tryptophan synthesis.

[0015] Preferably, the L-tryptophan recombinant bacteria also lack functional phosphoenolpyruvate-protein phosphotransferase and PTS system glucose-specific enzymes, including: deletion of the encoding gene for phosphoenolpyruvate-protein phosphotransferase (ptsI gene) and deletion of the encoding gene for PTS system glucose-specific enzyme (ptsG gene). The exogenous glucose-promoted diffusion transporter gene glf, the glucokinase gene glk, and the tryptophan operon gene trpEDCBA are introduced.

[0016] In a second aspect, the present invention provides a method for constructing the L-tryptophan-producing recombinant bacteria described in the first aspect, the method comprising:

[0017] Remove the bacterial strain and remove the functional amide phosphoribosyltransferase and 2-iminopropionate deaminase from it.

[0018] It is understood that the modification strategy designed based on this invention, as well as technical means that can achieve the corresponding effect, such as gene knockout, are all applicable to this invention and can be designed and selected according to actual needs.

[0019] Preferably, the construction method specifically includes: taking out the bacterial strain and knocking out or weakening the expression of the amide phosphoribosyltransferase encoding gene and the 2-iminopropionate deaminase encoding gene.

[0020] Thirdly, the present invention provides the application of the L-tryptophan-producing recombinant bacteria described in the first aspect in the production of L-tryptophan.

[0021] Fourthly, the present invention provides a method for producing L-tryptophan, the method comprising culturing an L-tryptophan-producing strain, purifying the product, and obtaining L-tryptophan; wherein the culture medium contains threonine.

[0022] In this invention, it was discovered that adding threonine to the culture medium can provide sufficient ATP for tryptophan synthesis, promote tryptophan synthesis, and thus increase the yield and conversion rate of tryptophan.

[0023] Preferably, the L-tryptophan-producing strain includes the L-tryptophan-producing recombinant strain described in the first aspect.

[0024] In this invention, a designed recombinant strain is used to provide nitrogen by adding exogenous threonine to the culture medium. Combined with the modification of the purF and ridA genes, the consumption of PRPP and glutamine during fermentation is reduced while promoting purine synthesis, increasing purine concentration, and avoiding a decrease in cell activity. This can further improve the yield and conversion rate of tryptophan.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects:

[0026] This invention employs a novel modification strategy to reconstruct metabolic flux in recombinant bacteria, reducing / blocking the consumption of PRPP and glutamine in the purine synthesis pathway. This allows more PRPP and glutamine to participate in the tryptophan synthesis pathway, redirecting the original purine synthesis towards threonine supply and replenishing purine synthesis metabolism. Consequently, under the condition of exogenous threonine addition, the recombinant bacteria promote purine synthesis, increase purine concentration, and further improve tryptophan yield and conversion rate. Attached Figure Description

[0027] Figure 1 This is a validation diagram of the purF gene knockout. M: DNA marker; E1: purF gene amplification result; E2: purF gene knockout validation result.

[0028] Figure 2 This is a validation diagram of the ridA gene knockout. M: DNA marker; E1: ridA gene amplification result; E2: ridA gene knockout validation result.

[0029] Figure 3 Figure showing the tryptophan production results of XJFF-151113S and its recombinant strain.

[0030] Figure 4 The graph shows the tryptophan conversion rate of XJFF-151113S and its recombinant strain.

[0031] Figure 5 The graph shows the tryptophan production results of XJFFtrp-1 and its recombinant strain.

[0032] Figure 6 The graph shows the tryptophan conversion rate of XJFFtrp-1 and its recombinant strain. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0034] 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 from legitimate channels.

[0035] This invention employs a novel gene modification strategy to construct a recombinant strain that produces high levels of tryptophan. It weakens or knocks out the expression of the purF gene, blocking the consumption of PRPP and glutamine in the purine synthesis pathway. Simultaneously, by weakening or inactivating the 2-iminopropionate deaminase encoding gene ridA and exogenously supplementing the culture medium with threonine, the 2-aminosuccinic acid generated from threonine metabolism is prevented from participating in the isoleucine synthesis pathway. This changes the primary amino group supply in the original purine synthesis pathway from glutamine to 2-aminosuccinic acid, which acts as a carrier. The synthesis involves 2-aminosuccinic acid, ribose pentaphosphate, and ammonium ions (NH4+). + Together, they participate in the synthesis of ribosamine 5-phosphate (PRA), which is the starting point for purine ring synthesis. During this process, 2-aminosuccinic acid (2-aminobutyric acid) is released as an amino group carrier, and this process is repeated. Exogenous addition of threonine promotes purine synthesis, increasing purine concentration and providing ATP for tryptophan synthesis. This not only effectively solves the problem of insufficient PRPP and glutamine in the tryptophan synthesis pathway but also addresses the decreased cell activity caused by knocking out or weakening the purF gene.

[0036] In this invention, strain XJFF-151113S can be derived from Escherichia coli (such as XJFF-151106S). Strain XJFF-151106S is classified as Escherichia coli, deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 17, 2015, with accession number CGMCC No. 11674, and located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0037] The XJFF-151113S strain had the ptsI and ptsG genes encoding the glucose-specific phosphotransferase system (PTS) knocked out, and the glucose-promoted diffusion transporter gene glf and the glucokinase gene glk were introduced exogenously from *Fermentomonas motilityis*. Furthermore, this strain overexpressed the tryptophan operon gene trpEDCBA (with trpGD and trpCF fused). The tryptophan operon is responsible for tryptophan biosynthesis in *E. coli*. Overexpression of the trpEDCBA gene enhances the enzyme activity of the tryptophan synthesis pathway, strengthening the cladose-to-tryptophan metabolic pathway. The catalysis of trpE and trpD depends on glutamine and PRPP. Overexpression of trpED can fully utilize the surplus glutamine and PRPP resulting from the inactivation of purF, further promoting tryptophan synthesis.

[0038] In this invention, "weakening" refers to a decrease in intracellular enzyme activity compared to the unmodified strain. Weakening occurs when the enzyme's activity is reduced compared to the microorganism's original enzyme activity due to mutations in the gene encoding the enzyme, or when the transcription or translation of the gene encoding the enzyme is inhibited. Enzyme activity decreases due to (translation) inhibition, resulting in an overall intracellular enzyme activity level lower than the unmodified strain; this concept also includes combinations of these situations. Furthermore, in this invention, "inactivation" refers to a state where the enzyme gene is not expressed at all compared to the wild-type strain or the unmodified strain, or even if expressed, it is inactive; or the gene is completely or partially knocked out, rendering it inactive; or the gene is inactivated through frameshift mutations or by introducing a stop codon, causing premature termination of translation. In a specific embodiment of this invention, the purF gene is partially replaced and knocked out using homologous recombination, inactivating amide phosphoribosyltransferase; however, this invention is not limited to this. In this invention, the weakening or inactivation of the ridA gene is achieved by replacing or knocking out the ridA gene through homologous recombination, thereby inactivating 2-iminopropionate deaminase. However, this invention is not limited to this method, and the weakening or inactivation of the ridA gene can also be achieved through other means in the art.

[0039] In this invention, "overexpression" refers to an increase in intracellular enzyme activity compared to the unmodified strain, which is the opposite of the "weakening" mentioned above.

[0040] Example 1

[0041] This embodiment describes the construction of recombinant bacterial strains.

[0042] (1) Construction of XJFF-151113S and its recombinant strain

[0043] The ptsI gene in strain XJFF-151106S was knocked out and replaced with the glf gene from *Cytomonas motile* using homologous recombination. The specific steps are as follows:

[0044] Competent cells of *E. coli* were prepared. The pKD46 plasmid was induced by arabinose stock solution and transformed into competent *E. coli* XJFF-151106S cells. Cells were then 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 culturing at 30℃, yielding a homologous recombination strain. The pKD46 temperature-sensitive plasmid was removed by culturing at 37℃, and the resulting strain was then transformed with the pCP20 plasmid (PpCP20-F). PCR amplification and verification using the / PpCP20-R primer pair expressed the flippant 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. The strain that grew normally in LB medium and did not grow in resistant medium was the strain with successfully knocked-out ptsI gene. Finally, the pCP20 temperature-sensitive plasmid was removed by 42℃ culture, and the ptsI gene knockout strain was obtained by sequencing verification and named XJFF-151106S-△ptsI. Using the nucleotide sequence of the glf gene in *F. motility fermentum* (Gene ID: 33073478) 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. The PCR amplification product was then transformed into *E. coli* XJFF-151106S-△ptsI. The target region was replaced by homologous recombination, and the glf gene was finally knocked in. Sequencing confirmed that the strain XJFF-151106S-△ptsI-glf was successfully obtained.

[0045] Following the above method, using Pkan-F-2 / Pkan-R-2 as primers, the ptsG gene in strain XJFF-151106S-△ptsI was knocked out via homologous recombination. Final sequencing verification yielded strain XJFF-151106S-△ptsI-△ptsG-glf. Using the glk nucleotide sequence (Gene ID: 33073808) from *Fermentation Monotrophus* as a template, homologous arm primers Pglk-F-1 / Pglk-R-1 were designed to introduce exogenous glk into strain XJFF-151106S-△ptsI-△ptsG-glf, resulting in strain XJFF-151106S-△ptsI-△ptsG-glf-glk.

[0046] The company synthesized 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). The pTrp plasmid was electroporated (electroplation parameters: 2.5 Kv, 5.8 ms) into the expression host strain XJFF-151106S-△ptsI-△ptsG-glf-glk). The successfully transformed strain was named XJFF-151113S, which contains the knockout of the ptsI and ptsG genes, as well as the exogenous introduction of the glf and glk genes from *Mammotrophic motility*. This strain also overexpresses the tryptophan operon gene trpEDCBA.

[0047] (2) Construction of purF gene inactivated strain

[0048] Referring to the method described in (1), the purF gene in strain XJFF-151113S was partially knocked out using Pkan-F-3 / Pkan-R-3 primers, resulting in gene inactivation. The knockout sequence is shown in SEQ ID NO.3, totaling 450 bp. PCR was performed using primer pair PpurF-F / PpurF-R to verify whether the target gene was successfully removed. The verification results are as follows: Figure 1 As shown, the strain with partial knockout of the purF gene was obtained and named XJFF-151113S-△purF.

[0049] (3) Construction of the ridA gene knockout strain

[0050] Using the method described in (1) with Pkan-F-4 / Pkan-R-4 as primers, homologous recombination was used to knock out the ridA gene in strain XJFF-151113S. PCR was then performed using primers PridA-F / PridA-R to verify whether the target gene was successfully removed. The verification results are as follows: Figure 2 As shown, the strain with the ridA gene knocked out was verified and named XJFF-151113S-△ridA. Using the primer pairs and methods described above, the ridA gene in the XJFF-151113S-△purF strain was replaced and knocked out, resulting in a strain with the ridA gene knocked out, named XJFF-151113S-△purF-△ridA.

[0051] (4) Construction of XJFFtrp-1 and its recombinant strains

[0052] According to the method described in (1), the ptsI and ptsG genes of Escherichia coli MG1655 were knocked out using the same method and primers, and the glf and glk genes from *Mammotrophic motility* were introduced exogenously. Furthermore, the tryptophan operon gene trpEDCBA was overexpressed in this strain to obtain strain XJFFtrp-1. Based on the primers described in (2) and (3), single and double knockouts of the purF and ridA genes were achieved, resulting in strains XJFFtrp-1-△purF, XJFFtrp-1-△ridA, and XJFFtrp-1-△purF-△ridA.

[0053] The PurF protein sequence in Escherichia coli (SEQ ID NO.1):

[0054] MCGIVGIAGVMPVNQSIYDALTVLQHRGQDAAGIITIDANNCFRLRKANGLVSDVFEARHMQRLQGNMGIGHVRYPTAGSSSASEAQPFYVNSPYGITLAHNGNLTNAHELRKKLFEEKRRHINTT SDSEILLNIFASELDNFRHYPLEADNIFAAIAATNRLIRGAYACVAMIIGHGMVAFRDPNGIRPLVLGKRDIDENRTEYMVASESVALDTLGFDFLRDVAPGEAIYITEEGQLFTRQCADNPVSNPC LFEYVYFARPDSFIDKISVYSARVNMGTKLGEKIAREWEDLDIDVVIPIPETSCDIALEIARILGKPYRQGFVKNRYVGRTFIMPGQQLRRKSVRRKLNANRAEFRDKNVLLVDDSIVRGTTSEQI IEMAREAGAKKVYLASAAPEIRFPNVYGIDMPSATELIAHGREVDEIRQIIGADGLIFQDLNDLIDAVRAENPDIQQFECSVFNGVYVTKDVDQGYLDFLDTLRNDDAKAVQRQNEVENLEMHNEG.

[0055] Nucleotide sequence of the purF gene in Escherichia coli (SEQ ID NO.2):

[0056]

[0057] purF gene knockout sequence (SEQ ID NO.3):

[0058] tttgcgtaaagcgaacgggctggtgagcgatgtatttgaagctcgccatatgcagcgtttgcagggcaatatgggcattggtcatgtgcgttaccccacggctggcagctccagcgcctctgaagcgcagccgttttacgttaactccccgtatggcattacgcttgcccacaacggcaatctgaccaacgctcacgagttgcgtaaaaaactgtttgaagaaaaacgccgccacatcaacaccacttccgactcggaaattctgcttaatatcttcgccagcgagctggacaacttccgccactacccgctggaagccgacaatattttcgctgccattgctgccacaaaccgcttaatccgcggcgcgtatgcctgtgtggcgatgattatcggccacggtatggttgctttccgcgatccaaacgggattcgtccgctggtactggg。

[0059] RidA protein sequence in Escherichia coli (SEQ ID NO.4):

[0060] MSKTIATENAPAAIGPYVQGVDLGNMIITSGQIPVNPKTGEVPADVAAQARQSLDNVKAIVEAAGLKVGDIVKTTVFVKDLNDFATVNATYEAFFTEHNATFPARSCVEVARLPKDVKIEIEAIAVRR。

[0061] Nucleotide sequence of ridA gene in Escherichia coli (SEQ ID NO.5):

[0062] atgagcaaaactatcgcgacggaaaatgcaccggcagctatcggtccttacgtacagggcgttgatctgggcaatatgatcatcacctccggtcagatcccggtaaatccgaaaacgggcgaagtaccggcagacgtcgctgcacaggcacgtcagtcgctggataacgtaaaagcgatcgtcgaagccgctggcctgaaagtgggcgacatcgttaaaactaccgtgtttgtaaaagatctgaacgacttcgcaaccgtaaacgccacttacgaagccttcttcaccgaacacaacgccaccttcccggcacgttcttgcgttgaagttgcccgtctgccgaaagacgtgaagattgagatcgaagcgatcgctgttcgtcgctaa。

[0063] Pkan-F-1(SEQ ID NO.6):

[0064] 5'-CCGGGTTCTTTTAAAAATCAGTCACAAGTAAGGTAGGGTTAGGCTGGAGCTGCTTC-3'。

[0065] Pkan-R-1(SEQ ID NO.7):

[0066] 5'-GATCTTCTCCTAAGCAGTAAATTGGGCCGCATCTCGTGGATCCGGGGATCCGTCGACC-3'。

[0067] Pglf-F-1(SEQ ID NO.8):

[0068] 5'-CCGGGTTCTTTTAAAAATCAGTCACAAGTAAGGTAGGGTTATGCGATTTTGTGTTGTTGGGG-3'。

[0069] Pglf-R-1(SEQ ID NO.9):

[0070] 5'-GATCTTCTCCTAAGCAGTAAATTGGGCCGCATCTCGTGGACTATAATGGATCAACAAAAAATGATGGC-3'。

[0071] Pkan-F-2 (SEQ ID NO.10):

[0072] 5'-GAACGTAAAAAAAGCACCCATACTCAGGAGCACTCTCAATTAGGCTGGAGCTGCTTC-3'。

[0073] Pkan-R-2 (SEQ ID NO.11):

[0074] 5'-GCAGCCATCTGGCTGCCTTAGTCTCCCCAACGTCTTACGGATCCGGGGATCCGTCGACC-3'。

[0075] Pglk-F-1(SEQ ID NO.12):

[0076] 5'-GAACGTAAAAAAAGCACCCATACTCAGGAGCACTCTCAATTATGGAAATTGTTGCGATTGACATC-3'。

[0077] Pglk-R-1(SEQ ID NO.13):

[0078] 5'-GCAGCCATCTGGCTGCCTTAGTCTCCCCAACGTCTTACGGATTATTCAACTTCAGAATTTGTTGGCAT-3'。

[0079] Pkan-F-3 (SEQ ID NO.14):

[0080] 5'-GCCGCCGGCATCATCACCATAGATGCCAATAACTGCTTCCGAGGCTGGAGCTGCTTC-3'。

[0081] Pkan-R-3 (SEQ ID NO.15):

[0082] 5'-GACCATATATTCTGTACGGTTCTCGTCAATATCACGTTTTTCCGGGGATCCGTCGACC-3'。

[0083] Pkan-F-4(SEQ ID NO.16):

[0084] 5'-AATGAGCCAGACTTTTTACCGCTGTAATAAAGGAGAAATCAGGCTGGAGCTGCTTC-3'。

[0085] Pkan-R-4 (SEQ ID NO.17):

[0086] 5'-AGACTTAATCCGGGCATGATAGCCCGGATTTCCATCAAGATCCGGGGATCCGTCGACC-3'.

[0087] PpCP20-F (SEQ ID NO.18):

[0088] 5'-ATGTCTGAATTAGTTGTTTTCAAAGCAAATGAAC-3'.

[0089] PpCP20-R (SEQ ID NO.19):

[0090] 5'-GATCCTTCCGTATTTAGCCAGTATGTTCT-3'.

[0091] PpurF-F (SEQ ID NO.20):

[0092] 5'-ATGTGCGGTATTGTCGGTATCGC-3'.

[0093] PpurF-R (SEQ ID NO.21):

[0094] 5'-TCATCCTTCGTTATGCATTTCGAGATTTTCCAC-3'.

[0095] PridA-F (SEQ ID NO. 22): 5'-TGCGGTTGGTAATAAAAGTCTGGCT-3'.

[0096] PridA-R (SEQ ID NO. 23): 5'-CCTGATAAGCGTAGCGCATCAGG-3'.

[0097] Example 2

[0098] This embodiment verifies the recombinant strain through fermentation in a 5L tank.

[0099] (1) Culture medium

[0100] Seed culture medium: glucose 30 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.

[0101] Fermentation medium: glucose 24 g / L, MgSO4·7H2O 1 g / L, KH2PO4 4 g / L, (NH4)2SO4 8 g / L, yeast extract 2 g / L, sodium citrate dihydrate 4 g / L, biotin 0.2 g / L, DL-calcium pantothenate 1 g / L and 10 mL of 100× reserve trace elements. This trace element solution contains 0.1 mol / L of Na₂MoO₄·2H₂O, 2.5 g / L of AlCl₃·6H₂O, 10 g / L of FeSO₄·7H₂O, 1.75 g / L of CoCl₂·6H₂O, 10 g / L of CaCl₂·2H₂O, 0.5 g / L of ZnSO₄·7H₂O, 0.25 g / L of CuCl₂·2H₂O, 0.125 g / L of boric acid, and 0.5 g / L of Na₂MoO₄·2H₂O.

[0102] (2) Fermentation production of L-tryptophan by recombinant strains

[0103] Eight bacterial strains, namely XJFF-151113S, XJFF-15113S-△purF, XJFF-151113S-△ridA, XJFF-151113S-△purF-△ridA, XJFFtrp-1, XJFFtrp-1-△purF, XJFFtrp-1-△ridA, and XJFFtrp-1-△purF-△ridA, were inoculated onto seed culture medium to obtain seed solutions. These seed solutions were then transferred to 5 L fermenters for fermentation. The glucose concentration was controlled by adding glucose feed solution during fermentation to maintain a glucose concentration of 10 g / L. The culture was conducted at 37℃ and pH 6.8, with the pH of the culture medium controlled by adding ammonia.

[0104] The seed cultures of these eight strains were simultaneously inoculated into fermentation medium supplemented with 1 g / L threonine, and fermentation was performed under the same conditions for verification. Finally, the tryptophan yield and conversion rate were measured using an amino acid analyzer (Hitachi, model LA8080) (the yield and conversion rate results for each strain were the average of three measurements). The conversion rate was calculated as follows: Conversion rate = Amount of amino acids produced (g / L) / (Amount of glucose input (g / L) - Amount of glucose remaining (g / L)) × 100%.

[0105] (3) Results of fermentation production of tryptophan by recombinant strain

[0106] The tryptophan yield and conversion rates of strains XJFF-151113S, XJFF-15113S-△purF, XJFF-151113S-△ridA, and XJFF-151113S-△purF-△ridA are as follows: Figure 3 and Figure 4 As shown, the tryptophan production and conversion rates of strains XJFFtrp-1, XJFFtrp-1-△purF, XJFFtrp-1-△ridA, and XJFFtrp-1-△purF-△ridA are respectively as follows: Figure 5 and Figure 6 As shown.

[0107] As shown in the figure, when threonine was not added to the fermentation medium, the tryptophan production and conversion rate of the strains XJFF-151113S-△purF and XJFF-151113S-△purF-△ridA, as well as XJFFtrp-1-△purF and XJFFtrp-1-△purF-△ridA, which had the purF gene knocked out, were significantly lower than those of the starting strains XJFF-151113S and XJFFtrp-1, respectively. The purF gene was inactivated, blocking the synthesis of PRPP and glutamine in purines. The consumption of the tryptophan synthesis pathway leads to a lack of purine energy supply, resulting in a significant decrease in the final tryptophan yield and conversion rate. In contrast, the tryptophan yield and conversion rate of strains XJFF-151113S-△ridA and XJFFtrp-1-△ridA are slightly improved compared to their respective starting strains. Although knocking out the ridA gene redirects the purine synthesis in the original pathway to threonine supply, the fermentation process still suffers from the same problem of large consumption of PRPP and glutamine as the starting strains, so the improvement in tryptophan yield and conversion rate is not significant.

[0108] After adding threonine to the fermentation medium, the tryptophan yield and conversion rate of strains XJFF-151113S-△purF and XJFF-151113S-△purF-△ridA were significantly improved compared with the original strain XJFF-151113S, with tryptophan yield increasing by 10.4% and 19.3%, respectively, and conversion rate increasing by 8.4% and 19.7%, respectively. The tryptophan yield and conversion rate of strains XJFFtrp-1-△purF and XJFFtrp-1-△purF-△ridA were significantly improved compared with the original strain XJFFtrp-1, with tryptophan yield increasing by 7.8% and 14.7%, respectively, and conversion rate increasing by 7.4% and 8.2%, respectively. This demonstrates that inactivating the purF gene while simultaneously adding exogenous threonine reduces the consumption of PRPP and glutamine during fermentation. The addition of exogenous threonine provides a nitrogen source, promoting partial purine synthesis and thus increasing tryptophan yield and conversion rate. Furthermore, knocking out both the purF and ridA genes and adding exogenous threonine further increases tryptophan yield and conversion rate because the ridA gene knockout redirects purine synthesis in the original pathway towards threonine supply, leading to a higher purine production rate. This reduces the consumption of PRPP and glutamine during fermentation while providing sufficient ATP for tryptophan synthesis, enhancing cell activity during fermentation and thus further increasing tryptophan yield and conversion rate.

[0109] After adding threonine to the fermentation medium, the yield and conversion rate of these eight strains were higher than those of tryptophan without the addition of threonine. This indicates that as long as threonine is added exogenously to the culture medium, it can promote the synthesis of purines, increase the concentration of purines, and provide sufficient ATP for the synthesis of tryptophan, thereby increasing the yield and conversion rate of tryptophan.

[0110] (4) Threonine content in the fermentation broth after fermentation of recombinant strain

[0111] The threonine content in the final fermentation broth of the fermentation medium supplemented with threonine was detected by high performance liquid chromatography (HPLC), and the results are shown in Table 1.

[0112] Table 1

[0113]

[0114] Combine Table 1 Figure 3 and Figure 4 Analysis shows that:

[0115] 1) The threonine content in the fermentation broth of the starting strains XJFF-151113S and XJFFtrp-1 decreased after fermentation, with 26.0% and 11.0% consumed, respectively. Some of the threonine was used to synthesize purine precursors, providing energy for the tryptophan synthesis pathway. Therefore, the yield and conversion rate of tryptophan were correspondingly improved compared to strains XJFF-151113S and XJFFtrp-1 in which threonine was not added to the culture medium.

[0116] 2) After fermentation, the remaining threonine content in the fermentation broth of strain XJFF-151113S-△purF-△ridA was 0.02 g / L, consuming 98%; after fermentation, the remaining threonine content in the fermentation broth of strain XJFFtrp-1-△purF-△ridA was 0.06 g / L, consuming 94%. This indicates that after the knockout of ridA, the direction of purine synthesis in the original pathway was redirected to threonine supply, and the exogenously added threonine was almost entirely utilized to synthesize purine precursors. This strain showed the highest increase in tryptophan yield and conversion rate after fermentation.

[0117] 3) After fermentation, the remaining amount of threonine in the fermentation broth of strain XJFF-151113S-△purF was 0.42 g / L, which consumed 58%; after fermentation, the remaining amount of threonine in the fermentation broth of strain XJFFtrp-1-△purF was 0.53 g / L, which consumed 47%. The XJFF-151113S-△purF and XJFFtrp-1-△purF strains did not knock out or weaken the ridA gene, but only blocked the consumption of PRPP and glutamine in the purine synthesis pathway. They did not redirect the purine synthesis direction in the original pathway to threonine supply. Therefore, only a portion of the exogenously added threonine was used to synthesize purine precursors. Compared with the XJFF-151113S-△purF-△ridA strain, the threonine consumption was lower. Correspondingly, the final yield and conversion rate of tryptophan fermentation were also lower than those of the XJFF-151113S-△purF-△ridA and XJFFtrp-1-△purF-△ridA strains, respectively.

[0118] 4) After fermentation, the remaining threonine content in the fermentation broth of strain XJFF-151113S-△ridA was 0.81 g / L, representing 19% consumption; after fermentation, the remaining threonine content in the fermentation broth of strain XJFFtrp-1-△ridA was 0.87 g / L, representing 13% consumption. Knockout of the ridA gene prevents 2-aminocrotonate from participating in the isoleucine synthesis pathway. Some of the threonine in the culture medium is used to generate purine precursors to provide energy for tryptophan synthesis. Compared to the starting strain, the ridA gene knockout utilizes a portion of the threonine produced during fermentation, thus minimizing the consumption of exogenously added threonine.

[0119] In summary, this invention employs a novel modification strategy to weaken / inactivate the purF gene, thereby reducing / blocking the consumption of PRPP and glutamine in the purine synthesis pathway. This effectively reduces excessive consumption of PRPP and glutamine during tryptophan production, allowing more PRPP and glutamine to participate in the tryptophan synthesis pathway. The weakening / inactivation of the ridA gene redirects purine synthesis in the original pathway towards threonine supply, reconstructing the metabolic flux in the recombinant bacteria. Furthermore, exogenous addition of threonine further promotes purine synthesis, increasing purine concentration and thus further improving tryptophan yield and conversion rate.

[0120] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A recombinant L-tryptophan-producing bacterium, characterized in that, The recombinant bacteria lack functional amide phosphoribosyltransferase, or, It lacks functional amide phosphoribosyltransferase and 2-iminopropionate deaminase.

2. The L-tryptophan-producing recombinant bacteria according to claim 1, characterized in that, The missing functional amide phosphoribosyltransferase and 2-iminopropionate deaminase include: deletion or weakened expression of the gene encoding amide phosphoribosyltransferase, and deletion or weakened expression of the gene encoding 2-iminopropionate deaminase.

3. The L-tryptophan-producing recombinant bacterium according to claim 1 or 2, characterized in that, The starting strain of the L-tryptophan-producing recombinant bacteria includes Escherichia coli.

4. The L-tryptophan-producing recombinant bacteria according to any one of claims 1-3, characterized in that, The L-tryptophan recombinant bacteria also lacked functional phosphoenolpyruvate-protein phosphotransferase and PTS system glucose-specific enzyme, and introduced exogenously glucose-promoted diffusion transporter, glucokinase and overexpressed tryptophan operon.

5. The L-tryptophan-producing recombinant bacteria according to claim 4, characterized in that, The L-tryptophan recombinant bacteria also lack functional phosphoenolpyruvate-protein phosphotransferase and PTS system glucose-specific enzymes, including: the coding gene for phosphoenolpyruvate-protein phosphotransferase is missing or its expression is weakened, and the coding gene for PTS system glucose-specific enzyme is missing or its expression is weakened.

6. The method for constructing L-tryptophan-producing recombinant bacteria according to any one of claims 1-5, characterized in that, The construction method includes: Remove the bacterial strain and remove the functional amide phosphoribosyltransferase and 2-iminopropionate deaminase from it.

7. The method for constructing L-tryptophan-producing recombinant bacteria according to claim 6, characterized in that, The construction method specifically includes: extracting the bacterial strain and knocking out or weakening the expression of the amide phosphoribosyltransferase encoding gene and the 2-iminopropionate deaminase encoding gene.

8. The use of the L-tryptophan-producing recombinant bacteria according to any one of claims 1-5 in the production of L-tryptophan.

9. A method for producing L-tryptophan, characterized in that, The method includes culturing L-tryptophan-producing strains, purifying the product, and obtaining L-tryptophan; The culture medium used for the culture contains threonine.

10. The method for producing L-tryptophan according to claim 9, characterized in that, The L-tryptophan-producing strain includes the L-tryptophan-producing recombinant strain as described in any one of claims 1-5.

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