Recombinant microorganism for producing L-tryptophan as well as construction method and fermentation process of recombinant microorganism

Abstract

The invention discloses a recombinant microorganism for producing L-tryptophan as well as a construction method and a fermentation process of the recombinant microorganism, and belongs to the technical field of genetic engineering. In order to solve the technical problems of low conversion rate and poor fermentation performance of L-tryptophan produced by a microbiological method in the prior art, the invention provides a recombinant microorganism WA-D for producing L-tryptophan, which is characterized in that CGMCC NO.11073 escherichia coli is used as an original strain, a purF gene is weakened, and glnA and prs genes are heterologously introduced at a ycap gene locus, so that the L-tryptophan is produced. Tryptophan synthesis precursors PRPP and glnA are increased and the amount of by-product glutamic acid is reduced. The recombinant microorganism WA-D provided by the invention is combined with a fermentation process, so that the yield and conversion rate of L-tryptophan are improved, and the yield of a byproduct glutamic acid is effectively reduced. The recombinant microorganism WA-D provided by the invention can be applied to large-scale industrial production of L-tryptophan.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and relates to a recombinant microorganism for producing L-tryptophan, a method for constructing the same, and a fermentation process. Background Art

[0002] L-tryptophan is one of the eight essential amino acids in the life activities of humans and animals, and plays an important role in the growth, development, and metabolism of humans and animals. It is widely used in the feed industry. The production of L-tryptophan was originally mainly dependent on protein hydrolysis method and chemical synthesis method. However, with the continuous in-depth research on the production of L-tryptophan by microbial method, the microbial method has become practical and is in the dominant position. The microbial method can be further divided into direct fermentation method, microbial transformation method, and enzymatic method. Currently, the production of L-tryptophan mainly relies on microbial fermentation method; the microbial fermentation method has the advantages of low raw material price, simple process control, and reliable product quality.

[0003] However, with the rapid development of the fermentation industry, the production of L-tryptophan by fermentation method has put forward higher requirements for the nutritional components of the culture medium and the rationality of fermentation regulation; excellent L-tryptophan-producing strains, reasonable culture medium composition, and appropriate fermentation regulation strategies are all beneficial to improving the acid production level of L-tryptophan.

[0004] Escherichia coli has the advantages of clear genetic background and simple operation, and is the main microorganism for industrial production of tryptophan. The production of L-tryptophan by Escherichia coli starts from DAHP (3-deoxy-D-arabino-heptulosonate-7-phosphate) produced by the polymerization of PEP (phosphoenolpyruvate) and E4P (erythrose-4-phosphate). PEP is an intermediate of glycolysis, and E4P is an intermediate of the pentose phosphate pathway. Then, L-tryptophan is biosynthesized from chorismic acid through the common aromatic biosynthesis pathway. There are several important precursors in the synthesis of L-tryptophan, such as: PEP, E4P, Ser (L-serine), PRPP (L-glutamine and 5-phosphoribosyl-1-pyrophosphate), Gln (L-glutamine), etc. However, there are few related reports on the production of L-tryptophan using the PRPP precursor in the prior art.

[0005] Those skilled in the art select Escherichia coli as the starting strain and develop a recombinant microorganism with high conversion rate and good fermentation performance based on the PRPP precursor for the production of L-tryptophan to meet the needs of large-scale industrial production. Summary of the Invention

[0006] The present invention provides a recombinant microorganism for producing L-tryptophan, a method for constructing the same, and a fermentation process to solve the technical problems of low conversion rate and poor fermentation performance in the production of L-tryptophan by microbial method in the prior art.

[0007] One of the objectives of the present invention is to provide a method for constructing a recombinant microorganism for producing L-tryptophan, and the construction method includes the following steps:

[0008] S1: Transfer the vector into the competent cells of the starting strain and prepare electrocompetent cells to obtain the strain to be transformed;

[0009] S2: Use the PKO3 suicide plasmid gene editing tool to weaken the start codon of the purF gene to obtain recombinant plasmid 1;

[0010] S3: Transform the recombinant plasmid obtained in S2 into the strain to be transformed obtained in S1 by electrotransformation to obtain recombinant microorganism 1;

[0011] S4: Use the CRISPR gene editing tool to design the sgRNA target sequence of the ycap gene, use the pGRB plasmid as a template, use the pamyL promoter, and insert the glnA and prs genes at the ycap gene locus to obtain recombinant plasmid 2;

[0012] S5: Transform the recombinant plasmid 2 obtained in S4 into the recombinant microorganism 1 obtained in S3 by electrotransformation to obtain a recombinant microorganism for producing L-tryptophan.

[0013] In a preferred embodiment of the present invention, the starting strain described in S1 is Escherichia coli CGMCC NO.11073.

[0014] In a preferred embodiment of the present invention, the nucleotide sequence of the purF gene described in S2 is shown as SEQ ID No.1, and the weakening of the start codon means replacing ATG with GTG.

[0015] In a preferred embodiment of the present invention, the sgRNA target sequence of the ycap gene described in S4 is shown as SEQ ID No.2, the nucleotide sequence of the promoter pamyL is shown as SEQ ID No.3, the nucleotide sequence of the glnA gene is shown as SEQ ID No.4, and the nucleotide sequence of the prs gene is shown as SEQ ID No.5.

[0016] Another objective of the present invention is to provide a recombinant microorganism WA-D for producing L-tryptophan, and the recombinant microorganism WA-D is obtained by using the above construction method.

[0017] Another objective of the present invention is to provide the application of the above recombinant microorganism WA-D and its fermentation product in the production of L-tryptophan.

[0018] A fourth object of the present invention is to provide a fermentation process for producing L-tryptophan. The fermentation process is to inoculate the above-mentioned recombinant microorganism WA-D into a plate solid slant medium and culture it at 36 °C for 16 h; inoculate the cultured WA-D into a seed liquid shake flask and culture it at 36 °C until the logarithmic growth phase; inoculate the WA-D cultured to the logarithmic growth phase into a fermentation medium and ferment and culture it at 36 °C, pH = 7.0, dissolved oxygen 25%-30%, tank pressure 0.02 MPa for 32 h-40 h. During the fermentation process, glucose with a concentration of 50%-60% is fed, and the residual sugar content is controlled at 0.04% to obtain an L-tryptophan fermentation broth.

[0019] In a preferred embodiment of the present invention, the slant medium includes: tryptone 10 g / L, beef extract 10 g / L, yeast powder 5 g / L, sodium chloride 5 g / L, anhydrous glucose 1-4 g / L, KH2PO4 1-5 g / L, the balance being water, pH = 6.8-7.2.

[0020] In a preferred embodiment of the present invention, the preparation method of the seed liquid medium: mix dipotassium hydrogen phosphate 20 g / L, potassium dihydrogen phosphate 12 g / L, yeast powder 18 g / L and ammonium sulfate 4 g / L, adjust the pH value of the above mixture to 7.15 with a sodium hydroxide solution with a mass fraction of 45%, then add an antifoaming agent, and sterilize it at 121 °C for 20 min to obtain a medium; use a magnesium sulfate solution with a concentration of 1.5 g / L and a glucose solution with a concentration of 35 g / L, sterilize it at 115 °C for 15 min to obtain a mixed solution of glucose and magnesium sulfate; dispense 45 mL of the above medium and 5 mL of the above mixed solution of glucose and magnesium sulfate into each baffle flask, wrap it for use to obtain a seed liquid medium.

[0021] In a preferred embodiment of the present invention, the fermentation medium includes: glucose 10-20 g / L, yeast powder 4-6 g / L, citric acid 1-4 g / L, (NH4)2SO4 2-8 g / L, KH2PO4 4-6 g / L, MgSO4·7H2O 1-2 g / L, FeSO4·7H2O 50-80 mg / L, VB1 5-7 mg / L, VH 0.2-0.6 mg / L, trace element mixed solution 1-2 mL / L;

[0022] The trace element mixed solution includes: 0.4 - 0.8 g / L of CoSO4·7H2O, 6 - 8 g / L of ZnSO4·7H2O, 4 - 6 g / L of CuSO4·5H2O, 2 - 4 g / L of Al2(SO4)3·18H2O, 4 - 6 g / L of MnSO4·H2O, 2 - 4 g / L of Na2MoO4·2H2O, 2 - 4 g / L of NiSO4·6H2O, and 1 - 2 g / L of H3BO3.

[0023] The beneficial effects of the present invention: A recombinant microorganism for producing L-tryptophan provided by the present invention uses Escherichia coli CGMCC NO.11073 as the starting strain, and introduces the purF gene (the nucleotide sequence is shown as SEQ ID No.1) after weakening the start codon. Weakening the start codon means replacing ATG with GTG, resulting in partial loss or inactivation of the function of the amidophosphoribosyltransferase encoded by the purF gene, increasing the contents of the tryptophan synthesis precursors PRPP and Gln, and reducing the content of the by-product glutamate, thereby increasing the yield of tryptophan; and heterologously introducing the glnA (the nucleotide sequence is shown as SEQ ID No.4) and prs (the nucleotide sequence is shown as SEQ ID No.5) genes. By heterologously expressing the glnA and prs genes, the purpose of increasing the contents of the tryptophan synthesis precursors glutamine and PRPP is achieved, thereby increasing the yield of tryptophan.

[0024] The recombinant microorganism WA-D provided by the present invention is combined with the fermentation process to maximize the ability of WA-D to produce L-tryptophan. The yield of L-tryptophan is 44.5 g / L, the productivity is 20.34%, and the yield of glutamate is 0.23 g / L; compared with the starting strain Escherichia coli CGMCC NO.11073, the recombinant microorganism WA-D increases the tryptophan content by 22.5%, increases the conversion rate by 25.8%, and reduces the glutamate content by 95.7%.

[0025] It can be seen that the recombinant microorganism WA-D provided by the present invention, and its corresponding fermentation process, increase the yield and conversion rate of L-tryptophan, effectively reduce the yield of the by-product glutamate, and can be applied to large-scale industrial production of L-tryptophan. Detailed implementation manners

[0026] Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described in this article without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with specific embodiments. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in this field, and those skilled in the art can obtain them through commercial channels without special instructions.

[0028] The starting strain Escherichia coli CGMCC NO.11073 used in the present invention is disclosed in the invention patent with the authorization publication number CN116376852B.

[0029] The recombinant microorganisms involved in the following embodiments:

[0030] Recombinant microorganism WA-A: Using Escherichia coli CGMCC NO.11073 as the starting strain, the purF gene was knocked out to obtain recombinant microorganism WA-A;

[0031] Recombinant microorganism WA-B: Using Escherichia coli CGMCC NO.11073 as the starting strain, the purF gene with weakened start codon was introduced. The weakening of the start codon means replacing ATG with GTG to obtain recombinant microorganism WA-B;

[0032] Recombinant microorganism WA-C: Using Escherichia coli CGMCC NO.11073 as the starting strain, the purF gene was overexpressed to obtain recombinant microorganism WA-C;

[0033] Recombinant microorganism WA-D: Using Escherichia coli CGMCC NO.11073 as the starting strain, the purF gene with weakened start codon was introduced. The weakening of the start codon means replacing ATG with GTG; the glnA and prs genes were inserted at the ycap gene locus to obtain recombinant microorganism WA-D.

[0034] Example 1: Preparation and transformation of competent cells

[0035] (1) Transformation of pREDCas9 plasmid

[0036] Take out the bacterial liquid of Escherichia coli CGMCC NO.11073 from the cryopreservation tube, streak it on an LB solid plate, and statically culture it overnight (12h - 16h) at 37°C; pick a single colony from the above LB solid plate and inoculate it into 5 mL of LB liquid medium, and shake it overnight (12h - 16h) at 30°C and 200 rpm; take 500 μL of the above overnight culture bacterial liquid and inoculate it into 500 mL of LB liquid medium, and shake it at 37°C and 200 rpm until OD 660= 0.3 - 0.4 (cultivation time is 2 - 3 h); for the above OD 600 Place the qualified bacterial liquid on ice for pre-cooling for 20 - 30 min, then transfer it to a pre-cooled 50 mL centrifuge tube, centrifuge at 4°C and 4500 rpm for 10 min, and carefully discard the supernatant; first add 5 mL of ice-cold 0.1 M CaCl2 to the obtained bacterial cell precipitate, gently pipette and mix evenly, then make up to 30 mL with 0.1 M CaCl2, centrifuge at 4°C and 4500 rpm for 10 min, and carefully discard the supernatant. Repeat the above steps twice; add 1 mL of ice-cold 0.1 M CaCl2 to the obtained precipitated bacteria to resuspend and mix evenly, add 1 mL of ice-cold 30% glycerol and mix evenly, aliquot into pre-cooled 1.5 mL EP tubes at a volume of 100 μL per tube, and use immediately or store at -80°C;

[0037] Transform the plasmid pREDCas9 containing the spectinomycin resistance gene into the above-obtained Escherichia coli CGMCC NO.11073 to obtain CGMCC NO.11073 competent cells; spread the above competent cells on an LB agar plate containing 100 mg / L spectinomycin resistance gene, and culture at 30°C to obtain a single colony resistant to spectinomycin; name the strain of this colony as CGMCC NO.11073-Cas9 strain.

[0038] (2) Preparation of electrocompetent cells of the strain

[0039] Take out the bacterial liquid of the CGMCC NO.11073-Cas9 strain obtained in (1) from the cryopreservation tube, streak on an LB solid plate, and statically culture overnight (12 - 16 h) at 37°C; pick a single colony from the above LB solid plate and inoculate it into 5 mL of LB liquid medium, and shake culture overnight (12 - 16 h) at 30°C and 200 rpm; take 500 μL of the above overnight-cultured bacterial liquid and inoculate it into 500 mL of LB liquid medium, and shake culture at 30°C and 220 rpm until OD 660 = 0.1, then add 0.1 M IPTG, and culture the strain until OD 600 = 0.7 - 0.9 (cultivation time is 4 - 6 h);

[0040] For the above OD 600The qualified bacterial solution was placed on ice for 20 - 30 min for pre - cooling, and then transferred to a pre - cooled 50 mL centrifuge tube. It was centrifuged at 4°C and 4500 rpm for 10 min, and the supernatant was carefully discarded. First, 5 mL of ice - cold sterile 10% glycerol was added to the obtained bacterial cell precipitate. After gently pipetting to mix evenly, it was made up to 30 mL with 10% glycerol. Then it was centrifuged at 4°C and 4500 rpm for 10 min, and the supernatant was carefully discarded. The above steps were repeated twice. 500 μL of ice - cold 10% glycerol was added to the obtained precipitated bacteria for resuspension, and it was aliquoted into pre - cooled 1.5 mL EP tubes at a volume of 90 μL per tube to obtain the electrotransformation competent cells of CGMCC NO.11073 - Cas9 strain, which could be used immediately or stored at - 80°C.

[0041] Example 2: Construction of recombinant microorganism WA - B

[0042] S1: The genomic sequence of Escherichia coli MG1655 was obtained through NCBI. By using the CRISPR / Cas9 gene editing technology and the high - fidelity enzyme 2×Phanta Flash Master Mix, with P11 (shown as SEQ ID No.7) and P12 (shown as SEQ ID No.8) as primers, the upstream gene of purF was amplified by PCR to obtain an amplified fragment of 522 bp. With P13 (shown as SEQ ID No.9) and P14 (shown as SEQ ID No.10) as primers, the downstream gene of purF was amplified by PCR to obtain an amplified fragment of 2019 bp, and a point mutation purF(A1G) was introduced. The nucleotide sequence of the purF gene is shown as SEQ ID No.1.

[0043] The PCR reaction system was: 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of plasmid DNA / template, 25 μL of PFU enzyme, and 19 μL of water; The reaction program was: 98°C for 30 s, 98°C for 10 s, 55°C for 10 s, 72°C for 5 s / kb, 72°C for 1 min, and 4°C ∞, for 35 cycles.

[0044] The DpnI digestion system was: 50 μL of plasmid PCR product, 5 μL of 10x Buffer, and 1 μL of DpnI, and it was digested at 37°C for 1 h to remove circular plasmid DNA.

[0045] S2: Using the pko3 plasmid backbone as a template, with the high-fidelity enzyme 2×Phanta Flash Master Mix, and using P9 (shown as SEQ ID No.11) and P10 (shown as SEQ ID No.12) as primers, amplify the editing plasmid for expressing the purF gene by PCR to obtain an amplification product with a length of 5681 bp. After DpnI enzyme digestion of the above amplification product, use a DNA purification kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to recover and obtain a linear pko3 vector; the DpnI digestion system is: 50 μL of plasmid PCR product, 5 μL of 10x Buffer, and 1 μL of DpnI, digest at 37°C for 1 h to remove circular plasmid DNA;

[0046] S3: Ligate the linear pko3 vector obtained in S2 with the purF upstream and downstream gene fragments obtained in S1 using the One Step Cloning Kit C115 from Novoprotein Scientific Inc. The reaction system is: 1 μL of pko3, 1 μL of the purF gene upstream fragment, 1 μL of the purF gene downstream fragment, 5 μL of 2×ClonExpress Mix, and 2 μL of water; the above ligation is carried out at 50°C for 0.5 h and then transformed into Escherichia coli DH5α competent cells to obtain the pko3-purF(A1G) plasmid;

[0047] S4: Transform the pko3-purF(A1G) plasmid obtained in S3 into the electrocompetent cells of the CGMCC NO.11073-Cas9 strain obtained in Example 1 by electroporation method. After centrifugally enriching the cells recovered and cultured after the above electroporation, coat them on a chloramphenicol-resistant LB solid plate and culture at 30°C; Pick 2 - 5 single colonies from the chloramphenicol-resistant LB solid plate cultured at 30°C above, and use 500 μL of chloramphenicol-resistant LB medium to serially dilute to 10 0 、10 -1 、10 -2 、10 -3 , respectively take 100 μL of each concentration dilution and coat it on a Cm-resistant LB solid plate, and culture overnight at 43°C and 30°C respectively;

[0048] S5: Pick 2 - 5 single colonies from the chloramphenicol-resistant LB solid plate cultured at 43°C in S4, and use 500 μL of chloramphenicol-resistant LB medium to serially dilute to 10 0 、10 -1 、10 -2 、10 -3, 100 μL of each concentration of the diluted solution was taken and spread on an LB solid plate containing 6% sucrose, and cultured overnight at 30 °C; another 100 μL of the bacterial solution of each concentration was spread on an LB solid plate resistant to chloramphenicol and cultured overnight at 43 °C; the probability of recombination in the second step was the ratio of the number of single colonies cultured on the sucrose plate at 30 °C to the number of single colonies cultured on the Cm-resistant plate at 43 °C;

[0049] S6: 20 - 30 single colonies were picked from the sucrose plate cultured at 30 °C in S5, and were streaked on the chloramphenicol-resistant plate and the sucrose plate in sequence, and cultured overnight at 37 °C;

[0050] In this example, a total of 48 monoclonal clones were picked, none of which grew on the chloramphenicol-resistant plate, while all grew on the sucrose plate; colonies without chloramphenicol resistance and insensitive to sucrose were picked, and F7 (shown as SEQ ID No. 13) and F8 (shown as SEQ ID No. 14) were used as primers to identify positive clones by PCR, and the recombinant microorganism WA-B was obtained.

[0051] Example 3: Construction of recombinant microorganism WA-D

[0052] S1: Using CRISPRRGEN Tools (http: / / www.rgenome.net / cas designer / ) to design the sgRNA target sequence of the ycap gene (shown as SEQ ID No. 2), and adding linearized pGRB vector homologous arm sequences at the 5' end and 3' end of the target sequence for constructing the sgRNA plasmid;

[0053] Using the pGRB plasmid as a template, and using F21 (shown as SEQ ID No. 15) and F22 (shown as SEQ ID No. 16) synthesized by Ruibo Xingke Company as primers, a target fragment with a length of 2281 bp was amplified using the high-fidelity enzyme 2×Phanta Flash Master Mix; after digesting the above amplification product with DpnI enzyme, the linearized pGRB vector was recovered using a DNA purification kit (purchased from Tiangen Biochemical Technology Co., Ltd.); then it was transformed into Escherichia coli DH5α competent cells to obtain the pGRB-△ycap::pamyL-glnA-prs sgRNA plasmid, and the promoter was pamyL (the nucleotide sequence is shown as SEQ ID No. 3);

[0054] S2: According to the Escherichia coli MG1655 genome sequence published by NCBI, a primer pair for amplifying the upstream and downstream homologous arms of the ycap gene was designed and synthesized by Ruibo Xingke Company;

[0055] Using the MG1655 genome as a template, primers F23 (shown as SEQ ID No. 17) and F24 (shown as SEQ ID No. 18); and primers F25 (shown as SEQ ID No. 19) and F26 (shown as SEQ ID No. 20) were used to amplify the upstream and downstream fragments of the △purF gene;

[0056] S3: Synthesize the puc57::PamyL-glnA-prs plasmid by GenScript Biotech Corporation. Using the puc57::PamyL-glnA-prs plasmid as a template, and F27 (shown as SEQ ID No. 21) and F28 (shown as SEQ ID No. 22) as primers, use the high-fidelity enzyme 2×Phanta Flash Master Mix to amplify the pamyL-glnA-prs homologous recombination fragment; the nucleotide sequence of the glnA gene is shown as SEQ ID No. 4, and the nucleotide sequence of the prs gene is shown as SEQ ID No. 5;

[0057] S4: Use overlap PCR to fuse the upstream and downstream homologous arms of the purF gene obtained in S2 and the pamyL-glnA-prs fragment obtained in S3 to obtain the △ycap::pamyL-glnA-prs homologous recombination fragment;

[0058] S5: For the recombinant microorganism WA-B obtained in Example 2, use the method for preparing and transforming competent cells described in Example 1 to prepare and transform WA-B into competent cells and transform the pREDCas9 plasmid to obtain the electrocompetent cells of the WA-B-Cas9 strain;

[0059] S6: By electrotransformation, simultaneously electrotransform the pGRB-△ycap::pamyL-glnA-prs plasmid obtained in S1 and the PamyL-glnA-prs homologous recombination fragment obtained in S3 into the electrocompetent cells of the WA-B-Cas9 strain obtained in S5; after centrifugally enriching the cells after resuscitation culture of the electrotransformation, coat them on an LB plate containing spectinomycin and kanamycin, and culture overnight at 30°C. Use F29 (shown as SEQ ID No. 23) and F30 (shown as SEQ ID No. 24) as primers to identify the positive transformants from the single colonies after the above overnight culture;

[0060] S7: Transfer the positive transformants identified in S6 to an LB medium containing 0.2% arabinose and spectinomycin and culture overnight, perform three-zone streaking on an LB plate containing spectinomycin, culture overnight at 30°C, spot-culture on an LB plate containing spectinomycin and an LB plate containing spectinomycin and kanamycin, and preserve the strains that only grow on the LB plate containing spectinomycin;

[0061] S8: Transfer the strains that only grow on the spectinomycin LB plate in S7 to the antibiotic-free LB medium and culture overnight at 37°C. Streak the cultures in three zones on the antibiotic-free LB plate and culture overnight at 37°C. Spot culture on the spectinomycin-containing LB plate and the antibiotic-free LB plate, and select the strains that do not grow on the spectinomycin plate but grow on the antibiotic-free plate for preservation to obtain the recombinant microorganism WA-D.

[0062] Comparative Example 1: Construction of the recombinant microorganism WA-A

[0063] S1: Use CRISPRRGEN Tools (http: / / www.rgenome.net / cas designer / ) to design the sgRNA target sequence of the purF gene (shown as SEQ ID No.1), and add the linearized pGRB vector homologous arm sequences to the 5' end and 3' end of the target sequence for constructing the sgRNA plasmid;

[0064] Using the pGRB plasmid as a template, and F1 (shown as SEQ ID No.25) and F2 (shown as SEQ ID No.26) synthesized by Ruibo Xingke Company as primers, use the high-fidelity enzyme 2×Phanta Flash Master Mix to amplify a target fragment with a length of 2281 bp; after digesting the above amplification product with DpnI enzyme, use a DNA purification kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to recover the linearized pGRB vector; then transform it into Escherichia coli DH5α competent cells to obtain the pGRB-ΔpurF plasmid.

[0065] S2: Using the MG1655 genome as a template, use primers F3 (shown as SEQ ID No.27) and F4 (shown as SEQ ID No.28); and primers F5 (shown as SEQ ID No.29) and F6 (shown as SEQ ID No.30) to amplify the upstream and downstream fragments of the ΔpurF gene; the nucleotide sequence of the purF gene is shown as SEQ ID No.1;

[0066] S3: By electroporation, simultaneously electroporate the pGRB-ΔpurF plasmid obtained in S1 and the upstream and downstream fragments of the ΔpurF gene obtained in S2 into the electrocompetent cells of the CGMCC NO.11073-Cas9 strain obtained in Example 1; after centrifugally enriching the cells recovered after electroporation and culturing, coat them on the LB plate containing spectinomycin and kanamycin, and culture overnight at 30°C. Use F7 (shown as SEQ ID No.13) and F8 (shown as SEQ ID No.14) as primers to identify the positive transformants of the above overnight cultured single colonies;

[0067] S4: Transfer the positive transformants identified in S3 to an LB medium containing 0.2% arabinose and spectinomycin and culture overnight. Perform three-zone streaking on a spectinomycin LB plate and culture overnight at 30°C. Spot culture on a spectinomycin LB plate and an LB plate containing spectinomycin and kanamycin, and preserve the strains that grow only on the spectinomycin LB plate;

[0068] S5: Transfer the strains that grow only on the spectinomycin LB plate in S4 to an antibiotic-free LB medium and culture overnight at 37°C. Perform three-zone streaking on an antibiotic-free LB plate and culture overnight at 37°C. Spot culture on a spectinomycin-containing LB plate and an antibiotic-free LB plate, and select and preserve the strains that do not grow on the spectinomycin plate but grow on the antibiotic-free plate to obtain the recombinant microorganism WA-A.

[0069] Comparative Example 2: Construction of recombinant microorganism WA-C

[0070] S1: Obtain the genomic sequence of Escherichia coli MG1655 through NCBI. Using the pRSFDuet-1-pamyL as the plasmid backbone by the CRISPR / Cas9 gene editing technology, the nucleotide sequence is shown as SEQ ID No.6; the promoter is pamyL (the nucleotide sequence is shown as SEQ ID No.3);

[0071] Using the pRSFDuet-1-pamyL plasmid as a template, and using P15 (shown as SEQ ID No.31) and P16 (shown as SEQ ID No.32) as primers, use the high-fidelity enzyme 2×Phanta Flash Master Mix to amplify a target fragment with a length of 4021 bp. After digesting the above amplification product with DpnI enzyme, use a DNA purification kit (purchased from Tiangen Biochemical Technology Co., Ltd.) to recover and obtain a linearized pRSFDuet-1-pamyL vector;

[0072] S2: Obtain the genomic sequence of Escherichia coli MG1655 through NCBI. Design and synthesize by Ruibo Xingke Co., Ltd. a primer pair F17 (shown as SEQ ID No.33) and F18 (shown as SEQ ID No.34) for amplifying the purF gene. Using the MG1655 genome as a template, amplify a purF gene fragment with a length of 1518 bp; the nucleotide sequence of the purF gene is shown as SEQ ID No.1;

[0073] S3: Ligate the linearized pRSFDuet-1-pamyL vector obtained in S1 with the purF gene fragment obtained in S2 using the One Step Cloning Kit C115 from Novoprotein Scientific Inc. The reaction system is as follows: 1 μL of pRSFDuet-1-pamyL, 1 μL of purF gene fragment, 4 μL of 5×CE II Buffer, 2 μL of xnase II, and 12 μL of water. The above ligation is carried out at 37 °C for 0.5 h and then transformed into Escherichia coli DH5α competent cells to obtain the pRSFDuet-1-pamyL-purF plasmid.

[0074] S4: By electroporation, the pRSFDuet-1-pamyL-purF plasmid (containing the kanamycin resistance gene) obtained in S3 is electroporated into the electrocompetent cells of the CGMCC NO.11073-Cas9 strain obtained in Example 1. The cells after electroporation and resuscitation culture are centrifuged and concentrated, and then spread on an LB plate containing 100 mg / L spectinomycin and cultured overnight at 30 °C to obtain kanamycin-resistant single colonies.

[0075] Using F19 (shown as SEQ ID No. 35) and F20 (shown as SEQ ID No. 36) as primers, identify the positive transformants from the above kanamycin-resistant single colonies. The positive transformants identified are the recombinant microorganism WA-B.

[0076] Effect experiment:

[0077] (1) Shake flask verification

[0078] Take 3 mL of each of the recombinant microorganism WA-B prepared in Example 2, the recombinant microorganism WA-D prepared in Example 3, the recombinant microorganism WA-A prepared in Comparative Example 1, the recombinant microorganism WA-C prepared in Comparative Example 2, and the CGMCC NO.11073 Escherichia coli, and place them respectively in fermentation medium shake flasks. Use a seed shake flask to supplement the above fermentation medium shake flasks to a final volume of 30 mL. Wrap the above fermentation medium shake flasks and place them in a shaker. Culture at 36 °C and 220 rpm for a fermentation cycle of 12.5 h. Ensure that the pH is within the appropriate range and the residual sugar is not exhausted when taking out the shake flasks to obtain L-tryptophan fermentation broth respectively.

[0079] Preparation of the seed shake flask: The shaker is sterilized and preheated with ultraviolet light in advance, and the cultured plate containing the recombinant microorganism WA-B prepared in Example 2, the recombinant microorganism WA-D prepared in Example 3, the recombinant microorganism WA-A prepared in Comparative Example 1, the recombinant microorganism WA-C prepared in Comparative Example 2, and CGMCC NO.11073 Escherichia coli is taken out, and a loop of bacteria is scraped with an inoculation loop under a sterile environment and inoculated into the seed shake flask containing the seed culture medium. After the inoculation, the bottle mouth is completely sealed with gauze and fixed with a rubber band, the ultraviolet light of the shaker is turned off, and the seed shake flask is placed in it, and cultured at 36°C and 170rpm; OD 660 =5-6 can be transplanted to fermentation shake flasks;

[0080] The seed liquid culture medium comprises: 20 g / L dipotassium hydrogen phosphate, 12 g / L potassium dihydrogen phosphate, 18 g / L yeast powder, 4 g / L ammonium sulfate, 45% sodium hydroxide solution is used to adjust the pH to 7.15, a defoamer is added, and the culture medium is obtained by sterilizing at 121° C. for 20 min; 1.5 g / L magnesium sulfate and 35 g / L glucose are dissolved in water, and the mixture is sterilized at 115° C. for 15 min to obtain a mixed solution of glucose and magnesium sulfate; 45 mL of the culture medium and 5 mL of the mixed solution of glucose and magnesium sulfate are dispensed into each baffle bottle, and the bottles are wrapped for standby use;

[0081] The fermentation medium comprises: 6 g / L dipotassium hydrogen phosphate, 6 g / L potassium dihydrogen phosphate, 5 g / L disodium hydrogen phosphate, 6 g / L ammonium sulfate, 0.1 g / L ferrous sulfate, 2 g / L citric acid, 1 mL / L trace element A, 1 mL / L trace element B, 50 g / L MOPS and 6 mL / L 2% phenol red dissolved in water, 45% sodium hydroxide solution is used to adjust the pH to 7.15, a defoaming agent is added, and the medium is sterilized at 121° C. for 20 min to obtain a medium; 3 g / L magnesium sulfate and 30 g / L glucose are dissolved in water, and the mixture is sterilized at 115° C. for 15 min to obtain a mixed solution of glucose and magnesium sulfate; 24 mL of the medium and 3 mL of the mixed solution of glucose and magnesium sulfate are filled into each baffle bottle, and the bottle is wrapped for standby use.

[0082] (2) Validation of 2L tank fermentation of recombinant microorganisms

[0083] The recombinant microorganism WA-B prepared in Example 2, the recombinant microorganism WA-D prepared in Example 3, the recombinant microorganism WA-A prepared in Comparative Example 1, the recombinant microorganism WA-C prepared in Comparative Example 2, and Escherichia coli CGMCC NO. 11073 were inoculated into a plate solid slant medium at an inoculum amount of 2 loops each and cultured at 36°C for 16 h; the above-mentioned cultured WA-D was inoculated into a seed liquid shake flask and cultured at 36°C until the logarithmic growth phase; the above-mentioned WA-D cultured to the logarithmic growth phase was inoculated into a fermentation medium and fermented and cultured at 36°C, pH = 7.0, dissolved oxygen 25%-30%, and tank pressure 0.02 MPa for 40 h. During the fermentation process, glucose with a concentration of 50%-60% was added dropwise, and the residual sugar content was controlled at 0.04% to obtain L-tryptophan fermentation broth respectively;

[0084] The slant medium includes: 10 g / L of tryptone, 10 g / L of beef extract, 5 g / L of yeast powder, 5 g / L of sodium chloride, 1-4 g / L of anhydrous glucose, 1-5 g / L of KH2PO4, and the balance is water, pH = 6.8-7.2.

[0085] The preparation method of the seed liquid medium: Mix 20 g / L of dipotassium hydrogen phosphate, 12 g / L of potassium dihydrogen phosphate, 18 g / L of yeast powder, and 4 g / L of ammonium sulfate, adjust the pH value of the above-mentioned mixed solution to 7.15 with a sodium hydroxide solution with a mass fraction of 45%, then add an antifoaming agent, and sterilize at 121°C for 20 min to obtain a medium; use a magnesium sulfate solution with a concentration of 1.5 g / L and a glucose solution with a concentration of 35 g / L, and sterilize at 115°C for 15 min to obtain a mixed solution of glucose and magnesium sulfate; dispense 45 mL of the above-mentioned medium and 5 mL of the above-mentioned mixed solution of glucose and magnesium sulfate into each baffle flask, wrap and set aside to obtain the seed liquid medium.

[0086] The fermentation medium includes: 10-20 g / L of glucose, 4-6 g / L of yeast powder, 1-4 g / L of citric acid, 2-8 g / L of (NH4)2SO4, 4-6 g / L of KH2PO4, 1-2 g / L of MgSO4·7H2O, 50-80 mg / L of FeSO4·7H2O, 5-7 mg / L of VB1, 0.2-0.6 mg / L of VH, and 1-2 mL / L of trace element mixed solution;

[0087] The trace element mixed solution includes: 0.4 - 0.8 g / L of CoSO4·7H2O, 6 - 8 g / L of ZnSO4·7H2O, 4 - 6 g / L of CuSO4·5H2O, 2 - 4 g / L of Al2(SO4)3·18H2O, 4 - 6 g / L of MnSO4·H2O, 2 - 4 g / L of Na2MoO4·2H2O, 2 - 4 g / L of NiSO4·6H2O, and 1 - 2 g / L of H3BO3.

[0088] Result analysis:

[0089] (1) Shake flask verification of recombinant microorganisms

[0090] In this example, the liquid chromatography method and Kjeldahl nitrogen analyzer were used to detect the L-tryptophan-related indicators of the fermentation broth obtained by fermenting the recombinant microorganisms WA-B prepared in Example 2, the recombinant microorganisms WA-D prepared in Example 3, the recombinant microorganisms WA-A prepared in Comparative Example 1, the recombinant microorganisms WA-C prepared in Comparative Example 2, and Escherichia coli CGMCC NO.11073. There were 3 parallels in each group.

[0091] The results are shown in Table 1. The recombinant microorganism WA-A showed an abnormal growth phenomenon in the shake flask verification, indicating that the purF gene is an essential gene for the production of L-tryptophan by recombinant microorganisms; the recombinant microorganism WA-B showed a positive effect in the shake flask verification, and the L-tryptophan yield was increased compared with the starting strain Escherichia coli CGMCC NO.11073, indicating that the purF gene after introducing the start codon weakening is beneficial to improving the L-tryptophan yield of recombinant microorganisms; the performance of the recombinant microorganism WA-C in the shake flask verification showed no obvious change compared with the starting strain Escherichia coli CGMCC NO.11073, indicating that the recombinant microorganism overexpressing the purF gene has no significant effect on the L-tryptophan yield.

[0092] It can be seen that in the present invention, the start codon of the purF gene is weakened and then introduced into the starting strain, achieving the purpose of improving the L-tryptophan yield of recombinant microorganisms.

[0093] Table 1

[0094]

[0095]

[0096] The results are shown in Table 2. The recombinant microorganism WA-D showed obvious positive effects in the shake flask verification. Compared with the recombinant microorganism WA-B, the L-tryptophan yield was increased to 3.88 g / L, an increase of 9.3%; compared with the starting strain Escherichia coli CGMCC NO.11073, the L-tryptophan yield was increased by 14.4%. It is indicated that the introduction of the purF gene with weakened start codon, as well as the insertion of the glnA and prs genes, has a significant effect on the increase of L-tryptophan yield.

[0097] It can be seen that in the present invention, the start codon of the purF gene is weakened, and the glnA and prs genes are inserted into the starting strain, and the obtained recombinant microorganism WA-D has a higher L-tryptophan yield.

[0098] Table 2

[0099] Shaking flask number Fermentation cycle pH <![CDATA[OD 660 > Tryptophan (g / L) Residual sugar (g / L) WA-B 12.5 6.32 13.23 3.52 <0.5 WA-B 12.5 6.39 13.12 3.52 <0.5 WA-B 12.5 6.33 13.22 3.47 <0.5 WA-D 12.5 6.21 12.42 3.85 <0.5 WA-D 12.5 6.28 12.31 3.88 <0.5 WA-D 12.5 6.24 12.28 3.79 <0.5

[0100] (2) Fermentation verification of the recombinant microorganism in a 2 L tank

[0101] In this example, high performance liquid chromatography and Kjeldahl apparatus were used to detect the contents of tryptophan, glutamate, acetic acid, citric acid, succinic acid and ammonia nitrogen in the fermentation broth obtained by fermenting the recombinant microorganism WA-B prepared in Example 2, the recombinant microorganism WA-D prepared in Example 3, the recombinant microorganism WA-A prepared in Comparative Example 1, the recombinant microorganism WA-C prepared in Comparative Example 2, and Escherichia coli CGMCC NO.11073. There were 5 parallels in each group.

[0102] Conversion rate = (volume of fermentation broth L × fermentation acid production content g / L) / amount of fermented glucose used g × 100%

[0103] The results are shown in Table 3. The recombinant microorganism WA-A could not grow normally during fermentation and the fermentation was terminated in advance. The L-tryptophan yield and conversion rate of the recombinant microorganism WA-C did not decrease significantly, while the glutamate content increased. Compared with the starting strain Escherichia coli CGMCC NO.11073, the recombinant microorganism WA-B increased the tryptophan content from 36.32 g / L to 39.87 g / L, increased the conversion rate from 16.17% to 18.75%, and decreased the glutamate content from 5.32 g / L to 2.24 g / L. It can be seen that the recombinant microorganism WA-B provided by the present invention has a significantly increased L-tryptophan yield and conversion rate compared with the starting strain, and a significantly decreased glutamate content, indicating that the purF gene after start codon weakening has the effect of reducing glutamate production while increasing the L-tryptophan yield and conversion rate.

[0104] Table 3

[0105]

[0106]

[0107] The results are shown in Table 4. Compared with the recombinant microorganism WA-B, the recombinant microorganism WA-D increased the tryptophan content by 12.5%, increased the conversion rate by 10.4%, and decreased the glutamic acid content by 90%. Compared with the starting strain Escherichia coli CGMCC NO.11073, the recombinant microorganism WA-D increased the tryptophan content by 22.5%, increased the conversion rate by 25.8%, and decreased the glutamic acid content by 95.7%. It can be seen that the recombinant microorganism WA-D provided by the present invention significantly improved the L-tryptophan yield and conversion rate compared with the starting strain, and significantly reduced the glutamic acid content, indicating that the heterologous introduction of the glnA and prs genes can increase the precursors for tryptophan synthesis and reduce the production of by-product glutamic acid, thereby improving the tryptophan yield and conversion rate. The recombinant microorganism WA-D provided by the present invention has a high L-tryptophan yield and conversion rate, and a low by-product glutamic acid content.

[0108] Table 4

[0109] Item CGMCC NO.11073 WA-B WA-D Tryptophan (g / L) 36.32 39.56 44.5 Conversion rate (%) 16.17 18.43 20.34 Glutamic acid (g / L) 5.32 2.31 0.23 OD 80.4 79.3 72.2 Cycle (h) 40 40 40

[0110] The content not described in detail in the specification of the present invention is well-known technology to those skilled in the art. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for constructing a recombinant microorganism for producing L-tryptophan, characterized in that, The construction method includes the following steps: S1: Transfer the vector into the competent cells of the starting strain, and prepare electrocompetent cells to obtain the strain to be modified; S2: Use the PKO3 suicide plasmid gene editing tool to weaken the start codon of the purF gene to obtain recombinant plasmid 1; S3: Transform the recombinant plasmid obtained in S2 into the strain to be modified obtained in S1 by electrotransformation to obtain recombinant microorganism 1; S4: Use the CRISPR gene editing tool to design the sgRNA target sequence of the ycap gene. Using the pGRB plasmid as a template and pamyL as a promoter, insert the glnA and prs genes at the ycap gene locus to obtain recombinant plasmid 2; S5: Transform the recombinant plasmid 2 obtained in S4 into the recombinant microorganism 1 obtained in S3 by electrotransformation to obtain a recombinant microorganism for producing L-tryptophan.

2. The construction method according to claim 1, wherein The starting strain described in S1 is Escherichia coli CGMCC NO. 11073.

3. The construction method according to claim 1, wherein The nucleotide sequence of the purF gene described in S2 is shown in SEQ ID No. 1, and the weakening of the start codon means replacing ATG with GTG.

4. The construction method according to claim 1, characterized in that The sgRNA target sequence of the ycap gene described in S4 is shown in SEQ ID No. 2, the nucleotide sequence of the promoter pamyL is shown in SEQ ID No. 3, the nucleotide sequence of the glnA gene is shown in SEQ ID No. 4, and the nucleotide sequence of the prs gene is shown in SEQ ID No.

5.

5. A recombinant microorganism WA-D for producing L-tryptophan, characterized in that, The recombinant microorganism WA-D is obtained by using the construction method described in any one of claims 1 to 4.

6. Use of the recombinant microorganism WA-D according to claim 5 and its fermentation product in the production of L-tryptophan.

7. A fermentation process for producing L-tryptophan, characterized in that, The fermentation process is to inoculate the recombinant microorganism WA-D described in claim 4 into a plate solid slant medium and culture it at 36°C for 16 h; inoculate the cultured WA-D into a seed liquid shake flask and culture it at 36°C until the logarithmic growth phase; inoculate the WA-D cultured to the logarithmic growth phase into a fermentation medium and ferment and culture it at 36°C, pH = 7.0, dissolved oxygen 25%-30%, tank pressure 0.02 MPa for 32 h-40 h. During the fermentation process, glucose with a concentration of 50%-60% is fed, and the residual sugar content is controlled at 0.04% to obtain an L-tryptophan fermentation broth.

8. The fermentation process according to claim 7, characterized in that, The slant medium includes: 10 g / L of tryptone, 10 g / L of beef extract, 5 g / L of yeast powder, 5 g / L of sodium chloride, 1-4 g / L of anhydrous glucose, 1-5 g / L of KH2PO4, and the balance is water, pH = 6.8-7.

2.

9. The fermentation process according to claim 7, wherein, The preparation method of the seed liquid medium: Mix 20 g / L of dipotassium hydrogen phosphate, 12 g / L of potassium dihydrogen phosphate, 18 g / L of yeast powder, and 4 g / L of ammonium sulfate. Use a sodium hydroxide solution with a mass fraction of 45% to adjust the pH value of the above mixture to 7.15, then add an antifoaming agent, and sterilize at 121 °C for 20 min to obtain the medium; use a magnesium sulfate solution with a concentration of 1.5 g / L and a glucose solution with a concentration of 35 g / L, and sterilize at 115 °C for 15 min to obtain a mixed solution of glucose and magnesium sulfate; dispense 45 mL of the above medium and 5 mL of the above mixed solution of glucose and magnesium sulfate into each baffle flask, wrap it for later use to obtain the seed liquid medium.

10. The fermentation process according to claim 7, characterized in that, The fermentation medium includes: 10 - 20 g / L of glucose, 4 - 6 g / L of yeast powder, 1 - 4 g / L of citric acid, 2 - 8 g / L of (NH4)2SO4, 4 - 6 g / L of KH2PO4, 1 - 2 g / L of MgSO4·7H2O, 50 - 80 mg / L of FeSO4·7H2O, 5 - 7 mg / L of VB1, 0.2 - 0.6 mg / L of VH, 1 - 2 mL / L of trace element mixed solution; The trace element mixed solution includes: 0.4 - 0.8 g / L of CoSO4·7H2O, 6 - 8 g / L of ZnSO4·7H2O, 4 - 6 g / L of CuSO4·5H2O, 2 - 4 g / L of Al2(SO4)3·18H2O, 4 - 6 g / L of MnSO4·H2O, 2 - 4 g / L of Na2MoO4·2H2O, 2 - 4 g / L of NiSO4·6H2O, 1 - 2 g / L of H3BO3.

Images