Recombinant escherichia coli for producing l-tyrosine and application thereof

Abstract

The application discloses a kind of recombinant escherichia coli for producing L-tyrosine and application, belong to genetic engineering and bioengineering technical field.The application knocks out aroP and tyrP gene, expresses the yddG gene of escherichia coli endogenous to modify the aromatic amino acid transport system of escherichia coli, then heterologous expression comes from bifidobacterium fpk and expresses the ppsA and tktA gene of escherichia coli endogenous to increase the precursor supply of shikimic acid pathway, again through the anti feedback gene expression aroG and tyrA 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase, branch acid mutase and prephenate dehydrogenase are removed feedback inhibition, increase the carbon flux of shikimic acid pathway.Knock out tyrR gene removes the repression of TyrR protein to shikimic acid pathway key enzyme, again knock out trpE and pheA gene to block partial side reaction of shikimic acid pathway, so that the synthesis flux of L-tyrosine increases.Finally knock out endogenous poxB gene to realize the modification of acetic acid pathway, realizes the stable fermentation performance of high glucose concentration.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of recombinant escherichia coli of L-tyrosine production and application, belong to genetic engineering and bioengineering field. BACKGROUND

[0002] L-tyrosine (L-Tyrosine, Tyr), as a kind of essential amino acid, is one of the 20 kinds of amino acids that constitute protein, is widely used in food, feed and medicine and other fields.L-tyrosine can promote catecholamine, thyroid hormone and melanin synthesis in human body, and has important role to the development and metabolism of human and animal.In medicine, L-tyrosine is the main raw material for the synthesis of a variety of drugs such as thyroid hormone, adrenaline and levodopa.

[0003] At present, the traditional L-tyrosine production method has protein hydrolysis method, chemical synthesis method, enzyme method and microbial fermentation method.Protein hydrolysis method, also known as extraction method, is to use natural protein resources such as casein, pig blood powder, animal hoof shell, horn and hair as raw materials, through hydrolysis, concentration, crystallization and decolorization steps to separate and extract L-tyrosine.Enzyme conversion method uses phenol, ammonia salt and pyruvic acid as precursor, and is converted by tyrosine enzyme, but enzyme is easy to inactivate, and reaction condition is strict, so enzyme method for preparing L-tyrosine cannot be industrialized on a large scale.Chemical synthesis method is to synthesize racemic DL-tyrosine through L-phenylalanine hydroxylation or through p-hydroxyphenylamine and hydantoin condensation, alkaline hydrolysis, transamination and other steps, which needs to further separate L-tyrosine, and the process is complex and the efficiency is low.Microbial fermentation method uses biomass raw materials to realize de novo synthesis of tyrosine, which greatly reduces the production cost.Compared with protein hydrolysis method and enzyme hydrolysis method, microbial fermentation method has the advantages of short cycle, high conversion rate and simple separation and purification steps.The existing tyrosine production strain has low yield, and it is difficult to realize industrial large-scale production.There is an urgent need to construct a recombinant strain for high-efficiency production of L-tyrosine and establish a microbial fermentation method with higher productivity. SUMMARY

[0004] In order to solve the above technical problems, the present application provides a recombinant escherichia coli for synthesizing L-tyrosine, which is improved in at least one of the following aspects based on the starting strain of escherichia coli:

[0005] (1) the endogenous aroP and tyrP are knocked out, which blocks the transport of L-tyrosine from extracellular to intracellular in escherichia coli;

[0006] (2) the endogenous yddG is overexpressed, which increases the ability of escherichia coli to transport L-tyrosine from intracellular to extracellular;

[0007] (3) Expressing phosphoketolase (fpk) derived from Bifidobacterium adolescentis and ppsA and tktA genes endogenous to E. coli, effectively guiding the carbon metabolic flow of glucose to the synthesis of L-tyrosine;

[0008] (4) Expressing aroG mutant from E. coli, which is a mutant of 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase that is relieved of aromatic amino acid feedback inhibition;

[0009] (5) Expressing tyrA mutant from E. coli, which effectively increases the synthesis flux of L-tyrosine in order to relieve the influence of L-tyrosine overaccumulation on chorismate mutase and prephenate dehydrogenase;

[0010] (6) Knocking out endogenous pheA and trpE, blocking part of the shikimic acid pathway synthesis pathway, so that more metabolic flow is used for the synthesis of L-tyrosine;

[0011] (7) Expressing ppsA and tktA genes endogenous to E. coli, increasing the front supply of the shikimic acid pathway, and increasing the synthesis flux of L-tyrosine;

[0012] (8) Knocking out the poxB gene, reducing the accumulation of acetic acid in the fermentation process, and realizing high glucose concentration fermentation;

[0013] The present application provides a recombinant E. coli for efficiently synthesizing L-tyrosine, wherein the recombinant E. coli is an E. coli strain, and any one of the following (a) to (d) is edited:

[0014] (a) Knocking out the genes pheA encoding a fused chorismate mutase / prephenate dehydratase, trpE encoding a subunit of anthranilate synthase TrpE, and tyrR encoding a DNA-binding transcriptional dual regulator TyrR on the genome of E. coli, and freely expressing the gene aroG encoding 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase fbr , the gene tyrA encoding chorismate mutase and prephenate dehydrogenase fbr ;

[0015] (b) Knocking out the genes pheA encoding a fused chorismate mutase / prephenate dehydratase, trpE encoding a subunit of anthranilate synthase TrpE, and tyrR encoding a DNA-binding transcriptional dual regulator TyrR on the genome of E. coli, and freely expressing the gene aroG encoding 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase fbr , the gene tyrA encoding chorismate mutase and prephenate dehydrogenase fbrand a phosphoketolase gene fpk;

[0016] (c) knocking out the fusion chorismate mutase / prephenate dehydratase gene pheA, the anthranilate synthase subunit TrpE gene trpE, the DNA-binding transcriptional dual regulator TyrR gene tyrR, and the pyruvate oxidase gene poxB on the genome of E. coli, and freely expressing the 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase gene aroG fbr , the chorismate mutase and prephenate dehydrogenase gene tyrA fbr , and the phosphoketolase gene fpk;

[0017] (d) knocking out the fusion chorismate mutase / prephenate dehydratase gene pheA, the anthranilate synthase subunit TrpE gene trpE, the DNA-binding transcriptional dual regulator TyrR gene tyrR, the pyruvate oxidase gene poxB, the aromatic amino acid transporter AroP permease gene aroP, and the tyrosine:H(+) symporter gene tyrP on the genome of E. coli, and freely expressing the 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthase gene aroG fbr , the chorismate mutase and prephenate dehydrogenase gene tyrA fbr , the phosphoketolase gene fpk, and the amino acid exporter YddG gene yddG.

[0018] In one embodiment, the recombinant E. coli freely expresses or integrally expresses the phosphoketolase synthase gene ppsA and the transketolase 1 gene tktA.

[0019] In one embodiment, the gene ppsA and the gene tktA are integrated at the ykgh-betA site on the genome of E. coli.

[0020] In one embodiment, the gene ppsA and the gene tktA are expressed by using the promoter PJ 231119 to initiate expression of the gene ppsA and the gene tktA.

[0021] In one embodiment, the E. coli uses the expression plasmid of heat-inducible expression vector pAP-B03 as the expression plasmid.

[0022] In one embodiment, the nucleotide sequence of the gene pheA is shown in SEQ ID NO. 1.

[0023] In one embodiment, the nucleotide sequence of the gene trpE is shown in SEQ ID NO. 2.

[0024] In an embodiment, the nucleotide sequence of the gene aroG is as shown in SEQ ID NO. 3.

[0025] In an embodiment, the nucleotide sequence of the gene tyrA is as shown in SEQ ID NO. 4.

[0026] In an embodiment, the nucleotide sequence of the gene tyrR is as shown in SEQ ID NO. 5.

[0027] In an embodiment, the nucleotide sequence of the gene ppsA is as shown in SEQ ID NO. 6.

[0028] In an embodiment, the nucleotide sequence of the gene tktA is as shown in SEQ ID NO. 7.

[0029] In an embodiment, the nucleotide sequence of the gene fpk is as shown in SEQ ID NO. 8.

[0030] In an embodiment, the nucleotide sequence of the gene poxB is as shown in SEQ ID NO. 9.

[0031] In an embodiment, the nucleotide sequence of the gene aroP is as shown in SEQ ID NO. 10.

[0032] In an embodiment, the nucleotide sequence of the gene tyrP is as shown in SEQ ID NO. 11.

[0033] In an embodiment, the nucleotide sequence of the gene yddG is as shown in SEQ ID NO. 12.

[0034] In an embodiment, the aroG, ppsA, tktA, fpk, yddG, tyrA are expressed by using the heat-induced expression vector pAP-B03 plasmid.

[0035] In an embodiment, the promoter PJ is integrated at the ykgh-betA site on the genome of the E. coli. 231119 The genes ppsA and tktA are initiated to express.

[0036] In an embodiment, the E. coli K12, E. coli BL21, E. coli DH5a, E. coli JM109 or E. coli WSH-Z06 is used as the starting strain.

[0037] The present application provides a method for producing L-tyrosine by using the recombinant E. coli to ferment L-tyrosine.

[0038] In an embodiment, the recombinant E. coli is inoculated into a fermentation system, cultured at 32-34℃ for 3-12h, and then fermented at 36-40℃, 200-220rpm for 48-60h.

[0039] In an embodiment, the fermentation system comprises glucose 30-40g / L, (NH4)2SO4 3-7g / L, KH2PO4 1-5g / L, MgSO4·7H2O 1-5g / L, sodium citrate 1-2g / L, NaCl 0.5-1.5g / L, vitamin B1 0.05-0.1g / L, FeSO4·7H2O 0.1-0.12g / L, yeast powder 1-3g / L, proteose peptone 2-6g / L, and trace element nutrient solution (TES) 1-2mL / L.

[0040] In an embodiment, the components of the TES include Al2(SO4)3·18H2O 2.0g / L, CoSO4·7H2O 0.75g / L, CuSO4·5H2O 2.5g / L, H3BO3 0.5g / L, MnSO4·H2O 24g / L, NiSO4·6H2O 2.5g / L, ZnSO4·7H2O 15g / L.

[0041] The present application provides the use of the recombinant E. coli in the production of L-tyrosine or a product containing L-tyrosine.

[0042] The present application has the following beneficial effects:

[0043] 1. The present application uses E. coli as a host, in order to increase the carbon flux of the synthesis pathway of tyrosine, the CM-PDT encoding gene pheA in E. coli is knocked out by CRISPR / Cas9 gene editing method, aroG fbr , tyrA fbr , tktA and ppsA are expressed by using heat-inducible plasmid pAP-B03, the xx gene trpE is further knocked out to block the synthesis of tryptophan, the tyrR gene is knocked out to relieve the repression of TyrR protein on the key enzymes of shikimic acid pathway, and tyrosine is accumulated to 5.6g / L in a shake flask after 48h fermentation.

[0044] 2. In order to further improve the utilization rate of glucose, fpk from Bifidobacterium is heterologously expressed to direct the flow of glucose to the shikimic acid pathway, the supply of precursors of the shikimic acid pathway is increased, and the genes tktA and ppsA are integrated and expressed on the E. coli genome, and the expression of the genes is initiated by the strong promoter PJ 231119 , and L-tyrosine is accumulated to 6.0g / L in a shake flask after 48h fermentation.

[0045] 3. The acetic acid pathway of E. coli is modified by knocking out the poxB gene encoding pyruvate oxidase (PoxB) on the genome of E. coli, effectively reducing the production of acetic acid, so that the amount of L-tyrosine is increased to 6.2 g / L.

[0046] 4. The aroP and tyrP genes are knocked out, the endogenous yddG gene of E. coli is expressed to modify the aromatic amino acid transport system of E. coli, then the phosphoketolase-encoding gene fpk is expressed, the ppsA and tktA genes are integrated and expressed to increase the precursor supply of the shikimic acid pathway, and the anti-feedback genes aroG and tyrA are expressed to relieve the feedback inhibition of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, chorismate mutase and prephenate dehydrogenase, thereby increasing the carbon flux of the shikimic acid pathway. The tyrR gene is knocked out to relieve the repression of TyrR protein on the key enzymes of the shikimic acid pathway, and the trpE and pheA genes are knocked out to block part of the side reactions of the shikimic acid pathway, thereby increasing the synthesis flux of tyrosine. Finally, the endogenous poxB gene is knocked out to modify the acetic acid pathway and achieve stable fermentation performance at high glucose concentration. The E. coli engineering strain obtained by the method of the present application is induced to ferment for 55 h, and the content of L-tyrosine in the fermentation broth is as high as 80.5 g / L, and the production intensity reaches 1.46 g / L / h, which provides a new idea for the industrial production of L-tyrosine. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The synthesis of L-tyrosine in E. coli is shown in the figure.

[0048] Figure 2 The L-tyrosine yield of E. coli in a 5 L fermenter is shown in the figure. DETAILED DESCRIPTION

[0049] (I) Culture medium

[0050] Seed culture medium (LB): 10 g / L of proteose peptone, 5 g / L of yeast extract, 5 g / L of sodium chloride; 2% (mass fraction) agar powder is added to the solid culture medium.

[0051] Fermentation medium (1 L): glucose 35 g, (NH4)2SO4 5 g, K2HPO4·3H2O 3 g, MgSO4·7H2O 3 g, sodium citrate 1.5 g, NaCl 1 g, vitamin B1 0.075 g, FeSO4·7H2O 0.1125 g, yeast powder 2 g, proteose peptone 4 g, trace element nutrient solution (TES) 1.5 mL, and appropriate antibiotics were added as required. A conical flask was added with 12 g of calcium carbonate for pH control; TES: Al2(SO4)3·18H2O 2.0 g / L, CoSO4·7H2O 0.75 g / L, CuSO4·5H2O 2.5 g / L, H3BO3 0.5 g / L, MnSO4·H2O 24 g / L, NiSO4·6H2O 2.5 g / L, ZnSO4·7H2O 15 g / L.

[0052] (ii) PCR reaction system and amplification conditions:

[0053] Forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 10-50 ng, 2×Phanta Max Master Mix 25 μL, and distilled water added to 50 μL. Amplification conditions: 95°C pre-denaturation for 3 min; then 30 cycles of 95°C for 15 s, 55°C for 15 s, 72°C for 15 s, and 72°C for 5 min.

[0054] (iii) Preparation of E. coli competence:

[0055] The E. coli K12 glycerol tube was streaked on a corresponding LB plate and incubated at 37°C overnight (about 12 h). After 12 h, a single colony was inoculated in a 50 mL shake flask containing 5 mL of LB medium, and incubated at 37°C at 220 rpm until the OD 600 = 0.6-0.8; the bacterial solution was transferred to a 50 mL centrifuge tube and placed on ice for about 15 min; centrifuged at 4000 rpm at 4°C for 5 min to remove the supernatant; 5 mL of solution A was added to resuspend the bacteria; centrifuged at 4000 rpm at 4°C for 5 min to remove the supernatant; 5 mL of solution B was added to resuspend the bacteria, and 100 μL per portion was aliquoted and stored at -80°C.

[0056] (iv) Transformation of E. coli:

[0057] Thaw the E. coli competent cells on ice; take 10 μL of the recombination product (plasmid 50 ng) and add it to 100 μL of the competent cells, mix gently and let stand on ice for 30 min; heat shock in a water bath at 42°C for 45 s, let stand on ice for 2 min; add 1 mL of LB medium, shake at 37°C and 220 rpm for 60 min; centrifuge at 4500 rpm for 2 min, remove the supernatant, resuspend the bacterial pellet with the remaining medium and spread on a resistant plate.

[0058] (v) L-tyrosine HPLC assay:

[0059] After the fermentation, take 1 mL of the fermentation broth, dilute it with 3M hydrochloric acid to the appropriate multiple, mix well by vigorous shaking, centrifuge at 14000 rpm for 10 min, take the supernatant, filter it through a 0.22 μm inorganic filter membrane and then use a Shimadzu LC-20A high-performance liquid chromatograph to detect the product. Use a Thermo Fisher C18 chromatographic column (4.6 mm x 250 mm, 5 μm) for chromatographic separation; set the column oven temperature to 30°C; the injection volume is 10 μL; the mobile phase is A: 0.1M sodium acetate (adjust the pH to 4.5 with glacial acetic acid) and B: pure methanol; the total flow rate is 1 mL / min, the volume percentage is 90% and 10%, and the detector wavelength is 280 nm.

[0060] (vi) Plasmids

[0061] The pAP-B03 plasmid involved in the following examples is described in the literature “Zhou, H., Liao, X., Wang, T., Du, G., Chen, J., 2010. Enhanced L-phenylalanine biosynthesis by co-expression of pheA fbr and aroF wt . Bioresource Technology. 101(11): 4151-4156.”; the pCas and p-Target plasmids involved in the following examples are described in the literature “Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S., 2015. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Applied and Environmental Microbiology. 81(7): 2506-2514.”; the recombinant plasmid pCDF-aroG fbr -tyrA fbrWu, J., Zhou, T., Du, G., Zhou, J., Chen, J., 2014. Modular optimization of heterologous pathways for de novo synthesis of (2S)-naringenin in Escherichia coli. PloS One. 9(7): 1-9. The strain E. coli HG involved in the following examples is the strain WSH-Z06 (pAP-B03) described in the literature "Zhou, H., Liao, X., Wang, T., Du, G., Chen, J., 2010. Enhanced L-phenylalanine biosynthesis by co-expression of pheA fbr andaroF wt . Bioresource Technology. 101(11): 4151-4156." and is designated E. coli HG in the present application.

[0062] (VII) The strain information is shown in Table 1:

[0063] Table 1 Strains and genes involved in the present application

[0064]

[0065] Example 1 : Construction of recombinant E. coli for L-tyrosine synthesis

[0066] (1) Preparation of the engineering strain HGA0

[0067] The plasmid-free strain HG0 was obtained by continuously passing the laboratory-preserved E. coli HG at 42°C. The pCas plasmid was transformed into the E. coli HG0 competent cells, and the obtained single colony was picked into 4 mL LB medium containing 50 μg / mL kanamycin and cultured at 30°C for 12 hours. Then, 2% of the bacterial solution was inoculated into 50 mL of LB medium, and 50 μg / mL of kanamycin and 10 mM of arabinose solution were added. After being cultured at 30°C and 220 rpm for 4-6 hours, the bacterial solution was transferred to a 50 mL centrifuge tube and placed on ice for 15 min. The supernatant was removed by centrifugation at 4000 rpm and 4°C for 10 min, and 10 mL of 10% glycerol was added for resuspension. The operation was repeated twice, and the cells were stored at -80°C in 100 μL / portion. Thus, the E. coli HG0-pCas competent cells containing the pCas plasmid were prepared and designated as E. coli HG0-pCas.

[0068] Escherichia coli HG0 was selected as the starting strain for the fermentation production of L-tyrosine. Firstly, as... Figure 1 As shown in the L-tyrosine synthesis pathway, to increase the carbon flux of the tyrosine synthesis pathway, the gene pheA encoding the branching acid mutase-prephenylate dehydratase (CM-PDT) in *E. coli* was knocked out using CRISPR / Cas9 gene editing. Using the *E. coli* K12 genome as a template, the upstream homologous arm U1 and downstream homologous arm D1 of the pheA gene were amplified using primer pairs F11 / R11 and F12 / R12, respectively, and the fragments were purified. Using the purified fragments U1 and D1 as templates, the knockout cassette UD1 was amplified using primer pair F11 / R12, and the fragment was purified. To obtain pTarget-pheA for knocking out pheA, pTarget stored in the laboratory was used as a template, amplified using primer pair F13 / R13, and the fragment was purified. The purified pTarget-pheA fragment was transformed into *E. coli* JM109, plasmid was extracted, and sequencing verification confirmed the correct recombinant vector pTarget-pheA.

[0069] 400 ng of recombinant vector pTarget-pheA and 1200 ng of knockout cassette UD1 were added to *E. coli* HG0-pCas electroporation competent cells. The cells were incubated on ice for 10 min, then transferred to a pre-chilled 1 mm electroporation cuvette and electroporated at 1.8 kV. After electroporation, 1 mL of LB broth was added, and the cells were incubated at 30 °C for 1.5 h. Colony PCR was performed using primer pair F14 / R14 to verify the colony. The confirmed single clones lost pTarget-pheA and pCas9, yielding the engineered *E. coli* strain HGA0.

[0070] (2) Preparation of overexpression plasmid pAP-aroG fbr -tyrA fbr pAP-aroG fbr -tyrA fbr -ppsA-tktA and engineered strain HGA

[0071] The heat-inducible plasmid framework was obtained from the pAP-B03 plasmid using primer pair F113 / R113, and includes the kanamycin gene, P... R P L Promoter (obtained using F116 / R116 primer pair) and p15A replicon. AroG fbr and tyrA fbr Gene from plasmid pCDF-aroG fbr -tyrA fbrF114 / R114 and F117 / R117, respectively. The tktA gene and the ppsA gene were amplified from the E. coli genome using primer pairs F115 / R115 and F118 / R118. The obtained heat-induced plasmid framework with the genes aroG fbr , tyrA fbr , tktA and ppsA were obtained using the Gibson assembly method to obtain plasmid pAP-aroG fbr -tyrA fbr and pAP-aroG fbr -tyrA fbr -ppsA-tktA.

[0072] The recombinant vector pAP-aroG fbr -tyrA fbr -ppsA-tktA was transformed into E. coli HGA0 to obtain the engineering strain HGA.

[0073] (3) Preparation of engineering strains HGB0 and HGB

[0074] Using the same method, the HGA0 electrotransformation competent cells HGA0-pCas containing the pCas plasmid were constructed, and the trpE gene was knocked out to block the synthesis of tryptophan using the same method. The E. coli K12 genome was used as a template, and the primer pairs F15 / R15 and F16 / R16 were used to amplify the homologous arms above and below the trpE, and the primer pair F15 / R16 was used to amplify the knockout cassette UD2. The p-Target was amplified using the primer pair F17 / R17 to prepare the recombinant vector pTarget-trpE. The knockout cassette UD2 and the recombinant vector pTarget-trpE were electrotransformed into HGA0-pCas, and colony PCR was performed using the primer pair F18 / R18. After verification, the correct single colony lost pTarget-trpE and pCas9 to obtain the engineering strain HGB0.

[0075] The recombinant vector pAP-aroG fbr -tyrA fbr -ppsA-tktA was transformed into E. coli HGB0 to obtain the engineering strain HGB.

[0076] (4) Preparation of engineering strains HGC0 and HGC

[0077] The same method was used to construct HGB0-pCas containing pCas plasmid by using engineering strain HGB0 as the starting strain; the same method was used to knock out tyrR gene by using E. coli K12 genome as the template, to release the repression of key enzymes in shikimic acid pathway caused by accumulation of amino acids, to amplify the homologous arms upstream and downstream of tyrR by using primer pair F19 / R19, and to amplify the knockout cassette UD3 by using primer pair F110 / R110; p-Target was amplified by using primer pair F111 / R111 to prepare recombinant vector pTarget-tyrR; the knockout cassette UD3 and the recombinant vector pTarget-tyrR were electroporated into HGB0-pCas, and colony PCR was performed by using primer pair F112 / R112 to verify the correct monoclonal loss of pTarget-tyrR and pCas9, and to obtain engineering bacteria HGC0.

[0078] The recombinant vector pAP-aroG fbr -tyrA fbr -ppsA-tktA was transformed into E. coli HGC0 to obtain engineering strain HGC.

[0079] The engineering strain HGC was inoculated into 50 mL seed culture medium, and cultured at 37°C and 220 rpm for 12 hours to obtain a seed liquid, which was then inoculated into fermentation medium containing kanamycin at a final concentration of 50 μg / mL at an inoculation amount of 2% (v / v), and cultured at 33°C and 220 rpm for 3 hours, and then the temperature was changed to 38°C. The synthesis of L-tyrosine was induced and carried out at 38°C and 220 rpm, and the fermentation time was 48 h, and the tyrosine could be accumulated to 5.6 g / L in a shake flask.

[0080] All primer sequences are listed in Table 2.

[0081] Table 2 Primer sequences

[0082]

[0083]

[0084] Example 2: Exogenous introduction of fpk to improve L-tyrosine synthesis

[0085] In order to improve the utilization rate of glucose, fpk from Bifidobacterium was heterologously expressed to direct the flow of glucose to the shikimic acid pathway and increase the supply of precursors of the shikimic acid pathway. In order to prevent the influence of too long plasmid on the expression efficiency of genes, ppsA and tktA genes were connected with strong promoter PJ 231119 The promoter was integrated on the E. coli genome.

[0086] (1) Preparation of engineering strain HGD0

[0087] The engineering strain HGC0 constructed in Example 1 was used as the starting strain, and the same method was used to construct HGC0-pCas containing pCas plasmid; the same method as gene knockout was used to integrate tktA gene, and E. coli K12 genome was used as the template, tktA gene was amplified by primer pair F23 / R23, and dadx-cvra upstream and downstream homologous arms were amplified by primer pair F24 / R24 and F25 / R25. The tktA gene and dadx-cvra upstream and downstream homologous arms were used as the templates, and the knock-in cassette UTD of tktA and upstream and downstream homologous arms was amplified by primer pair F24 / R25. p-Target was amplified by primer pair F26 / R26 to prepare the recombinant vector pTarget-dadx-cvra; the knock-in cassette UTD and the recombinant vector pTarget-dadx-cvra were electroporated into HGC0-pCas, and colony PCR was performed by primer pair F27 / R27 to verify that the correct monoclonal lost pTarget-dadx-cvra and pCas9, and the engineering strain HGD0 was obtained.

[0088] (2) Preparation of overexpression plasmid pAP-aroG fbr -tyrA fbr -fpk and engineering strains HGE0, HGE

[0089] The same method was used to construct HGD0-pCas containing pCas plasmid; the same method was used to integrate ppsA gene, and E. coli K12 genome was used as the template, ppsA gene was amplified by primer pair F28 / R28, ykgh-betA upstream and downstream homologous arms were amplified by primer pair F29 / R29 and F210 / R210, and ppsA and upstream and downstream homologous arms were obtained by amplification by primer pair F29 / R210 to obtain the knock-in cassette UPD. p-Target was amplified by primer pair F211 / R211 to prepare the recombinant vector pTarget-ykgh-betA; the knock-in cassette UPD and the recombinant vector pTarget-ykgh-betA were electroporated into HGD0-pCas, and colony PCR was performed by primer pair F212 / R212 to verify that the correct monoclonal lost pTarget-ykgh-betA and pCas9, and the engineering strain HGE0 was obtained.

[0090] The synthesized fpk was used as the template, and primer pair F21 / R21 was used for amplification, and the product was purified and recovered; the recombinant vector pAP-aroG fbr -tyrA fbr was used as the template, and primer pair F22 / R22 was used for amplification, and the product was recovered. The fragments fpk and the vector pAP-aroG fbr -tyrA fbrThe backbone recombination obtained a recombination vector, the recombination vector was transformed into E. coli JM109, plasmid was extracted and sequencing verification was performed, and the correct recombination vector pAP-aroG was obtained fbr -tyrA fbr -fpk.

[0091] The pAP-aroG fbr -tyrA fbr -fpk was transformed into E. coli HGE0, and the engineering strain HGE was obtained.

[0092] The engineering strain HGE was inoculated into 50 mL seed medium, and was cultured at 37°C, 220 rpm for 12 hours to obtain a seed liquid, and then was inoculated into a fermentation medium with a final concentration of 50 μg / mL kanamycin at a inoculation amount of 2% (v / v). After being cultured at 33°C, 220 rpm for 3 hours, the temperature was changed to 38°C. The synthesis of L-tyrosine was induced and carried out at 38°C, 220 rpm, and the fermentation time was 48 hours. The shake flask could accumulate 6.0 g / L of L-tyrosine.

[0093] All primer sequences are listed in Table 3.

[0094] Table 3 Primer sequences

[0095]

[0096] Example 3: Modification of acetate pathway to improve glucose utilization

[0097] When the HGE strain constructed in Example 2 was subjected to shake flask fermentation, it was found that 1.2 g / L of acetic acid accumulated in the shake flask within 48 hours, causing serious waste of carbon resources. Therefore, the acetic acid pathway of E. coli was modified. The poxB gene encoding pyruvate oxidase (PoxB) in E. coli was knocked out.

[0098] Using E. coli K12 genome as a template, the upstream homologous arm U1 and the downstream homologous arm D1 of the gene poxB were amplified by primer pairs F31 / R31 and F32 / R32, respectively, and the fragments were purified; using the purified fragments U1 and D1 as templates, the knockout cassette UD1 was obtained by amplification with primer pair F31 / R32, and the fragment was purified. In order to obtain pTarget-poxB for knocking out poxB, p-Target preserved in the laboratory was used as a template, and primer pair F33 / R33 was used for amplification, and the fragment was purified; the purified fragment was transformed into E. coli JM109, plasmid was extracted and sequencing verification was performed, and the correct recombination vector pTarget-poxB was obtained.

[0099] The same method was used to construct HGE-pCas containing pCas plasmid by using the engineering strain HGE as the starting strain. The poxB gene of E. coli HGE was knocked out by using the same experimental method in Example 1, and the correct monoclonal was verified by colony PCR using primer pair F34 / R34, and then pTarget-poxB and pCas9 were lost to obtain the engineering strain HGF0.

[0100] The recombinant vector pAP-aroG fbr -tyrA fbr -fpk was transformed into E. coli HGF0 to obtain the engineering strain HGF.

[0101] The engineering strain HGF was inoculated into 50 mL seed medium and cultured at 37°C, 220 rpm for 12 hours to obtain the seed liquid, and then inoculated into fermentation medium containing kanamycin with a final concentration of 50 μg / mL at an inoculation amount of 2% (v / v). After 3 hours of culture at 33°C, 220 rpm, the temperature was changed to 38°C. The synthesis of L-tyrosine was induced and carried out at 38°C, 220 rpm, and the fermentation time was 48 h. The amount of L-tyrosine accumulated in the shake flask was 6.2 g / L, and the accumulation of acetic acid was only 0.45 g / L, which was effectively reduced by 62.5%.

[0102] All primer sequences are listed in Table 4.

[0103] Table 4 Primer sequences

[0104]

[0105] Example 4: Modification of aromatic amino acid transport system

[0106] (1) Preparation of engineering strains HGG0 and HGG

[0107] The content of intracellular tyrosine of strain HGF in Example 3 was determined, and it was found that the intracellular tyrosine concentration of HGF was 972.7% higher than that of the control wild-type E. coli K12. Therefore, the aromatic amino acid transport system of HGF was modified. The coding gene aroP of the intracellular aromatic amino acid transporter AroP permease of E. coli was knocked out. The upstream homologous arm U1 and the downstream homologous arm D1 of the gene aroP were amplified by primer pair F41 / R41 and F42 / R42, respectively, using the E. coli K12 genome as a template, and the fragments were purified; the knockout cassette UD1 was obtained by amplifying the purified fragments U1 and D1 using primer pair F41 / R42, and the fragment was purified. In order to obtain pTarget-aroP for knocking out aroP, p-Target preserved in the laboratory was used as a template, and primer pair F43 / R43 was used for amplification, and the fragment was purified; the purified fragment was transformed into E. coli JM109, and the plasmid was extracted and verified by sequencing, and the correct recombinant vector pTarget-aroP was obtained.

[0108] Using the same method, the HGF0 electrotransformation competent cells HGF0-pCas containing pCas plasmid were constructed using the engineering strain HGF0 as the starting strain, and the aroP gene of E. coli HGF0 was knocked out using the same experimental method in Example 1. Colony PCR was performed using primer pair F44 / R44, and the correct monoclonal was verified, and pTarget-aroP and pCas9 were lost, and the engineering strain E. coli HGG0 was obtained.

[0109] The recombinant vector pAP-aroG fbr -tyrA fbr -fpk in Example 2 was transformed into E. coli HGG0, and the engineering strain HGG was obtained.

[0110] (2) Preparation of overexpression plasmid pAP-aroG fbr -yddG-tyrA fbr -fpk and engineering strains HGH0 and HGH

[0111] The same method was used to knock out the tyrP gene to prevent the synthesis of tyrosine-specific transporter protein, and the same method was used to construct HGG0-pCas containing pCas plasmid. The same method was used to knock out the tyrP gene to block the synthesis of tryptophan, and the genome of E. coli K12 was used as a template. The upstream and downstream homologous arms of tyrP were amplified by primers F45 / R45 and F46 / R46, and the knockout cassette UD1 was amplified by primers F45 / R46. The recombinant vector pTarget-tyrP was prepared by amplifying p-Target with primers F47 / R47. The knockout cassette UD1 and the recombinant vector pTarget-tyrP were electroporated into HGG0-pCas, and colony PCR was performed with primers F48 / R48. The correct monoclonal colonies were verified by losing pTarget-tyrP and pCas9, and the engineered bacteria HGH0 were obtained.

[0112] The genome of E. coli K12 was used as a template, and the yddG fragment was amplified by PCR with primers F49 / R49. The pAP-aroG fbr -tyrA fbr -fpk vector as a template, and the product was purified. The yddG fragment and the pAP-aroG fbr -tyrA fbr -fpk backbone were recombined by the Gibson assembly method to obtain a recombinant vector. The recombinant vector was transformed into E. coli JM109, and the plasmid was extracted and sequenced to verify that the correct recombinant vector pAP-aroG fbr -yddG-tyrA fbr -fpk was obtained. The recombinant vector pAP-aroG fbr -yddG-tyrA fbr -fpk was transformed into E. coli HGH0 to obtain the engineered strain HGH.

[0113] The engineered strain HGH was inoculated into 50 mL seed medium and cultured at 37°C, 220 rpm for 12 hours to obtain a seed solution. Then, it was inoculated into fermentation medium containing kanamycin at a final concentration of 50 μg / mL at an inoculation amount of 2% (v / v). After 3 hours of culture at 33°C, 220 rpm, the temperature was changed to 38°C. The synthesis of L-tyrosine was induced and carried out at 38°C, 220 rpm, and the fermentation time was 48 h. The shake flask could accumulate tyrosine at 6.9 g / L.

[0114] All primer sequences are listed in Table 5.

[0115] Table 5 Primer sequences

[0116]

[0117]

[0118] Example 5: Fermentation optimization in 5-L fermenter

[0119] The engineered strain HGH constructed in Example 4 was inoculated into 50 mL seed medium and cultured at 37°C, 220 rpm for 12 h to obtain primary seed liquid, then inoculated into 50 mL secondary seed liquid at 2% (v / v) inoculation amount, and the secondary seed liquid was inoculated into 2.5 L fermentation medium with kanamycin at a final concentration of 50 μg / mL at 2% (v / v) inoculation amount. The initial rotation speed was controlled at 300 rpm, and the culture was incubated at 33°C for 12 h to OD 600 After 20-23, the temperature was raised to 38°C, and the culture was further incubated to 48-55 h to obtain fermentation broth; during the whole fermentation process, the pH was controlled at 6.4-6.6 by feeding 50% ammonia water, and when the DO decreased to 20%, the rotation speed or aeration was gradually increased to maintain the DO above 20%; when the glucose concentration in the medium was depleted, the feeding program was started to feed 750 g / L glucose for fed-batch fermentation, and the glucose concentration was maintained at about 5-8 g / L. After the fermentation, the tyrosine content was determined. Finally, as shown in Table 1, the engineered strain HGH accumulated tyrosine 80.5 g / L in 5 L fermenter for 55 h, and the production intensity reached 1.46 g / L / h. Figure 2

[0120] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the application, therefore, the protection scope of the application should be defined by the claims.​

Claims

1. A recombinant Escherichia coli strain for efficient L-tyrosine synthesis, characterized in that, The recombinant Escherichia coli was derived from Escherichia coli WSH-Z06, by knocking out the gene encoding a fusion branching acid mutase / prephenolic acid dehydratase in the E. coli genome. pheA Gene encoding the TrpE subunit of anthranilate synthase trpE TyrR gene, which encodes a DNA-binding transcriptional dual regulator. tyrR The gene encoding 3-deoxy-D-arabinohepenolate-7-phosphate synthase is expressed in free form. aroG fbr Genes encoding branched acid mutase and prephenyl acid dehydrogenase tyrA fbr The recombinant *E. coli* strain expresses the phosphoenolpyruvate synthase gene either freely or integratedly. ppsA and the gene encoding transketolase 1 tktA The gene aroG fbr ,Gene tyrA fbr ,Gene ppsA and genes tktA The nucleotide sequences are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.7, respectively.

2. A recombinant Escherichia coli strain that efficiently synthesizes L-tyrosine, characterized in that, Starting with Escherichia coli WSH-Z06, the gene encoding a fusion branching acid mutase / prephenolic acid dehydratase in the E. coli genome was knocked out. pheA Gene encoding the TrpE subunit of anthranilate synthase trpE TyrR gene, which encodes a DNA-binding transcriptional dual regulator. tyrR Integrated expression of the gene encoding transketolase 1 tktA and phosphoenolpyruvate synthase gene ppsA It also expressed the gene encoding 3-deoxy-D-arabinohepenolate-7-phosphate synthase in a free state. aroG fbr Genes encoding branched acid mutase and prephenyl acid dehydrogenase tyrA fbr and the gene encoding phosphatidylcholinesterase fpk ;Gene aroG fbr ,Gene tyrA fbr ,Gene ppsA ,Gene tktA and genes fpk The nucleotide sequences are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

3. The recombinant Escherichia coli according to claim 2, characterized in that, On the E. coli genome ykgh-betA site integration genes ppsA and genes tktA .

4. The recombinant Escherichia coli according to claim 2, characterized in that, Knock out the gene encoding pyruvate oxidase in the E. coli genome poxB and freely express the gene encoding phosphatidylcholinesterase. fpk .

5. The recombinant Escherichia coli according to claim 2, characterized in that, Knock out the gene encoding pyruvate oxidase in the E. coli genome poxB Gene encoding AroP permease, an aromatic amino acid transporter aroP and genes encoding tyrosine:H(+) cotransporters tyrP and freely express the gene encoding phosphatidylcholinesterase. fpk and the gene encoding the amino acid export protein YddG yddG The gene yddG The nucleotide sequence is shown in SEQ ID NO.12; the gene tyrP The nucleotide sequence is shown in SEQ ID NO.

11.

6. The recombinant Escherichia coli according to any one of claims 1 to 5, characterized in that, The heat-inducible expression vector pAP-B03 was used as the expression plasmid.

7. A method for producing L-tyrosine, characterized in that, L-tyrosine is produced by fermentation using the recombinant Escherichia coli according to any one of claims 1 to 6.

8. The method according to claim 7, characterized in that, The recombinant Escherichia coli according to any one of claims 1 to 6 is inoculated into the fermentation system and cultured at 32 to 34°C for 3 to 12 h, and then fermented at 36 to 40°C and 200 to 220 rpm for 48 to 60 h.

9. The method according to claim 8, characterized in that, The fermentation system includes 30-40 g / L glucose, 3-7 g / L (NH4)2SO4, 1-5 g / L KH2PO4, 1-5 g / L MgSO4·7H2O, 1-2 g / L sodium citrate, 0.5-1.5 g / L NaCl, 0.05-0.1 g / L vitamin B1, 0.1-0.12 g / L FeSO4·7H2O, 1-3 g / L yeast extract, 2-6 g / L peptone, and 1-2 mL / L trace element nutrient solution.

10. The use of the recombinant Escherichia coli according to any one of claims 1 to 6 or the method according to any one of claims 7 to 9 in the preparation of L-tyrosine or products containing L-tyrosine.

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