Construction and application of a plasmid-free recombinant escherichia coli for L-tyrosine production

Abstract

The application discloses a plasmid-free L-tyrosine-producing recombinant Escherichia coli strain and an application thereof. The application takes a high-yield L-tyrosine engineering strain Escherichia coli Tyr10 disclosed in the prior art as a starting strain, integrates three copies of an anti-feedback inhibition type prebenzoic acid dehydrogenase coding gene tyrA fbr , two copies of a shikimic acid kinase II coding gene aroL , two copies of a 3-dehydroquinate synthase coding gene aroB and one copy of a phosphoenolpyruvate synthase coding gene ppsA at multiple neutral sites in the genome, and adopts a temperature-triggered temperature control switch to regulate the expression of a phenylalanine synthesis key enzyme gene pheA , to obtain a recombinant strain. The obtained recombinant strain is fermented in a 5 L fermenter to produce L-tyrosine, and the yield reaches 79.6 g / L. The construction method adopts a genome multi-site integration mode, has good genetic stability, a stable fermentation process and a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the construction and application of a plasmid-free recombinant Escherichia coli for producing L-tyrosine, belonging to the field of industrial microbiology and metabolic engineering technology. Background Technology

[0002] L-Tyrosine is an important aromatic amino acid with wide applications in food additives, pharmaceutical intermediates, and fine chemicals. Traditional L-tyrosine production methods mainly include chemical synthesis and protein hydrolysis extraction. Chemical synthesis typically produces a D / L racemic mixture, requiring further separation and purification, demanding high production conditions, and potentially causing environmental pollution. Protein hydrolysis extraction uses animal hair, hooves, and other protein resources as raw materials, obtaining L-tyrosine through hydrolysis, decolorization, concentration, and crystallization. However, it suffers from low raw material utilization, high production costs, and a heavy environmental burden, making it difficult to meet the needs of large-scale production. With the development of synthetic biology and metabolic engineering, L-tyrosine biosynthesis based on microbial fermentation has gradually become an important technological route for industrial production. L-tyrosine biosynthesis uses glucose as a carbon source, generating aromatic amino acid precursors via the shikimic acid pathway, which are then further converted into L-tyrosine.

[0003] With the development of metabolic engineering technology, L-tyrosine biosynthesis based on microbial fermentation has become an important technical route for industrial production. L-tyrosine biosynthesis involves the shikimic acid pathway and its downstream aromatic amino acid branches, with multiple key enzymes working synergistically to convert carbon flux into end products. In the construction of existing engineered bacteria, metabolic flux is typically increased by enhancing the expression of key enzymes. However, L-tyrosine biosynthesis involves multiple metabolic nodes, and there is a coupling relationship between the expression levels of upstream and downstream enzymes. Simply overexpressing multiple genes sequentially may lead to carbon flux imbalance, precursor accumulation, or metabolic bottleneck shift, thus affecting overall production capacity. Therefore, based on existing engineered bacteria, how to hierarchically regulate and optimize the expression intensity and ratio of key enzymes in different metabolic segments to achieve a synergistic effect of flux enhancement and metabolic balance is a crucial technical problem that needs to be solved to further improve L-tyrosine production capacity. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a new L-tyrosine-producing strain and a method for producing L-tyrosine, in order to improve L-tyrosine yield, efficiency, and production intensity.

[0005] This invention is achieved through the following technical solution: The first objective of this invention is to provide a plasmid-free recombinant Escherichia coli that produces L-tyrosine, wherein, compared to the starting strain, the recombinant Escherichia coli overexpresses one or more of the following genes: (1) Feedback-resistant mutant encoding prephenyl acid dehydrogenase with three copies tyrA fbrM53I / A354V Gene; (2) Two copies encoding shikimate kinase II aroL Gene; (3) Two copies encoding 3-dehydroquinic acid synthase aroB Gene; (4) One copy encodes phosphoenolpyruvate synthase ppsA Gene.

[0006] In one embodiment of the present invention, the recombinant Escherichia coli further includes a gene for regulating the key enzyme in phenylalanine synthesis using a temperature-triggered temperature control switch. pheA The expression; the temperature-triggered temperature control switch is a CI857-P that incorporates TetR as an intermediate control layer. R / P L The system, with P R / P L The expression of the tetR gene is controlled by P. LtetO1 Promoter reverse regulation pheA The expression, in which P LtetO1 The promoter is inhibited by TetR.

[0007] In one embodiment of the present invention, the regulation specifically involves CI857 binding to P under low-temperature conditions. R / P L The promoter inhibits tetR transcription, thereby relieving TetR's effect on P. LtetO1 The inhibition, making pheA CI857 is maintained during the growth phase; upon entering the production phase and increasing the temperature, CI857 is inactivated and releases P. R / P L Promoter, induce tetR Enhanced expression, which in turn inhibits P LtetO1 Startup, implementation pheA Dynamic shutdown.

[0008] In one embodiment of the present invention, the starting strain is Escherichia coli Tyr10, which is disclosed in the patent application document with publication number CN118086161A.

[0009] In one embodiment of the present invention, the NCBI accession number of the shikimate kinase II is NP_414922.1; the NCBI accession number of the 3-dehydroquinic acid synthase is NP_417848.1; the NCBI accession number of the phosphoenolpyruvate synthase is NP_416217.1; the gene tyrA fbrM53I / A354VThe amino acid sequence is shown in SEQ ID NO.1.

[0010] In one embodiment of the present invention, the nucleotide sequences encoding the genes for shikimate kinase II, 3-dehydroquinic acid synthase, and phosphoenolpyruvate synthase are shown as Gene IDs: 945031, 947927, and 946209, respectively.

[0011] In one embodiment of the present invention, promoter P is used. j23119 The expression of the genes for prebenzoic acid dehydrogenase, shikimate kinase II, 3-dehydroquinic acid synthase, and phosphoenolpyruvate synthase is driven to enhance the enzyme activity of the enzymes and / or the expression level of their encoding genes.

[0012] In one embodiment of the present invention, the promoter P j23119 The nucleotide sequence is shown in SEQ ID NO.2.

[0013] In one embodiment of the invention, the genes encoding the prebenzoic acid dehydrogenase, shikimate kinase II, 3-dehydroquinic acid synthase, and phosphoenolpyruvate synthase are integrated into the genome of the starting strain.

[0014] In one embodiment of the present invention, the three copies encoding the prephenyl acid dehydrogenase gene are each integrated into a genomic neutral site. ylbE , ilvG and gatZ Site.

[0015] In one embodiment of the present invention, two copies encoding the shikimate kinase II gene are respectively integrated into a genomic neutral site. ydjK and fdrA Site.

[0016] In one embodiment of the present invention, two copies encoding the 3-dehydroquinic acid synthase gene are respectively integrated into a genomic neutral site. yghX and yeeL Site.

[0017] In one embodiment of the present invention, a copy encoding the phosphoenolpyruvate synthase gene is integrated into a genomic neutral site. elfC Site.

[0018] A second objective of this invention is to provide a method for producing L-tyrosine, wherein the recombinant Escherichia coli is fermented using glucose as a substrate.

[0019] In one embodiment of the present invention, the method involves aerobic fermentation of the recombinant Escherichia coli in a fermentation medium to obtain a fermentation broth containing L-tyrosine.

[0020] In one embodiment of the present invention, during the aerobic fermentation process, the dissolved oxygen is controlled at 30% or higher.

[0021] In one embodiment of the present invention, during the aerobic fermentation process, when the glucose in the initial culture medium is depleted, glucose is added to control the glucose concentration at 0.1-1.0 g / L.

[0022] In one embodiment of the present invention, the pH is controlled at 6.4-6.6 during the aerobic fermentation process.

[0023] In one embodiment of the present invention, the method includes the following steps: (1) Seed culture: The recombinant bacteria are inoculated into a seed culture medium and cultured to obtain seed liquid; (2) Fermentation: The seed liquid obtained in step (1) is transferred to the fermentation medium to produce L-tyrosine.

[0024] In one embodiment of the present invention, the seed culture medium comprises: 5-15 g / L peptone, 4-6 g / L yeast extract, and 5-15 g / L sodium chloride.

[0025] In one embodiment of the present invention, the fermentation medium contains: 20-30 g / L glucose and 5 g / L yeast extract. 8 g / L, magnesium sulfate 1-3 g / L, potassium dihydrogen phosphate 2-4 g / L, sodium citrate 0.5-1.5 g / L, ammonium sulfate 6-7 g / L, manganese sulfate 15-25 mg / L, ferrous sulfate 30-40 mg / L, L Methionine 0.5-1.5 g / L, L Tryptophan 0.05-0.15 g / L, Biotin 0.4-0.6 mg / L, Vitamin B1 0.4-0.6 mg / L, Vitamin B3 0.4-0.6 mg / L, Vitamin B5 0.4-0.6 mg / L, and a mixture of trace elements 1-3 mL / L.

[0026] In one embodiment of the present invention, the metal element mixture contains: Na2MoO4·2H2O 2-3 g / L, NiSO4·6H2O 1-2 g / L, CoCl2·6H2O 1.5-2 g / L, CaCl2·2H2O 8-12 g / L, ZnSO4·7H2O 0.5-1 g / L, H3BO3 0.1-0.2 g / L, Al2(SO4)3·18H2O 2-3 g / L, and CuSO4·5H2O 0.3-0.5 g / L.

[0027] The beneficial effects of this invention are: This invention uses the publicly disclosed high-yield L-tyrosine engineered strain Escherichia coli Tyr10 (publication number CN118086161A) as the starting strain. By implementing differentiated copy number integration expression of key segments of the L-tyrosine biosynthesis pathway, an expression intensity gradient structure is constructed to achieve hierarchical enhancement and overall coordinated optimization of metabolic flux.

[0028] Specifically, this invention integrates three copies of the anti-feedback inhibition prephenylate dehydrogenase encoding gene at different neutral sites in the genome. tyrA fbr Strengthening key downstream rate-limiting steps; integrating two copies of the shikimate kinase II encoding gene. aroL and two copies of the gene encoding 3-dehydroquinic acid synthase. aroB It increases the flux of the shikimic acid pathway and integrates a one-copy gene encoding phosphoenolpyruvate synthase. ppsA This enhances precursor supply capacity. The combination and integration of genes from different regions forms an expression intensity gradient structure, enabling a more rational allocation of carbon flux within the synthetic pathway. Furthermore, a temperature-triggered thermostatic switch is used to regulate key enzyme genes involved in phenylalanine synthesis. pheA The expression of [the substance] was used to obtain recombinant engineered strains.

[0029] The engineered strain constructed using this invention achieved an L-tyrosine yield of 79.6 g / L in a 5 L fermenter, representing an increase of approximately 20.8% compared to the starting strain Tyr10 (65.9 g / L). This further improvement under high-yield conditions demonstrates excellent fermentation performance and production stability. Therefore, this invention establishes a regulatory model for the differential copy number integration expression of different metabolic regions, providing a stable and scalable construction strategy for the efficient production of aromatic amino acids. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The results of fed-batch fermentation of recombinant strain TYR-4 in a 5 L fermenter.

[0032] Figure 2 Based on Cl587-P R / P L and tetR-P LtetO1 A schematic diagram of the dynamic control of the circuit's temperature rise.

[0033] Figure 3Based on Cl587-P R / P L A schematic diagram of the cooling-type dynamic control of the circuit. Detailed Implementation

[0034] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0035] Materials and methods involved in the embodiments of the present invention: (1) Culture medium: Solid culture medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 20 g / L. Seed culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0036] Fermentation medium: The fermentation medium contains: glucose 25 g / L, yeast extract 6 g / L, magnesium sulfate 2 g / L, potassium dihydrogen phosphate 3 g / L, sodium citrate 1 g / L, ammonium sulfate 6.5 g / L, manganese sulfate 20 mg / L, ferrous sulfate 35 mg / L, L Methionine 1 g / L, L Tryptophan 0.1 g / L, Biotin 0.5 mg / L, Vitamin B1 0.5 mg / L, Vitamin B3 0.5 mg / L, Vitamin B5 0.5 mg / L, and a trace element mixture of 2.0 mL / L.

[0037] Metal ionic liquids: Na2MoO4·2H2O 2.50 g / L, NiSO4·6H2O 1.60 g / L, CoCl2·6H2O 1.80 g / L, CaCl2·2H2O 10.00 g / L, ZnSO4·7H2O 0.75 g / L, H3BO3 0.14 g / L, Al2(SO4)3·18H2O 2.25 g / L, CuSO4·5H2O 0.4 g / L.

[0038] (2) Glucose determination: Centrifuge the fermentation broth at 12,000 rpm for 10 min and collect the supernatant. Dilute to an appropriate ratio and use an M-100 biosensor analyzer to detect the glucose concentration of the fermentation broth.

[0039] (3) Determination of L-tyrosine: Mobile phase A consisted of 3.01 g anhydrous sodium acetate, 200 μL triethylamine, and 5 mL tetrahydrofuran dissolved in 995 mL ultrapure water, with the pH adjusted to 7.2 using 10% acetic acid. Mobile phase B consisted of 3.01 g anhydrous sodium acetate dissolved in 200 mL ultrapure water, with the pH adjusted to 7.2 using 10% acetic acid, followed by the addition of 400 mL acetonitrile and 400 mL methanol. The chromatographic column was an Aglient ZORBAX SB-Aq 250 × 4.6 mm, 5 μm. Pre-column online derivatization was performed using OPA as the derivatizing agent. The column temperature was 40 °C, and gradient elution was performed at a flow rate of 1.0 mL / min. The UV detection wavelength was UV 338 nm.

[0040] The method for calculating glucose yield is: L-tyrosine yield × lower tank volume / glucose consumption.

[0041] Fermentation broth pretreatment: Take 1 mL of fermentation broth, dilute it with 3M hydrochloric acid to an appropriate ratio, shake vigorously to mix, centrifuge at 14000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm inorganic filter membrane, and then use HPLC to detect it. Substitute the obtained peak area into the linear regression equation, and multiply the result by the dilution factor to obtain the L-tyrosine concentration in the fermentation broth.

[0042] Plasmid loss method: The strain was inoculated into LB medium and IPTG was added for induction to eliminate the pTargetF targeting plasmid containing gRNA-N20. The strain was cultured in LB medium at 42℃ to eliminate the pCas9 plasmid.

[0043] The pCas plasmid used in the gene knockout in the following examples was purchased from addgene, catalog number 60847.

[0044] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0045] Example 1: Feedback-resistant mutant tyrA fbr Gene multicopy integration construction The recombinant Escherichia coli Tyr10, which was constructed and preserved in the early stage, was rationally metabolically engineered as a chassis cell (disclosed in patent: CN118086161A). The target gene was integrated and replaced using CRISPR / Cas9 gene editing technology, and all materials used were commercially available reagents, plasmids and other materials.

[0046] To further enhance the metabolic flux of the L-tyrosine master synthesis pathway, a three-copy feedback resistance mutant was integrated into the genome of the TYR-10 starting strain. tyrAfbr Genes. The aforementioned tyrA fbr The gene encodes prebenzoic acid dehydrogenase, containing the M53I / A354V mutation, which is used to relieve feedback inhibition of the final product. The three copies... tyrA fbr Integrating into genomic neutral sites respectively ylbE , ilvG and gatZ The primers for constructing the recombinant strain TYR-1.3 were determined by identifying the target site. See Table 1 for the primers used in the construction.

[0047] In order to build on ylbE Site integration tyrA fbr The recombinant strain, using the genomic DNA of Escherichia coli W3110 as a template, was synthesized through a pair of... ylbE- up-f / ylbE -up-r primers were used to amplify the upstream homologous arm fragment; through a pair of -up-r primers... ylbE -dn-f / ylbE -dn-r primers were used to amplify downstream homologous arm fragments; using a mutant containing feedback resistance... tyrA fbr Using the gene plasmid as a template, through tyrA fbr -f / tyrA fbr -r primer amplification obtained tyrA fbr Expression cassette fragment. The above fragments were fused to obtain the integrated donor DNA fragment. Using the same method, the fusion fragment and pTargetF plasmid were co-transformed into Tyr10 strain carrying the Cas9 plasmid. After culture, antibiotic selection, and PCR verification, the expression cassette fragment was obtained. ylbE Site integration tyrA fbr Recombinant strains.

[0048] PCR reaction procedure: Prime Star MIX polymerase was used as the polymerase for the following PCR amplification: denaturation at 95℃ for 3 minutes; denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, polymerization at 72℃ for 1 minute, for 29 cycles; and polymerization at 72℃ for 5 minutes. A fusion fragment was obtained by homologous recombination of the three fragments obtained from PCR amplification using Gibson assembly. The fusion fragment and pTargetF plasmid were transformed into Tyr10 strain carrying Cas9 plasmid by electroporation. Colonies were screened on LB plates containing Kan and Spe resistance at 30℃ to verify the growth of the target strain. After IPTG induction and incubation at 42℃ for 16 hours, growth was confirmed on LB solid medium but not on LB solid medium containing Kan or LB solid medium containing Spe. The recombinant strain TYR-1.1 was obtained.

[0049] Using the same method, in ilvG The site further integrates the second copy. tyrA fbr ;exist gatZ The site further integrates the third copy. tyrA fbr After cultivation, screening, and validation, a genome containing three copies was obtained. tyrA fbr The recombinant strain TYR-1.3.

[0050] After the above strains were constructed, the accumulation of L-tyrosine was verified using shake-flask methods (Table 2). Table 1: Relevant Primer Sequences

[0051] Table 2. Effects of gene expression on tyrosine accumulation.

[0052] Example 2: Shikimate kinase II gene aroL Multi-copy integration Using the *Escherichia coli* TYR-1.3 constructed in Example 1 as the starting strain, the shikimic acid kinase II gene was overexpressed twice. aroL The recombinant strain TYR-2.2 was constructed. (Gene) aroL The gene ID is 945031, which is integrated into the genomic neutral site. ydjK and fdrA The primers used to construct the strain are shown in Table 3.

[0053] In order to build on ydjK Site integration aroLThe recombinant strain of the gene, using the genomic DNA of Escherichia coli W3110 as a template, through a pair of ydjK -up-f / ydjK -up-r primers were used to amplify the upstream homologous arm fragment; through a pair of -up-r primers... ydjK -dn-f / ydjK Downstream homologous arm fragments were obtained by amplification using -dn-r primers; using E. coli genomic DNA as a template, through... aroL -f / aroL -r primer amplification obtained aroL Expression cassette fragment. The above fragments are fused to obtain the integrated donor DNA fragment.

[0054] The fusion fragment and pTargetF plasmid were co-transformed into the TYR-1.3 strain carrying the Cas9 plasmid. After culture, resistance selection, and PCR verification, the desired Cas9 plasmid was obtained. ydjK Site integration aroL Recombinant strains of genes.

[0055] Using the same method, in fdrA The site further integrates the second copy. aroL Genes. After culturing, screening, and validation, genes containing two copies of each gene were obtained. aroL The recombinant strain TYR-2.2.

[0056] After the above strains were constructed, the accumulation of L-tyrosine was verified using shake-flask methods (Table 4).

[0057] Table 3: Relevant Primer Sequences

[0058] Table 4. Effects of gene expression on tyrosine accumulation

[0059] Example 3: 3-Dehydroquinic acid synthase gene aroB Multi-copy integration Using the *E. coli* TYR-2.2 strain constructed in Example 2 as the starting strain, the enzyme encoding 3-dehydroquinic acid synthase was overexpressed twice. aroB Gene ID: 947927, integrated into a neutral site in the genome. yghX and yeeL The recombinant strain TYR-3.2 was constructed. The primers used to construct the strain are shown in Table 5.

[0060] In order to build on yghX Site integration aroB The recombinant strain of the gene, using the genomic DNA of Escherichia coli W3110 as a template, through a pair of yghX -up-f / yghX -up-r primers were used to amplify the upstream homologous arm fragment; through a pair of -up-r primers... yghX -dn-f / yghX Downstream homologous arm fragments were obtained by amplification using -dn-r primers; using E. coli genomic DNA as a template, through... aroB -f / aroB -r primer amplification obtained aroB Expression cassette fragment. The above fragments are fused to obtain the integrated donor DNA fragment.

[0061] The fusion fragment and pTargetF plasmid were co-transformed into the TYR-2.2 strain carrying the Cas9 plasmid. After culture, resistance selection, and PCR verification, the desired fusion fragment was obtained. yghX Site integration aroB Recombinant strains of genes.

[0062] Using the same method, in yeeL The site further integrates the second copy. aroB Genes. After culturing, screening, and validation, genes containing two copies of each gene were obtained. aroB The recombinant strain TYR-3.2.

[0063] After the above strains were constructed, the accumulation of L-tyrosine was verified using shake-flask methods (Table 6).

[0064] Table 5: Relevant Primer Sequences

[0065] Table 6. Effects of gene expression on tyrosine accumulation

[0066] Example 4: Phosphoenolpyruvate synthase gene ppsA Integrated expression Using the *E. coli* TYR-3.2 strain constructed in Example 3 as the starting strain, an enzyme encoding phosphoenolpyruvate synthase was integrated. ppsA Gene (Gene ID: 946209) to genomic neutral site elfC The recombinant strain TYR-4 was constructed. The primers used for constructing the strain are shown in Table 7.

[0067] In order to build on elfC Site integration ppsA The recombinant strain of the gene, using the genomic DNA of Escherichia coli W3110 as a template, through a pair of elfC -up-f / elfC -up-r primers were used to amplify the upstream homologous arm fragment; through a pair of -up-r primers... elfC -dn-f / elfCDownstream homologous arm fragments were obtained by amplification using -dn-r primers; using E. coli genomic DNA as a template, through... ppsA -f / ppsA -r primer amplification obtained ppsA Expression cassette fragment. The above fragments are fused to obtain the integrated donor DNA fragment.

[0068] The fusion fragment and pTargetF plasmid were co-transformed into the TYR-3.2 strain carrying the Cas9 plasmid. After culture, resistance selection, and PCR verification, the desired fusion fragment was obtained. elfC Site integration ppsA The recombinant strain TYR-4.

[0069] After the above strains were constructed, the accumulation of L-tyrosine was verified at the shake-flask level. The TYR-4 strain accumulated 5.71 g / L of L-tyrosine at the shake-flask level.

[0070] Table 7: Relevant Primer Sequences

[0071] Example 5: Dynamic Regulation of Metabolic Flow Distribution by Temperature Control Switch Because the chassis cells used in this invention have had the key gene for L-phenylalanine synthesis knocked out. pheA The basal cells exhibited a clear L-phenylalanine auxotrophic pattern, requiring an additional 1 g / L of L-phenylalanine to maintain growth.

[0072] In CI857-P R / P L Based on the existing system, TetR was introduced as an intermediate control layer to construct a P-based system. R / P L control tetR Expressed, and by P LtetO1 Promoter reverse regulation pheA The "heat-triggered" control system Figure 2 In this system, CI857 binds to P under low temperature (30℃) conditions. R / P L Promoter suppression tetR Transcription, thereby relieving TetR from P LtetO1 The inhibition, making pheA CI857 is maintained during the growth phase; upon entering the production phase, the temperature is increased to 37 °C, causing CI857 to be inactivated and releasing P. R / P L Promoter, induce tetR Enhanced expression, which in turn inhibits P LtetO1 Startup, implementation pheA Dynamic shutdown.

[0073] Based on strain TYR-4, this system was introduced to construct strain TYR-5. The specific steps are as follows: Using Escherichia coli TYR-4 constructed in Example 4 as the starting strain, a model based on CI857–P was constructed in its genome. R / P L A temperature-triggered dynamic regulation system based on the TetR regulatory loop was used to obtain the recombinant strain TYR-5. The primers used to construct the strain are shown in Table 8.

[0074] In order to construct P LtetO1 Promoter reverse regulation pheA The recombinant strain, using the genomic DNA of Escherichia coli W3110 as a template, was synthesized through a pair of... pheA -up-f / pheA -up-r primers were used to amplify the upstream homologous arm fragment of the promoter; through a pair of... pheA -dn-f / pheA -dn-r primers were used to amplify the downstream homologous arm fragment of the promoter; using the synthesized DNA fragment as a template, P... LtetO1 -f / P LtetO1 -r amplification to obtain P LtetO1 Promoter expression cassette fragment. The above fragments are fused to obtain an integrated donor DNA fragment.

[0075] The fusion fragment and pTargetF plasmid were co-transformed into the TYR-4 strain carrying the Cas9 plasmid. After culture, resistance selection, and PCR verification, P-resistant strains were obtained. LtetO1 Promoter reverse regulation pheA The recombinant strain TYR-4.1.

[0076] In order to build on ygaY Site integration P R / P L - tetR The recombinant strain of the gene, using the genomic DNA of Escherichia coli W3110 as a template, through a pair of ygaY -up-f / ygaY -up-r primers were used to amplify the upstream homologous arm fragment of the promoter; through a pair of... ygaY -dn-f / ygaY -dn-r primers were used to amplify the downstream homologous arm fragment of the promoter; using the synthesized DNA fragment as a template, P... R / P L - tetR -f and P R / P L - tetR -r primer amplification to obtain P R / P L - tetRExpression cassette fragment. The above fragments are fused to obtain the integrated donor DNA fragment.

[0077] The fusion fragment and pTargetF plasmid were co-transformed into the TYR-4.1 strain carrying the Cas9 plasmid. After culture, resistance selection, and PCR verification, the desired Cas9 plasmid was obtained. ygaY Site integration P R / P L - tetR The recombinant strain TYR-4.2.

[0078] In order to build on ydfT Site integration P RM -The recombinant strain of the CI857 gene, using the genomic DNA of Escherichia coli W3110 as a template, through a pair of ydfT -up-f / ydfT -up-r primers were used to amplify the upstream homologous arm fragment of the promoter; through a pair of... ydfT -dn-f / ydfT -dn-r primers were used to amplify the downstream homologous arm fragment of the promoter; using the synthesized DNA fragment as a template, P... RM -CI857-f / P RM -CI857-r primers were used to amplify P R / P L - tetR Expression cassette fragment. The above fragments are fused to obtain the integrated donor DNA fragment.

[0079] The fusion fragment and pTargetF plasmid were co-transformed into the TYR-4.2 strain carrying the Cas9 plasmid. After culture, resistance selection, and PCR verification, the desired Cas9 plasmid was obtained. ydfT Site integration P RM -The recombinant strain TYR-5 with the CI857 gene.

[0080] During fermentation, the growth stage temperature was set at 30℃. After entering the production stage (approximately 10 hours), the temperature was increased to 37℃. Solubility characterization experiments showed that, compared to 30℃, restoring the temperature to 37℃ effectively alleviated the problem of limited L-tyrosine solubility under low-temperature conditions, thereby improving the extracellular physical microenvironment. Fermentation evaluation results showed that after adopting the temperature control strategy, the L-tyrosine yield reached 6.44 g / L, an increase of 12.7% compared to strain TYR-4.

[0081] Table 8: Relevant Primer Sequences

[0082] Example 6: Fed-batch fermentation of recombinant strain TYR-5 in a fermenter The recombinant strain TYR-5 was fermented in a 5 L fermenter under the following conditions: (1) Seed activation and culture The engineered strain TYR-5 constructed in Example 5 was streaked onto a solid plate and cultured at 37°C for 12 h. A single colony was picked using an inoculation needle, and a loopful was inoculated into 50 mL / 500 mL seed culture medium for further culture. The culture was then carried out at 37°C with shaking at 200 rpm for 8 h. OD 600 Value at 10 Between 1 and 2.

[0083] (2) Fermentation culture in a 5 L fermenter The seed culture prepared in step (1) was inoculated at an inoculation rate of 10% (v / v) into a 5 L fermenter containing 2.5 L of initial fermentation medium. Initial fermentation conditions were: temperature 37℃, pH 7.0, airflow rate 2 vvm, constant tank pressure 0.03 MPa, and initial rotation speed 350 r / min. During fermentation, dissolved oxygen was maintained above 30% by adjusting the aeration rate and stirring speed, with a maximum airflow rate of 3 vvm and a maximum stirring speed of 1000 r / min. Ammonia was added to maintain the pH at 6.4. 6.6. When the initial glucose in the fermentation medium is completely depleted, the glucose concentration is controlled below 1.0 g / L by feeding in a 720 g / L glucose solution until fermentation is complete.

[0084] The results showed that the method described in this embodiment was effective in producing L from strain TYR-5 via fermentation. Tyrosine, fermented for 48 h, L Tyrosine production reached 79.6 g / L, and glucose yield was 23.7%.

[0085] After fermentation of strain TYR-5 was completed, the cell survival rate was tested. The results showed that the cell mortality rate of the engineered strain of this invention at the end of fermentation was 46.3%, which was not significantly higher than that of the original strain Tyr10 (43.5%).

[0086] The above results demonstrate that the engineered strain constructed by multi-site integration in this invention increases L-tyrosine production without significantly adversely affecting cell viability, and the fermentation process remains stable.

[0087] Comparative Example 1: To further verify the rationality of the integration copy number of each key gene, comparative experiments were conducted to further increase the integration copy number of each gene during the construction of the engineered strain of this invention.

[0088] (1) The TYR-1.3 strain constructed in Example 1 ( tyrAfbr Based on the three copies, another copy is then integrated. tyrA fbr Genes were extracted to obtain strain TYR-1.4. The L strain was validated using shake-flask methods. Regarding the accumulation of tyrosine, strain TYR-1.4 accumulated 4.02 g / L of L-tyrosine at the shake flask level, which was lower than the yield of strain TYR-1.3 (4.3 g / L).

[0089] (2) The TYR-2.2 strain constructed in Example 2 ( aroL Based on two copies, then integrate one more copy. aroL Genes were extracted to obtain strain TYR-2.3. The L strain was validated using shake-flask methods. Regarding the accumulation of tyrosine, strain TYR-2.3 accumulated 4.8 g / L of L-tyrosine at the shake flask level, which was not significantly higher than the yield of strain TYR-2.2 (4.86 g / L).

[0090] (3) The TYR-3.2 strain constructed in Example 3 ( aroB Based on two copies, then integrate one more copy. aroB Genes were extracted to obtain strain TYR-3.3. The L strain was validated using shake-flask methods. Regarding the accumulation of tyrosine, strain TYR-3.3 accumulated 5.12 g / L of L-tyrosine at the shake flask level, which was lower than the yield of strain TYR-3.2 (5.33 g / L).

[0091] (4) The TYR-4 strain constructed in Example 4 ( ppsA Based on a single copy, then integrate another copy. ppsA Genes were extracted to obtain strain TYR-4.3. The L strain was validated using shake-flask methods. Regarding the accumulation of tyrosine, strain TYR-4.3 accumulated 5.3 g / L of L-tyrosine at the shake flask level, which was lower than the yield of strain TYR-4 (5.7 g / L).

[0092] Comparative results show that a higher integration copy number for each key gene is not necessarily more beneficial; different gene segments require specific copy number combinations to achieve optimal fermentation performance. The present invention determines... tyrA fbr Three copies aroL Two copies, aroB Two copies and ppsA Combinations of single copies exhibit the best yield levels.

[0093] Comparative Example 2: Utilizing the temperature-sensitive inhibitory protein CI857 derived from bacteriophages and its corresponding P R / PL A starter was constructed, creating a "cooling-triggered" control switch, which was then used for control. pheA The expression ( Figure 3 In this system, the CI857 conformation is unstable at 37°C and cannot bind P. R / P L promoter, thus allowing pheA Normal transcription; when the temperature is lowered to 30°C, CI857 restores DNA-binding activity and inhibits P. R / P L promoter, thereby enabling pheA The goal was to dynamically shut down the temperature control to limit the competition of L-phenylalanine synthesis for aromatic precursor flux during the production stage. Based on this strategy, strain TYR-5.1 was constructed. However, fermentation results showed that the final L-tyrosine yield after adopting the temperature control strategy was only 5.53 g / L, failing to achieve the expected increase.

[0094] The sequences used in the examples are as follows: tyrA fbr (M53I / A354V) amino acid sequence (SEQ ID NO.1): MVAELTALRDQIDEVDKALLNLLAKRLELVAEVGEVKSRFGLPIYVPEREASILASRRAEAEALGVPPDLIEDVLRRVMRESYSSENDKGFKTLCPSLRPVVIVGGGGQMGRLFEKMLTLSGYQVRILEQHDWDRAADIVADAGMVIVSVPIHVTEQVIGKLPPLPKDCILVDLASVKNGPLQA MLVAHDGPVLGLHPMFGPDSGSLAKQVVVWCDGRKPEAYQWFLEQIQVWGARLHRISAVEHDQNMAFIQALRHFATFAYGLHLAEENVQLEQLLALSSPIYRLELAMVGRLFAQDPQLYADIIMSSERNLALIKRYYKRFGEAIELLEQGDKQAFIDSFRKVEHWFGDYVQRFQSESRVLLRQANDNRQ* promoter P j23119 The nucleotide sequence (SEQ ID NO.2) is: TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC.

[0095] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A recombinant *Escherichia coli* strain for plasmid-free production of L-tyrosine, characterized in that, Compared to the starting strain, the recombinant Escherichia coli overexpressed one or more of the following genes: (1) Feedback-resistant mutant encoding prephenyl acid dehydrogenase with three copies tyrA fbrM53I / A354V Gene; (2) Two copies encoding shikimate kinase II aroL Gene; (3) Two copies encoding 3-dehydroquinic acid synthase aroB Gene; (4) One copy encodes phosphoenolpyruvate synthase ppsA Gene.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The recombinant E. coli also includes a gene for a key enzyme in phenylalanine synthesis regulated by a temperature-triggered temperature control switch. pheA The expression; the temperature-triggered temperature control switch is a CI857-P that incorporates TetR as an intermediate control layer. R / P L The system, with P R / P L The expression of the tetR gene is controlled by P. LtetO1 Promoter reverse regulation pheA The expression, in which P LtetO1 The promoter is inhibited by TetR.

3. The recombinant Escherichia coli according to claim 1, characterized in that, The starting strain includes Escherichia coli Tyr10.

4. The recombinant Escherichia coli according to claim 1, characterized in that, The NCBI accession number for shikimate kinase II is NP_414922.1; the NCBI accession number for 3-dehydroquinic acid synthase is NP_417848.1; the NCBI accession number for phosphoenolpyruvate synthase is NP_416217.1; the gene tyrA fbrM53I / A354V The amino acid sequence is shown in SEQ ID NO.

1.

5. The recombinant Escherichia coli according to claim 1, characterized in that, The recombinant E. coli uses promoter P j23119 This drives the expression of the genes for prebenzoic acid dehydrogenase, shikimate kinase II, 3-dehydroquinic acid synthase, and phosphoenolpyruvate synthase.

6. The recombinant Escherichia coli according to claim 5, characterized in that, The promoter P j23119 The nucleotide sequence is shown in SEQ ID NO.

2.

7. The recombinant Escherichia coli according to claim 1, characterized in that, The genes for prephenylacetic acid dehydrogenase, shikimate kinase II, 3-dehydroquinic acid synthase, and phosphoenolpyruvate synthase were integrated into the genome of the starting strain; among them... The three copies encoding the prephenylacetic acid dehydrogenase gene were each integrated into a neutral site in the genome. ylbE , ilvG and gatZ site; The two copies encoding the shikimate kinase II gene were each integrated into a neutral site in the genome. ydjK and fdrA site; The two copies encoding the 3-dehydroquinic acid synthase gene were each integrated into a neutral site in the genome. yghX and yeeL site; One copy encoding the phosphoenolpyruvate synthase gene was integrated into a neutral site in the genome. elfC Site.

8. A method for producing L-tyrosine, characterized in that, The recombinant Escherichia coli according to any one of claims 1-7 is fermented using glucose as a substrate.

9. The method according to claim 8, characterized in that, The method involves aerobic fermentation of the recombinant Escherichia coli in a fermentation medium to obtain a fermentation broth containing L-tyrosine. During the aerobic fermentation process, dissolved oxygen is controlled at 30% or higher, and pH is controlled at 6.4-6.

6. During the aerobic fermentation process, when glucose in the initial culture medium is depleted, glucose is added to control the glucose concentration at 0.1-1.0 g / L.

10. The method according to claim 9, characterized in that, The fermentation medium contains: glucose 20-30 g / L, yeast extract 5 g / L. 8 g / L, magnesium sulfate 1-3 g / L, potassium dihydrogen phosphate 2-4 g / L, sodium citrate 0.5-1.5 g / L, ammonium sulfate 6-7 g / L, manganese sulfate 15-25 mg / L, ferrous sulfate 30-40 mg / L, L Methionine 0.5-1.5 g / L, L Tryptophan 0.05-0.15 g / L, Biotin 0.4-0.6 mg / L, Vitamin B1 0.4-0.6 mg / L, Vitamin B3 0.4-0.6 mg / L, Vitamin B5 0.4-0.6 mg / L, and a mixture of trace elements 1-3 mL / L.

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