Genetically engineered bacterium capable of producing tyrosine at high yield based on self-adaptive regulation and construction method and application of genetically engineered bacterium

Abstract

The invention relates to the production field of a compound biotechnology and a fermentation engineering technology, in particular to a genetic engineering bacterium capable of producing tyrosine at high yield based on self-adaptive regulation and a construction method and application of the genetic engineering bacterium. Wild E.coli.W3110 is used as a chassis microorganism, and a promoter participating in flagellum construction is used for replacing a branch pathway promoter; the branching pathway of the tyrosine precursor is weakened, and the supply of the tyrosine precursor is enhanced; the biosynthesis pathway of tyrosine is enhanced; carrying out site-specific mutagenesis on a key enzyme in a tyrosine synthesis pathway, and knocking out a tyrosine regulatory protein gene tyrR to relieve feedback inhibition; by adopting a fed-batch fermentation method, the yield of tyrosine can reach 86.4 g / L in a 5L fermentation tank for 40h, the sugar-acid conversion rate is 26.3%, and the method has a relatively good industrial prospect.

Description

Technical Field

[0001] The present invention relates to the field of compound biotechnology and fermentation engineering technology production, in particular to a genetic engineering bacterium based on adaptive regulation and high tyrosine production, and a construction method and application thereof. Background Art

[0002] Tyrosine is a conditionally essential amino acid and a ketogenic and glucogenic amino acid for the human body. It is composed of a benzene ring structure connected by a carboxyl group and an amino group through covalent bonds. The main functions of tyrosine in the body involve protein synthesis and metabolic pathways. It is a precursor of catecholamines such as dopamine, norepinephrine and epinephrine, and is involved in the synthesis and transmission of neurotransmitters. In addition, tyrosine is also a precursor for the synthesis of thyroid hormones and melanin. These metabolic pathways are essential for maintaining the function of the nervous system, temperature regulation, and pigmentation of the skin and hair.

[0003] Tyrosine is widely used in medicine, biotechnology and food industry. In medicine, it can be used to treat neurological diseases such as Parkinson's disease and depression because it is a precursor of dopamine and adrenaline, which can regulate emotions and improve concentration. In the field of biotechnology, tyrosine is used to study protein function and metabolic engineering. By modifying metabolic pathways, high value-added products such as natural pigments and flavors can be produced. In the food industry, tyrosine is used as a nutritional supplement to improve energy metabolism and cognitive function. It is often used in functional foods and dietary supplements, and can also enhance flavor.

[0004] The main production methods of tyrosine include chemical synthesis and microbial fermentation. Chemical synthesis usually synthesizes tyrosine from simple precursor compounds through multi-step organic reactions. The advantage of this method is that the purity of the product can be controlled, but the disadvantages are complicated steps, expensive reagents, harsh reaction conditions, and low yield. It usually produces a large amount of waste and is not environmentally friendly. In contrast, the microbial fermentation method uses metabolically engineered microorganisms to convert precursor substances into tyrosine through a fermentation process. The main advantage of this method is that the reaction conditions are mild, extreme conditions such as high temperature and high pressure are not required, the production process is more environmentally friendly, and the cost is low. By optimizing the strain and fermentation process, the fermentation method can achieve efficient tyrosine production. In addition, microbial fermentation can be carried out in large-scale industrial production, and there are relatively few by-products, which is suitable for the needs of modern green chemical industry.

[0005] Microbial fermentation has been adopted as the mainstream method for large-scale production of most natural amino acid products. Therefore, constructing a tyrosine-producing strain with a clear genetic background and relatively high yield is an urgent problem to be solved. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a genetic engineering bacterium based on adaptive regulation and high production of tyrosine, and a construction method and application thereof. A genetic engineering bacterium Ltyr-14 that synthesizes tyrosine from scratch is constructed by the method of the present invention. The fermentation production of tyrosine by the engineering bacterium is simple to operate, and tyrosine can be directly synthesized with a cheap carbon source, which has a good industrial application prospect.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a genetically engineered bacterium for high tyrosine production based on adaptive regulation, namely Ltyr-14, which is obtained by the following genetic modification method: in the genome of the starting strain wild-type E. coli W3110, the natural promoter of the prephenate dehydratase gene pheA is replaced with a promoter P involved in the adaptive regulation of flagella construction. fliC , strain Ltyr-01 was obtained; the natural promoters of the anthranilate synthase genes trpD and trpE were replaced with promoters involved in the adaptive regulation of flagella construction P fliC , and obtained strain Ltyr-02; knocked out the tyrosine regulatory protein gene tyrR to obtain strain Ltyr-03; introduced the mutated 3-deoxy-7-phosphoheptanoate synthase gene aroG and prephenate dehydrogenase gene pyrA at the ygaY site of the genome and connected them with the ribosome binding site RBS sequence for overexpression, and the strain Ltyr-03 was obtained by P trc promoter to initiate transcription, and strain Ltyr-04 was obtained; 3-dehydroquinate synthase gene aroB, 3-deoxy-7-phosphoheptanoate synthase genes aroF and aroH, 3-dehydroquinic acid dehydratase gene aroD, shikimate dehydrogenase gene aroE, shikimate kinase genes aroL and aroK, 5-enolpyruvylshikimate-3-phosphate synthase gene aroA, chorismate synthase gene aroC, tyrosine aminotransferase gene tyrB were introduced into the yghX, gapC, ilvG, yeeL, ycgH, yciQ, mbhA, rph, yghE, and yjiP sites of the genome, respectively. trc The promoter was activated to obtain strains Ltyr-05, Ltyr-06, Ltyr-07, Ltyr-08, Ltyr-09, Ltyr-10, Ltyr-11, Ltyr-12, Ltyr-13, and Ltyr-14. The Ltyr-14 strain was the tyrosine production strain obtained after the final transformation.

[0009] Preferably, the starting strain of the genetically engineered bacteria for adaptively regulating high tyrosine production is wild-type E. coli W3110 (ATCC 27325).

[0010] Preferably, in the genetically engineered bacteria for high tyrosine production based on adaptive regulation, the nucleotide sequence of the prephenate dehydratase gene pheA is shown in SEQ ID NO.1 (pheA; NCBI-GeneID: 947081); the nucleotide sequence of the anthranilate synthase gene trpD is shown in SEQ ID NO.2 (trpD; NCBI-GeneID: 945109); the nucleotide sequence of the anthranilate synthase gene trpE is shown in SEQ ID NO.3 (trpE; NCBI-GeneID: 945846).

[0011] Preferably, in the genetically engineered bacteria for high tyrosine production based on adaptive regulation, the nucleotide sequence of the 3-dehydroquinoline synthase gene aroB is shown in SEQ ID NO.4 (aroB; NCBI-GeneID: 947927); the nucleotide sequence of the 3-deoxy-7-phosphoheptanoate synthase gene aroF is shown in SEQ ID NO.5 (aroF; NCBI-GeneID: 947084); the nucleotide sequence of the 3-deoxy-7-phosphoheptanoate synthase gene aroH is shown in SEQ ID NO.6 (aroH; NCBI-GeneID: 946229); the nucleotide sequence of the 3-dehydroquinoline dehydratase gene aroD is shown in SEQ ID NO.7 (aroD; NCBI-GeneID: 946210); the nucleotide sequence of the shikimate dehydrogenase gene aroE is shown in SEQ ID NO.8 (aroE; NCBI-GeneID: 947776); the nucleotide sequence of the shikimate kinase gene aroL is shown in SEQ ID NO. NO.9 (; NCBI-GeneID: 945031); the nucleotide sequence of the shikimate kinase gene aroK is shown in SEQ ID NO.10 (aroK; NCBI-GeneID: 2847759); the nucleotide sequence of the 5-enolpyruvylshikimate-3-phosphate synthase gene aroA is shown in SEQ ID NO.11 (aroA; NCBI-GeneID: 945528); the nucleotide sequence of the chorismate synthase gene aroC is shown in SEQ ID NO.12 (aroC; NCBI-GeneID: 946814); the nucleotide sequence of the tyrosine aminotransferase gene tyrB is shown in SEQ ID NO.13 (tyrB; NCBI-GeneID: 948563).

[0012] Preferably, in the genetically engineered bacteria for high tyrosine production based on adaptive regulation, the nucleotide sequence of the overexpressed 3-deoxy-7-phosphoheptanoate synthase gene aroG is shown in SEQ ID NO.14 (aroG; NCBI-GeneID: 945605); the nucleotide sequence of the prephenate dehydrogenase gene pyrA is shown in SEQ ID NO.15 (pyrA; NCBI-GeneID: 4985706).

[0013] Further preferably, the mutation mode of the aroG gene and the pyrA gene is point mutation; the point mutation of the aroG gene is to mutate aspartic acid at position 146 to asparagine, and the point mutation of the pyrA gene is to mutate alanine at position 53 to valine.

[0014] Preferably, in the genetically engineered bacteria for high tyrosine production based on adaptive regulation, the nucleotide sequence of the tyrosine regulatory protein gene tyrR is as shown in SEQ ID NO.16 (tyrR; NCBI-GeneID: 945879).

[0015] Preferably, in the genetically engineered bacteria for high tyrosine production based on adaptive regulation, the nucleotide sequence of the ribosome binding site RBS is as shown in SEQ ID NO.17; trc The nucleotide sequence of the promoter is shown in SEQ ID NO.18; the promoter P involved in the adaptive regulation of flagella construction fliC The nucleotide sequence is shown in SEQ ID NO.19.

[0016] In a second aspect, the present invention provides a method for constructing a genetically engineered bacterium Ltyr-14 with high tyrosine production based on adaptive regulation, and the specific steps are as follows:

[0017] (1) In the genome of E. coli W3110, the natural promoter of the prephenate dehydratase gene pheA (nucleotide sequence as shown in SEQ ID NO.1) was replaced with the promoter P involved in the adaptive regulation of flagellar construction. fliC , and obtained strain Ltyr-01;

[0018] (2) The natural promoters of the anthranilate synthase genes trpD (nucleotide sequence as shown in SEQ ID NO.2) and trpE (nucleotide sequence as shown in SEQ ID NO.3) were replaced with promoters involved in the adaptive regulation of flagella construction. fliC (the nucleotide sequence is shown in SEQ ID NO. 19), and strain Ltyr-02 was obtained;

[0019] (3) knocking out the tyrosine regulatory protein gene tyrR (nucleotide sequence as shown in SEQ ID NO. 16) to obtain strain Ltyr-03;

[0020] (4) by introducing a mutated 3-deoxy-7-phosphoheptanoate synthase gene aroG (nucleotide sequence as shown in SEQ ID NO.14) and a prephenate dehydrogenase gene pyrA (nucleotide sequence as shown in SEQ ID NO.15) at the ygaY site of the genome and connecting them with a ribosome binding site RBS (nucleotide sequence as shown in SEQ ID NO.17) sequence for overexpression, trc (the nucleotide sequence is shown in SEQ ID NO. 18) promoter to initiate transcription, resulting in strain Ltyr-04;

[0021] (5) by introducing 3-dehydroquinoline synthase gene aroB (nucleotide sequence as shown in SEQ ID NO.4), 3-deoxy-7-phosphoheptanoate synthase gene aroF (nucleotide sequence as shown in SEQ ID NO.5) and aroH (nucleotide sequence as shown in SEQ ID NO.6), 3-dehydroquinoline dehydratase gene aroD (nucleotide sequence as shown in SEQ ID NO.7), shikimate dehydrogenase gene aroE (nucleotide sequence as shown in SEQ ID NO.8), shikimate kinase gene aroL (nucleotide sequence as shown in SEQ ID NO.9) and aroK (nucleotide sequence as shown in SEQ ID NO.10), 5-enolpyruvylshikimate-3-phosphate synthase gene aroA (nucleotide sequence as shown in SEQ ID NO.11), chorismate synthase gene aroC (nucleotide sequence as shown in SEQ ID NO.12) into the yghX, gapC, ilvG, yeeL, ycgH, yciQ, mbhA, rph, yghE, and yjiP sites of the genome, respectively. ID NO.12), tyrosine aminotransferase gene tyrB (nucleotide sequence as shown in SEQ ID NO.13), both of which are P trc (The nucleotide sequence is shown in SEQ ID NO.18) promoter was started to obtain strains Ltyr-05, Ltyr-06, Ltyr-07, Ltyr-08, Ltyr-09, Ltyr-10, Ltyr-11, Ltyr-12, Ltyr-13, and Ltyr-14; the Ltyr-14 strain was the genetically engineered bacterium with high tyrosine production based on adaptive regulation obtained after the final transformation.

[0022] In a third aspect, the present invention provides an application of the above-mentioned genetically engineered bacteria with high tyrosine production based on adaptive regulation in the fermentation production of tyrosine.

[0023] Preferably, in the above application, the method for producing tyrosine by fermentation comprises shake flask fermentation and fermenter culture.

[0024] Preferably, the specific method of shake flask fermentation is: inoculating the seed liquid into the fermentation medium at an inoculation rate of 10%-20%, maintaining the temperature at 37±0.2°C, and shaking the culture at 200-220r / min; maintaining the pH at 7.0-7.2 by adding 25% ammonia water during the fermentation process; adding 60% glucose solution to supplement the carbon source required by the bacteria, and the fermentation cycle is 24-30h.

[0025] Preferably, the specific method of the fermentation tank culture is:

[0026] S1. Activation of strains: transfer the strains from the glycerol tube to the slant culture medium for activation and culture for 11-13 hours; transfer the strains from the slant culture medium to the eggplant-shaped flask culture medium for continued activation and expansion culture for 10-12 hours, culture temperature: 37°C;

[0027] S2. Seed culture: During the culture process, the pH was maintained at 6.7±0.2, the temperature was maintained at 37±0.2℃, the dissolved oxygen was maintained at 35%-50%, and the OD was maintained at 4.5. 600nm At 20 o'clock, the mixture was transferred to a fermentation tank for fermentation culture;

[0028] S3, fermentation culture: 20% of the seed liquid was inoculated into the fermentation medium for fermentation culture, the fermentation pH was maintained at 6.7±0.2, the temperature was maintained at 37±0.2°C, and the dissolved oxygen was maintained at 30%-50%; during the culture process, 80% of the seed liquid was added by feeding.

[0029] (m / v) glucose solution was used to maintain the carbon source required by the bacteria during the fermentation process, and the pH was adjusted by adding 25% ammonia water.

[0030] Preferably, the seed culture medium in the shake flask fermentation and fermentation tank culture is: glucose 25g / L, yeast extract 5g / L, citric acid 2g / L, (NH4)2SO4 3g / L, MgSO4·7H2O 2g / L, KH2PO4 3g / L, FeSO4·7H2O 5mg / L, MnSO4 1.2mg / L, Vb 1、 Vb 3、 Vb 5、 Vb 12 0.5 mg / L each, VH 1 mg / L, and the rest is water.

[0031] Preferably, the fermentation medium in the shake flask fermentation and fermenter culture is: glucose 30g / L, yeast extract 6g / L, citric acid 2g / L, (NH4)2SO4 5g / L, MgSO4·7H2O 2g / L, KH2PO4 4g / L, FeSO4·7H2O 20mg / L, MnSO4 10mg / L, Vb 1、 Vb 3、 Vb 5、 Vb 12 0.5 mg / L each, VH 1 mg / L, methionine 1 g / L, phenylalanine 0.5 g / L, and the rest is water.

[0032] Preferably, the culture medium used for bacterial activation in step S1 is a seed culture medium added with 2% agar powder.

[0033] The above culture media can be prepared by conventional standard methods.

[0034] Compared with the prior art, the genetically engineered bacteria for high tyrosine production based on adaptive regulation provided by the present invention has the following advantages:

[0035] The genetically engineered bacteria Ltyr-14 based on adaptive regulation and high tyrosine production provided by the present invention analyzes the tyrosine metabolic synthesis pathways of E. coli and introduces P involved in flagella construction. fliC The promoter weakens the branching pathway of the precursor and reduces carbon consumption; the key enzyme genes of the tyrosine biosynthesis pathway are integrated into the genome to improve the efficiency of tyrosine synthesis; by mutating some key enzymes and knocking out the tyrosine regulatory protein gene, feedback inhibition is relieved, and a genetically engineered bacterium Ltyr-14 that synthesizes tyrosine from scratch is successfully constructed. The genetically engineered bacterium Ltyr-14 has the advantages of clear genetic background, sustainable transformation, and no defects. The fermentation production of tyrosine by this strain is simple to operate and can directly synthesize tyrosine with cheap carbon sources, which has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The present invention is a diagram illustrating the construction method of the genetically engineered bacterium Ltyr-14 based on adaptive regulation and high tyrosine production;

[0037] Figure 2 The figure is a fermentation growth curve of the 5L fermenter of the genetically engineered bacteria Ltyr-14 with high tyrosine production based on adaptive regulation according to the present invention. DETAILED DESCRIPTION

[0038] The present invention is further illustrated below through the description of specific implementation methods, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the protection scope of the present invention.

[0039] In the following examples, reagents not specially described are conventional reagents and can be purchased from conventional reagent production and sales companies. The methods used are all prior arts unless otherwise specified.

[0040] In the following examples, the percentage sign "%" refers to mass percentage unless otherwise specified. The percentage of a solution refers to the number of grams of a solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0041] In the following examples, the starting strain used is the wild-type E. coli W3110, deposited with ATCC 27325, and the corresponding promoters and genes are shown in the sequence listing.

[0042] Example 1

[0043] The gene editing method used is based on the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). The engineering plasmid pGRB involved in this method is based on pUC18 as the backbone, including the promoter J23100, the gRNA-Cas9 binding region sequence and the terminator sequence and ampicillin resistance (working concentration: 100 mg / L). The professional terms involved in the following examples can be explained in this article. The primers used in the strain construction process are shown in Table 1.

[0044] Table 1 Primers involved in strain construction

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] 1.1 Adaptive regulation of the pheA gene

[0052] Using pGRB-pheA-s and pGRB-pheA-a as primers, pGRB-pheA plasmid was constructed. fliC pheA-dn-sP fliC , pheA-dn-a as primers, and the genome of E. coli W3110 as a template to obtain the upstream homology arm pheA-up and the downstream homology arm pheA-dn; then, pheA-up-s and pheA-dn-a as primers, and the upstream homology arm pheA-up and the downstream homology arm pheA-dn as templates, the fusion fragment P was obtained by overlapping PCR. fliC -pheA. Finally, the pGRB-pheA plasmid was combined with the overlapping fragment P fliC -pheA was electroporated into the electroporation competent cells of E. coli W3110; then pheA-up-s and pheA-dn-a were used as identification primers to screen positive transformants to obtain strain Ltyr-01.

[0053] 1.2 Adaptive regulation of trpD and trpE genes

[0054] Using pGRB-trp-s and pGRB-trp-a as primers, the pGRB-trp plasmid was constructed. fliC ,trp-DN-sP fliC , trp-DN-a as primers, and then the genome of E. coli W3110 as a template to obtain the upstream homology arm trp-up and the downstream homology arm trp-DN; then trp-up-s and trp-DN-a as primers, the upstream homology arm trp-up and the downstream homology arm trp-DN as templates, and the fusion fragment P was obtained by overlapping PCR. fliC -trp. Finally, the pGRB-trp plasmid was combined with the overlapping fragment P fliC -trp was electroporated into the electroporation competent cells of the starting strain Ltyr-01; then trp-up-s and trp-DN-a were used as identification primers to screen positive transformants to obtain strain Ltyr-02.

[0055] 1.3 Integration of aroB gene

[0056] Using pGRB-yghX-s and pGRB-yghX-a as primers, the pGRB-yghX plasmid was constructed. Then, using yghX-up-s, yghX-up-a, aroB-s, aroB-a, yghX-DN-s, and yghX-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homology arm yghX-up, the downstream homology arm yghX-DN, and the target gene aroB were obtained; and then, using yghX-up-s and yghX-DN-a as primers and the upstream homology arm yghX-up and the downstream homology arm yghX-DN as templates, the fusion fragment yghX::aroB was obtained by overlapping PCR. Finally, the pGRB-yghX plasmid and the overlapping fragment yghX::aroB were electroporated into the electroporation competent cells of Ltyr-02; yghX-up-s and yghX-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-03.

[0057] 1.4 Integration of the aroF gene

[0058] Using pGRB-gapC-s and pGRB-gapC-a as primers, the pGRB-gapC plasmid was constructed. Then, using gapC-up-s, gapC-up-a, aroF-s, aroF-a, gapC-DN-s, gapC-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homology arm gapC-up, the downstream homology arm gapC-DN, and the target gene aroF were obtained; and then using gapC-up-s and gapC-DN-a as primers and the upstream homology arm gapC-up, the downstream homology arm gapC-DN and the target gene aroF as templates, the fusion fragment gapC::aroF was obtained by overlapping PCR. Finally, the pGRB-gapC plasmid and the overlapping fragment gapC::aroF were electroporated into the electroporation competent cells of Ltyr-03; gapC-up-s and gapC-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-04.

[0059] 1.5 Integration of the aroH gene

[0060] Using pGRB-ilvG-s and pGRB-ilvG-a as primers, the pGRB-ilvG plasmid was constructed. Then, using ilvG-up-s, ilvG-up-a, aroH-s, aroH-a, ilvG-DN-s, and ilvG-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homology arm ilvG-up, the downstream homology arm ilvG-DN, and the target gene aroH were obtained; and then, using ilvG-up-s and ilvG-DN-a as primers and the upstream homology arm ilvG-up, the downstream homology arm ilvG-DN, and the target gene aroH as templates, the fusion fragment ilvG::aroH was obtained by overlapping PCR. Finally, the pGRB-ilvG plasmid and the overlapping fragment ilvG::aroH were electroporated into the electroporation competent cells of Ltyr-04; ilvG-up-s and ilvG-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-05.

[0061] 1.6 Integration of the aroD gene

[0062] Using pGRB-yeeL-s and pGRB-yeeL-a as primers, the pGRB-yeeL plasmid was constructed. Then, using yeeL-up-s, yeeL-up-a, aroD-s, aroD-a, yeeL-DN-s, and yeeL-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homology arm yeeL-up, the downstream homology arm yeeL-DN, and the target gene aroD were obtained; and then, using yeeL-up-s and yeeL-DN-a as primers and the upstream homology arm yeeL-up, the downstream homology arm yeeL-DN, and the target gene aroD as templates, the fusion fragment yeeL::aroD was obtained by overlapping PCR. Finally, the pGRB-yeeL plasmid and the overlapping fragment yeeL::aroD were electroporated into the electroporation competent cells of Ltyr-05; yeeL-up-s and yeeL-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-06.

[0063] 1.7 Integration of the aroE gene

[0064] Using pGRB-ycgH-s and pGRB-ycgH-a as primers, the pGRB-ycgH plasmid was constructed. Then, using ycgH-up-s, ycgH-up-a, aroE-s, aroE-a, ycgH-DN-s, and ycgH-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homology arm ycgH-up, the downstream homology arm ycgH-DN, and the target gene aroE were obtained; and then, using ycgH-up-s and ycgH-DN-a as primers, the upstream homology arm ycgH-up, the downstream homology arm ycgH-DN, and the target gene aroE were used as templates, and the fusion fragment ycgH::aroE was obtained by overlapping PCR. Finally, the pGRB-ycgH plasmid and the overlapping fragment ycgH::aroE were electroporated into the electroporation competent cells of Ltyr-06; ycgH-up-s and ycgH-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-07.

[0065] 1.8 Integration of the aroL gene

[0066] Using pGRB-yciQ-s and pGRB-yciQ-a as primers, the pGRB-yciQ plasmid was constructed. Then, using yciQ-up-s, yciQ-up-a, aroL-s, aroL-a, yciQ-DN-s, and yciQ-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homology arm yciQ-up, the downstream homology arm yciQ-DN, and the target gene aroL were obtained; and then, using yciQ-up-s and yciQ-DN-a as primers, the upstream homology arm yciQ-up, the downstream homology arm yciQ-DN, and the target gene aroL were used as templates, the fusion fragment yciQ::aroL was obtained by overlapping PCR. Finally, the pGRB-yciQ plasmid and the overlapping fragment yciQ::aroL were electroporated into the electroporation competent cells of Ltyr-07; yciQ-up-s and yciQ-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-08.

[0067] 1.9 Integration of the aroK gene

[0068] Using pGRB-mbhA-s and pGRB-mbhA-a as primers, the pGRB-mbhA plasmid was constructed. Then, using mbhA-up-s, mbhA-up-a, aroK-s, aroK-a, mbhA-DN-s, and mbhA-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homology arm mbhA-up, the downstream homology arm mbhA-DN, and the target gene aroK were obtained; and then, using mbhA-up-s and mbhA-DN-a as primers, the upstream homology arm mbhA-up, the downstream homology arm mbhA-DN, and the target gene aroK were used as templates, and the fusion fragment mbhA::aroK was obtained by overlapping PCR. Finally, the pGRB-mbhA plasmid and the overlapping fragment mbhA::aroK were electroporated into the electroporation competent cells of Ltyr-08; mbhA-up-s and mbhA-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-09.

[0069] 1.10 Integration of the aroA gene

[0070] Using pGRB-rph-s and pGRB-rph-a as primers, the pGRB-rph plasmid was constructed. Then, using rph-up-s, rph-up-a, aroA-s, aroA-a, rph-DN-s, and rph-DN-a as primers, and the genome of E. coli W3110 as a template, the upstream homology arm rph-up, the downstream homology arm rph-DN, and the target gene aroA were obtained; then, using rph-up-s and rph-DN-a as primers, the upstream homology arm rph-up, the downstream homology arm rph-DN, and the target gene aroA were used as templates, and the fusion fragment rph::aroA was obtained by overlapping PCR. Finally, the pGRB-rph plasmid and the overlapping fragment rph::aroA were electroporated into the electroporation competent cells of Ltyr-09; and rph-up-s and rph-DN-a were used as identification primers to screen positive transformants and obtain the strain Ltyr-10.

[0071] 1.11 Integration of the aroC gene

[0072] Using pGRB-yghE-s and pGRB-yghE-a as primers, the pGRB-yghE plasmid was constructed. Then, using yghE-up-s, yghE-up-a, aroC-s, aroC-a, yghE-DN-s, and yghE-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homology arm yghE-up, the downstream homology arm yghE-DN, and the target gene aroC were obtained; and then, using yghE-up-s and yghE-DN-a as primers, the upstream homology arm yghE-up, the downstream homology arm yghE-DN, and the target gene aroC as templates, the fusion fragment yghE::aroC was obtained by overlapping PCR. Finally, the pGRB-yghE plasmid and the overlapping fragment yghE::aroC were electroporated into the electroporation competent cells of Ltyr-10; yghE-up-s and yghE-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-11.

[0073] 1.12 Integration of the tyrB gene

[0074] Using pGRB-yjiP-s and pGRB-yjiP-a as primers, the pGRB-yjiP plasmid was constructed. Then, using yjiP-up-s, yjiP-up-a, tyrB-s, tyrB-a, yjiP-DN-s, and yjiP-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homology arm yjiP-up, the downstream homology arm yjiP-DN, and the target gene tyrB were obtained; and then, using yjiP-up-s and yjiP-DN-a as primers, the upstream homology arm yjiP-up, the downstream homology arm yjiP-DN, and the target gene tyrB were used as templates, the fusion fragment yjiP::tyrB was obtained by overlapping PCR. Finally, the pGRB-yjiP plasmid and the overlapping fragment yjiP::tyrB were electroporated into the electroporation competent cells of Ltyr-11; yjiP-up-s and yjiP-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-12.

[0075] 1.13 Integration of aroG and pyrA mutants

[0076] Using pGRB-ygaY-s and pGRB-ygaY-a as primers, the pGRB-ygaY plasmid was constructed. Then, using ygaY-up-s and ygaY-up-a, aroG-1-a and aroG-1-s, aroG-2-s and aroG-2-s, tyrA-1-s and tyrA-1-s, tyrA-2-s and tyrA-2-sa, ygaY-DN-s and ygaY-DN-a as primers and the genome of E. coli W3110 as a template, the upstream homologous arm ygaY-up, the target fragment aroG-1, the target fragment aroG-2, the target fragment tyrA-1, the target fragment tyrA-2, and the downstream homologous arm ygaY-DN were obtained respectively; then, using ygaY-up-s and ygaY-DN-a as primers and the upstream homologous arm ygaY-up, the downstream homologous arm ygaY-DN and the target genes aroG-1, aroG-2, tyrA-1, and tyrA-2 as templates, the fusion fragment aroG-tyrA was obtained by overlapping PCR. Finally, the pGRB-ygaY plasmid and the overlapping fragment aroG-tyrA were electroporated into the electroporation competent cells of Ltyr-12; ygaY-up-s and ygaY-DN-a were used as identification primers to screen positive transformants and obtain strain Ltyr-03.

[0077] 1.14 Knockout of the tyrR gene

[0078] The pGRB-tyrR plasmid was constructed using pGRB-tyrR-s and pGRB-tyrR-a as primers. Then, the upstream homology arm tyrR-up and the downstream homology arm tyrR-dn were obtained using tyrR-up-s, tyrR-up-a, tyrR-DN-s, and tyrR-DN-a as primers and the genome of E. coli W3110 as a template; then, the fusion fragment ΔtyrR was obtained by overlapping PCR using tyrR-up-s and tyrR-DN-a as primers and the upstream homology arm tyrR-up and the downstream homology arm tyrR-dn as templates. Finally, the pGRB-tyrR plasmid and the overlapping fragment ΔtyrR were electroporated into the electroporation competent cells of Ltyr-13; and then, tyrR-up-s and tyrR-dn-a were used as identification primers to screen positive transformants and obtain the strain Ltyr-14.

[0079] Example 2

[0080] The genetically engineered bacteria Ltyr-14 described in Example 1 was used to produce tyrosine by shake flask fermentation.

[0081] 1. Culture medium

[0082] 1.1 Seed culture medium

[0083] Glucose 25g / L, Yeast extract powder 5g / L, Citric acid 2g / L, (NH4)2SO4 3g / L, MgSO4·7H2O 2g / L, KH2PO4 3g / L, FeSO4·7H2O 5mg / L, MnSO4 1.2mg / L, Vb 1、 Vb 3、 Vb 5、 Vb 12 0.5 mg / L each, VH 1 mg / L, and the rest is water.

[0084] 1.2 Fermentation medium

[0085] Glucose 30g / L, Yeast extract powder 6g / L, Citric acid 2g / L, (NH4)2SO4 5g / L, MgSO4·7H2O 2g / L, KH2PO4 4g / L, FeSO4·7H2O 20mg / L, MnSO4 10mg / L, Vb 1、 Vb 3、 Vb 5、 Vb 12 0.5 mg / L each, VH 1 mg / L, methionine 1 g / L, phenylalanine 0.5 g / L, and the rest is water.

[0086] 2. Shake flask culture protocol

[0087] 2.1 Bacterial Activation

[0088] The genetically engineered bacteria Ltyr-14 for producing tyrosine described in Example 1 stored at -80°C was streaked and inoculated onto an activated slant, cultured at 37°C for 12 hours, and passaged once.

[0089] 2.2 Seed culture

[0090] Use an inoculating loop to scrape a ring of slant seeds and inoculate it into a 500mL Erlenmeyer flask containing 30mL of seed culture medium. Seal the flask with nine layers of gauze and culture at 37℃, 220r / min for 10h.

[0091] 2.3 Fermentation culture

[0092] The seed liquid prepared after the activated strain is inoculated at a rate of 10%-15% into a triangular flask containing fermentation medium, and the bottle is sealed with nine layers of gauze. The temperature is maintained at 37±0.2°C, and the culture is shaken at 220r / min. During the fermentation process, 25% ammonia water is added to maintain the pH at 7.0-7.2; 60% glucose solution is added to supplement the carbon source required by the bacteria, and the fermentation cycle is 24h.

[0093] After 24 hours of shake flask fermentation, the tyrosine production was detected to be up to 16.4 g / L.

[0094] Example 3

[0095] The genetically engineered bacteria Ltyr-14 described in Example 1 was used to carry out 5 L scale-up fermentation to produce tyrosine. The fermentation medium is shown in Example 2.

[0096] S1. Activation of bacterial strains: transfer the bacterial strains from the glycerol tube to the slant culture medium for activation and culture for 11-13 hours; transfer the bacterial strains from the slant culture medium to the eggplant flask culture medium for continued activation and expansion culture for 10-12 hours; culture temperature: 37°C; the slant culture medium and the eggplant flask culture medium are seed culture mediums with 2% agar powder added;

[0097] S2. Seed culture: During the culture process, the pH was maintained at 6.7±0.2, the temperature was maintained at 37±0.2℃, and the dissolved oxygen was maintained between 35% and 50%. The culture was continued until OD 600nm At 20 o'clock, the mixture was transferred to a fermentation tank for fermentation culture;

[0098] S3, fermentation culture: 20% of the seed liquid was inoculated into the fermentation medium for fermentation culture, the fermentation pH was maintained at 6.7±0.2, the temperature was maintained at 37±0.2°C, and the dissolved oxygen was maintained between 30% and 50%; during the culture process, 80% (m / v) glucose solution was added to maintain the carbon source required by the fermentation process, and 25% ammonia water was added to adjust the pH.

[0099] After 40 hours of fermentation in a 5L fermenter, the tyrosine production was detected to be 86.4 g / L, and the sugar-acid conversion rate was 26.3%. Figure 2 shown.

[0100] It can be seen that the genetically engineered bacteria Ltyr-14 described in Example 1 can use cheap glucose as a substrate to synthesize tyrosine with higher value from scratch, and has the advantages of stable genetic performance, clear genetic background, no defects, no plasmid, high production efficiency, etc.; it has good industrial prospects.

[0101] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A genetically engineered bacterium for high tyrosine production based on adaptive regulation, characterized in that: The invention is obtained by the following gene modification method: in the genome of wild-type E. coli W3110, the promoters of the pheA gene for prephenate dehydratase and the trpD and trpE genes for anthranilate synthase are replaced with the promoter P involved in flagella construction. fliC ; Overexpression of 3-dehydroquinoline synthase gene aroB, 3-deoxy-7-phosphoheptanoate synthase genes aroF and aroH, 3-dehydroquinoline dehydratase gene aroD, shikimate dehydrogenase gene aroE, shikimate kinase genes aroL and aroK, 5-enolpyruvylshikimate-3-phosphate synthase gene aroA, chorismate synthase gene aroC, tyrosine aminotransferase gene tyrB, all from P trc The promoter initiates transcription; the 3-deoxy-7-phosphoheptanoate synthase gene aroG and the prephenate dehydrogenase gene pyrA are mutated and connected using the ribosome binding site RBS sequence for overexpression. trc The promoter starts transcription; the tyrosine regulatory protein gene tyrR is knocked out to obtain the successfully transformed target bacterium Ltyr-14.

2. The genetically engineered bacteria for high tyrosine production based on adaptive regulation according to claim 1, characterized in that: The deposit number of the wild type E. coli W3110 is ATCC 27325.

3. The genetically engineered bacteria for high tyrosine production based on adaptive regulation according to claim 1 or 2, characterized in that: The nucleotide sequence of the pheA gene is shown in SEQ ID NO.1; the nucleotide sequence of the anthranilate synthase gene trpD is shown in SEQ ID NO.2; the nucleotide sequence of the anthranilate synthase gene trpE is shown in SEQ ID NO.3; the nucleotide sequence of the 3-dehydroquinate synthase gene aroB is shown in SEQ ID NO.4; the nucleotide sequence of the 3-deoxy-7-phosphoheptanoate synthase gene aroF is shown in SEQ ID NO.5; the nucleotide sequence of the 3-deoxy-7-phosphoheptanoate synthase gene aroH is shown in SEQ ID NO.6; the nucleotide sequence of the 3-dehydroquinic acid dehydratase gene aroD is shown in SEQ ID NO.7; the nucleotide sequence of the shikimate dehydrogenase gene aroE is shown in SEQ ID NO.8; the nucleotide sequence of the shikimate kinase gene aroL is shown in SEQ ID NO.9; the nucleotide sequence of the shikimate kinase gene aroK is shown in SEQ ID NO.

10. NO.10; the nucleotide sequence of the 5-enolpyruvylshikimate-3-phosphate synthase gene aroA is shown in SEQ ID NO.11; the nucleotide sequence of the chorismate synthase gene aroC is shown in SEQ ID NO.12; the nucleotide sequence of the tyrosine transaminase gene tyrB is shown in SEQ ID NO.13; the nucleotide sequence of the 3-deoxy-7-phosphoheptanoate synthase gene aroG is shown in SEQ ID NO.14; the nucleotide sequence of the prephenate dehydrogenase gene pyrA is shown in SEQ ID NO.15; the nucleotide sequence of the tyrosine regulatory protein gene tyrR is shown in SEQ ID NO.

16.

4. The genetically engineered bacteria for high tyrosine production based on adaptive regulation according to claim 1 or 2, characterized in that: The mutation modes of the aroG gene and the pyrA gene are point mutations; the point mutation of the aroG gene is to mutate the 146th aspartic acid to asparagine, and the point mutation of the pyrA gene is to mutate the 53rd alanine to valine.

5. The genetically engineered bacteria for high tyrosine production based on adaptive regulation according to claim 1 or 2, characterized in that: The nucleotide sequence of the ribosome binding site RBS is shown in SEQ ID NO.17; trc The nucleotide sequence of the promoter is shown in SEQ ID NO.18; the promoter P involved in flagella construction fliC The nucleotide sequence is shown in SEQ ID NO.

19.

6. A method for constructing a genetically engineered bacterium capable of producing high tyrosine production based on adaptive regulation according to any one of claims 1 to 5, characterized in that: The specific steps are as follows: (1) In the genome of E. coli W3110, the natural promoter of the pheA gene was replaced with the promoter P involved in the adaptive regulation of flagellar construction. fliC , and obtained strain Ltyr-01; (2) The natural promoters of the anthranilate synthase genes trpD and trpE were replaced with promoters involved in the adaptive regulation of flagellar construction. fliC , and obtained strain Ltyr-02; (3) The tyrosine regulatory protein gene tyrR was knocked out to obtain strain Ltyr-03; (4) By introducing the mutated 3-deoxy-7-phosphoheptanoate synthase gene aroG and the prephenate dehydrogenase gene pyrA at the ygaY site of the genome and connecting them with the RBS sequence for overexpression, the P trc The promoter initiated transcription, resulting in strain Ltyr-04; (5) The 3-dehydroquinoline synthase gene aroB, 3-deoxy-7-phosphoheptanoate synthase genes aroF and aroH, 3-dehydroquinoline dehydratase gene aroD, shikimate dehydrogenase gene aroE, shikimate kinase genes aroL and aroK, 5-enolpyruvylshikimate-3-phosphate synthase gene aroA, chorismate synthase gene aroC, and tyrosine aminotransferase gene tyrB were introduced into the yghX, gapC, ilvG, yeeL, ycgH, yciQ, mbhA, rph, yghE, and yjiP sites of the genome, respectively. trc The promoter was activated to obtain strains Ltyr-05, Ltyr-06, Ltyr-07, Ltyr-08, Ltyr-09, Ltyr-10, Ltyr-11, Ltyr-12, Ltyr-13, and Ltyr-14; the Ltyr-14 strain was the genetically engineered bacterium with high tyrosine production based on adaptive regulation obtained after the final transformation.

7. Use of the genetically engineered bacteria for high tyrosine production based on adaptive regulation according to any one of claims 1 to 5 in the fermentation production of tyrosine.

8. The use according to claim 7, characterized in that: The method for producing tyrosine by fermentation includes shake flask fermentation and fermenter culture.

9. The use according to claim 8, characterized in that: The specific method of the shake flask fermentation is: inoculating the seed liquid into the fermentation medium at an inoculation rate of 10%-20%, maintaining the temperature at 37±0.2°C, and shaking the culture at 200-220r / min; maintaining the pH at 7.0-7.2 by adding 25% ammonia water during the fermentation process; adding 60% glucose solution to supplement the carbon source required by the bacteria, and the fermentation cycle is 24-30h.

10. The use according to claim 8, characterized in that: The specific method of the fermentation tank culture is: S1. Activation of strains: transfer the strains from the glycerol tube to the slant culture medium for activation and culture for 11-13 hours; transfer the strains from the slant culture medium to the eggplant-shaped flask culture medium for continued activation and expansion culture for 10-12 hours, culture temperature: 37°C; S2. Seed culture: During the culture process, the pH was maintained at 6.7±0.2, the temperature was maintained at 37±0.2℃, the dissolved oxygen was maintained at 35%-50%, and the OD was maintained at 4.

5. 600nm At 20 o'clock, the mixture was transferred to a fermentation tank for fermentation culture; S3. Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 20% for fermentation culture. The fermentation pH is maintained at 6.7±0.2, the temperature is maintained at 37±0.2°C, and the dissolved oxygen is maintained at 30%-50%. During the culture process, 80% glucose solution is added to maintain the carbon source required by the fermentation process, and 25% ammonia water is added to adjust the pH.

Images