Genetically engineered bacteria overexpressing ydeD gene and method for producing l-phenylalanine

Abstract

The present application belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a genetically engineered bacterium with overexpression of a gene and a method for producing L-phenylalanine by the genetically engineered bacterium. ydeD The present application belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a genetically engineered bacterium with overexpression of a gene and a method for producing L-phenylalanine by the genetically engineered bacterium.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to overexpression. Dey Genetically engineered bacteria and their methods for producing L-phenylalanine. Background Technology

[0002] L-Phenylalanine, first discovered in yellow lupins, is an essential amino acid in mammals. It is an important precursor to substances such as adrenaline, melanin, and the semi-essential amino acid tyrosine. It also serves as an intermediate and good carrier for some antihypertensive and anticancer drugs, finding wide application in the pharmaceutical industry. Furthermore, L-phenylalanine can be used as a precursor to aspartame and chiral compounds in feed, food, and chemical industries. Production methods for L-phenylalanine include natural protein hydrolysis, chemical synthesis, enzymatic methods, and microbial fermentation. Natural protein hydrolysis, chemical synthesis, and enzymatic methods suffer from drawbacks such as high cost, low yield, complex processes, environmental pollution, and difficulty in industrialization. Therefore, microbial fermentation has become the mainstream production method. Microbial fermentation combines advanced biotechnology and offers advantages such as ease of control, low cost, high yield, short cycle time, environmental friendliness, and ease of large-scale industrialization. However, it also faces challenges such as imprecise fermentation control in large-scale production, low sugar-acid conversion rates, and insufficient understanding of the balance between microbial metabolic networks. Therefore, to increase yield, it is necessary to balance the metabolic network and systematically modify it.

[0003] Currently, rational modification through metabolic engineering remains the mainstream method for constructing strains, generally involving the supply of precursor substances, relieving feedback inhibition of key enzymes, strengthening pathway enzymes, supplying relevant cofactors, and modifying the efflux system. For L-phenylalanine-producing strains, there is still room for modification of the transport system. In *E. coli*, the intrinsic PheP, AroP, and YddG proteins are the most widely used L-phenylalanine transporters. PheP is a protein responsible for the specific intracellular transport of L-phenylalanine; AroP is an aromatic amino acid permease capable of transporting phenylalanine and tyrosine with high affinity; YddG is an aromatic amino acid efflux protein with affinity for all three aromatic amino acids, and YddG is currently the most widely reported transporter protein in aromatic amino acid-producing strains. Among these, Liu Qian et al. overexpressed it in plasmid form. Gdy Gene acquisition E. coli The TRTH-Y strain, verified in a 30 L fermenter, achieved a yield of 36.3 g / L, a 12.6% increase compared to the control strain. Liu Shuangping and others used genetic engineering techniques to... Gdy The gene was introduced into host E. coli in plasmid form and verified by fermentation to contain [the gene]. GdyThe strain with the gene showed improved extracellular transport capacity of L-phenylalanine, ultimately achieving a yield of 62 g / L. Other studies used the *E. coli* tryptophan-producing strain SV164 (pGH5) as the parent strain, and further developed a strain with a stronger promoter-controlled extracellular transport capacity of L-phenylalanine. Gdy Genes were introduced into cells to obtain the SV164PL-yddG(pGH5) strain. This strain was cultured in 20×200 mm test tubes at 37°C with shaking for 48 h, and the final L-tryptophan yield was 4.17 g / L, an increase of approximately 12% compared to the starting strain.

[0004] The above cases show that overexpression Gdy The gene can enhance the yield of aromatic amino acids, but the membrane protein encoded by this gene has varying affinities for different aromatic amino acids, resulting in poor transport efficiency and affecting bacterial growth. As an intrinsic transport protein, it is easily subject to the self-regulation of the bacterial cell, thus failing to perform its efficient transport function and making it difficult to meet the requirements of industrial production. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a genetically modified Escherichia coli and a method for producing L-phenylalanine.

[0006] The technical solution of this invention is summarized as follows:

[0007] Firstly, the present invention provides a genetically engineered bacterium for producing L-phenylalanine, specifically, belonging to *Escherichia coli*, and more specifically, the genetically engineered bacterium is an *Escherichia coli* strain overexpressing... Dey The genes were modified.

[0008] The overexpression Dey A gene is a gene whose expression intensity is significantly enhanced after modification compared to the original expression intensity of the target gene.

[0009] In a second aspect, the present invention provides a method for producing L-phenylalanine using genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-phenylalanine; and collecting the L-phenylalanine from the genetically engineered bacteria and / or the culture medium.

[0010] As described in the second aspect of the present invention, the genetically engineered bacteria have been overexpressed. Dey The genes were modified to make them comparable to unmodified E. coli. Dey The intensity of gene expression is increased or the expression level is raised.

[0011] The beneficial effects of this invention are as follows:

[0012] DeyThe gene encodes a putative permease, the relationship and role of which in the L-phenylalanine synthesis pathway are not yet clear. This invention is the first to discover, through overexpression... Dey Gene modification can significantly increase the production of L-phenylalanine in Escherichia coli and enhance the growth ability of the strain; especially in strains that have been overexpressed. Gdy When the gene enhances the ability of L-phenylalanine to efflux and further enhances its effect on strain growth, overexpression Dey The gene can further enhance the production capacity of L-phenylalanine and improve the growth capacity of the strain. Attached Figure Description

[0013] Figure 1 The pREDCas9 and pGRB plasmid maps used in the examples.

[0014] Figure 2 : The pSTV28-ydeD plasmid map used in the examples. Detailed Implementation

[0015] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0016] Firstly, the present invention provides a genetically engineered bacterium for producing L-phenylalanine, specifically, belonging to *Escherichia coli*, and more specifically, the genetically engineered bacterium is an *Escherichia coli* strain overexpressing... Dey The genes were modified.

[0017] According to a first aspect of the present invention, the genetically engineered bacterium is an overexpression of *Escherichia coli*. Dey The genes were modified to make them comparable to unmodified E. coli. Dey Increased gene expression levels, or Dey The total enzyme activity of the gene expression product (such as an enzyme) is increased, for example, by 150% or more, 200% or more, 300% or more.

[0018] According to a first aspect of the present invention, the *Escherichia coli* can be any type of *Escherichia coli*. Escherichia coli (), can be a wild-type strain, a model strain, or a genetically engineered strain, for example E. coli MG1655 E. coli W3110 E. coliBL21 E. coli BW25113 E. coli Genetically engineered bacteria derived from DH5α and its basis can be overexpressed. Dey It can be modified through genetic means.

[0019] According to a first aspect of the invention, overexpression Dey Genetic methods can be used by introducing and / or adding genes into the bacterial genome. Dey The copy number of genes (e.g., increased by autonomously replicating plasmids such as pET28a, pTrc99a, and pSTV28) Dey The copy number of genes, or increasing the number of genes in bacterial chromosomes through gene editing or other methods. Dey (copy number of genes), or modifications Dey Gene expression regulatory sequences (e.g., promoters, ribosome binding sites, etc.), or a combination of the above methods.

[0020] In some implementations, overexpression Dey The gene is integrated into the *E. coli* genome using gene editing methods. The integration site is selected based on conventional knowledge in the field of genetics, choosing pseudogene sites that will not significantly affect bacterial growth and basal metabolism, for example... yciQ, mbhA, yeeP , ygiP , yghX, ygaY , yjiT , ycjV , ycgH, ygaY, yeeL, ilvG All gene loci are selectable. In other embodiments, the gene is also linked to a promoter, which can be arbitrarily selected from P. trc Examples of promoters include BBa-J23100, BBA-J23106, and T7; in other embodiments, the promoter is a P sequence as shown in SEQ ID NO: 5. yddG Promoter.

[0021] According to a first aspect of the invention, the Dey The gene originates from Bacillus subtilis.

[0022] According to a first aspect of the invention, the Dey The gene is not limited to the nucleotide sequence shown in SEQ ID NO: 1, but may also include a mutant nucleotide sequence that is a variant of the sequence shown in SEQ ID NO: 1 or a gene that is homologous to the sequence shown in SEQ ID NO: 1 and encodes the YdeD protein. DeyGenes can be variant nucleotide sequences due to the degeneracy of the genetic code, such as nucleotide sequences encoding proteins as shown in SEQ ID NO: 2.

[0023] In a second aspect, the present invention provides a method for producing L-phenylalanine using genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-phenylalanine; and collecting the L-phenylalanine from the genetically engineered bacteria and / or the culture medium.

[0024] As described in the second aspect of the present invention, the genetically engineered bacteria have been overexpressed. Dey The genes were modified to make them comparable to unmodified E. coli. Dey The intensity of gene expression is increased or the expression level is raised.

[0025] According to a second aspect of the invention, the L-phenylalanine includes not only L-phenylalanine in its free form, but may also include salts or hydrates of L-phenylalanine.

[0026] According to a second aspect of the invention, the culture of the genetically engineered bacteria can be carried out using methods conventional in the art. The culture medium used for the production of L-phenylalanine can be a synthetic or natural culture medium, such as a typical culture medium containing a carbon source, nitrogen source, sulfur source, inorganic ions, and other required organic and inorganic components.

[0027] According to a second aspect of the invention, the genetically engineered bacteria can be cultured under aerobic conditions for 12 to 72 hours, or 24 to 60 hours, or 36 to 48 hours; the culture temperature can be controlled at 30 to 45°C, or 30 to 37°C; and the pH can be adjusted between 5.0 and 8.0, or 6.0 and 7.5, or 6.8 and 7.2. The pH can be adjusted by using inorganic or organic acidic or alkaline substances, as well as ammonia.

[0028] In some embodiments, the method further includes maintaining a constant culture temperature of 37°C; in other embodiments, the method further includes maintaining a pH of approximately 7.0.

[0029] After cultivation, solids, such as cells and cell debris, can be removed from the liquid culture medium using conventional techniques (e.g., centrifugation, membrane filtration). L-phenylalanine can then be recovered from the fermentation broth using any combination of conventional techniques (e.g., concentration, ion exchange chromatography, crystallization).

[0030] Before inoculation and fermentation, bacteria can undergo strain activation, seed culture, etc., depending on their storage state. Appropriate conditions and culture media can be selected according to conventional techniques in this field. For example, the seed culture medium can use the same composition as the fermentation culture medium, or it can be appropriately adjusted based on this.

[0031] Other specific operational methods involving molecular biology and genetic engineering can be implemented using technical manuals, textbooks, or literature reports readily available to those skilled in the art, and need not be described in detail here. Furthermore, specific bacterial strains were selected as hosts in the following examples, and therefore specific gene integration sites, target genes, and primers were chosen based on the host. However, this does not mean that the purpose of the present invention can only be achieved through these specific selections, and should not be construed as limiting the scope of the invention. The essence and scope of the present invention are defined only by the claims.

[0032] The present invention will be described in more detail below through specific embodiments.

[0033] 1. Methods of gene editing

[0034] The gene editing method used in this embodiment was performed in accordance with the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genomeediting. Metabolic engineering, 2015, 31: 13-21.). The two plasmid maps used in this method are shown in [link to relevant documentation]. Figure 1 The pREDCas9 plasmid carries the elimination system of the gRNA expression plasmid pGRB, the Red recombination system of λ phage, and the Cas9 protein expression system. It is resistant to zirconia (working concentration: 100 mg / L) and cultured at 32℃. pGRB uses pUC18 as its backbone and includes the promoter J23100, the gRNA-Cas9 binding region sequence, and the terminator sequence. It is resistant to ampicillin (working concentration: 100 mg / L) and cultured at 37℃.

[0035] The specific steps of this method are as follows:

[0036] 1.1 Construction of pGRB plasmid

[0037] The purpose of constructing plasmid pGRB is to transcribe the corresponding gRNA, thereby forming a complex with the Cas9 protein, and to achieve a double-strand break in the target DNA through base pairing and PAM recognition of the target gene site. The pGRB plasmid is constructed using a recombination method involving a DNA fragment containing the target sequence and a linearized vector fragment.

[0038] 1.1.1 Target Sequence Design

[0039] The target sequence (PAM: 5'-NGG-3') was designed using CRISPR RGEN Tools.

[0040] 1.1.2 Preparation of DNA fragments containing the target sequence

[0041] Primer design: 5'-linearized vector terminal sequence (15 bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector terminal sequence (15 bp)-3' and its reverse complementary primer. DNA fragments containing the target sequence were prepared by annealing single-stranded DNA. Reaction conditions: pre-denaturation 95℃, 5 min; annealing 30-50℃, 1 min. The annealing system is shown in Table 1.

[0042] Table 1

[0043]

[0044] 1.1.3 Preparation of linear carriers

[0045] The vector was linearized using reverse PCR amplification.

[0046] 1.1.4 Recombination reaction

[0047] The recombination system is shown in the table below. All recombinant enzymes used were from the ClonExpress® II One Step Cloning Kit series. Recombination conditions: 37℃, 30 min. The recombination system is shown in Table 2.

[0048] Table 2

[0049]

[0050] 1.1.5 Plasmid Transformation

[0051] Take 10 μL of reaction solution and add it to 100 mL of DH5α-transformed competent cells. Gently mix and incubate on ice for 20 min. Heat shock at 42℃ for 45-90 s, then immediately incubate on ice for 2-3 min. Add 900 μL of SOC and incubate at 37℃ for 1 h. Centrifuge at 8000 rpm for 2 min, discard part of the supernatant, and resuspend the bacterial cells in about 200 μL. Spread the resuspended cells onto a plate containing 100 mg / L ampicillin. Invert the plate and incubate overnight at 37℃. After single cells have grown on the plate, identify them by colony PCR and select positive recombinants.

[0052] 1.1.6 Cloning Identification

[0053] PCR-positive colonies were inoculated into LB medium containing 100 mg / L ampicillin and cultured overnight for preservation. Plasmids were then extracted and identified by enzyme digestion.

[0054] 1.2 Preparation of Recombinant DNA Fragments

[0055] For Dey Recombinant fragments of overexpressed genes are generated by Dey The gene's upstream and downstream homologous arms are composed (upstream homologous arm - downstream homologous arm). Primer design software Primer5 was used to... Dey Using the upstream and downstream sequences of the gene as templates, primers for the upstream and downstream homologous arms were designed. The upstream and downstream homologous arms were amplified separately by PCR, and then the recombinant fragment was prepared by overlap PCR. The PCR amplification system is shown in Table 3.

[0056] Table 3

[0057]

[0058] The systems for overlap PCR are shown in Table 4:

[0059] Table 4

[0060]

[0061] Note: The template consists of equimolar amounts of amplified fragments from upstream and downstream homologous arms and the target gene, and the total amount does not exceed 10 ng.

[0062] PCR reaction conditions (Takara Bio PrimeSTAR HS enzyme): Pre-denaturation (95℃) for 5 min; then 30 cycles: denaturation (98℃) for 10 s, annealing ((Tm-3 / 5)℃) for 15 s, extension at 72℃ (this enzyme activity extends by about 1 kb per min); continue extension at 72℃ for 10 min; maintain (4℃).

[0063] 1.3 Transformation of plasmids and recombinant DNA fragments

[0064] 1.3.1 Conversion of pREDCas9

[0065] The pREDCas9 plasmid was electroporated into the electroporation competent cells of the starting strain. After cell resuscitation and culture, the cells were plated on LB agar plates containing zirconia and incubated overnight at 32°C. Single colonies growing on the antibiotic-resistant plates were subjected to colony PCR using identification primers to screen for positive recombinants.

[0066] 1.3.2 Preparation of electrotransformation competent cells of the target strain containing pREDCas9

[0067] Incubate at 32℃ until OD 600 When the concentration reaches 0.1–0.2, add 0.1 M IPTG (to bring the final concentration to 0.1 mM) and continue culturing until OD reaches 0.2. 600 Competent cells were prepared when the pH was 0.6–0.7. The purpose of adding IPTG was to induce the expression of the recombinase on the pREDCas9 plasmid. The culture medium and preparation process for competent cells followed standard operating procedures.

[0068] 1.3.3 Transformation of pGRB and recombinant DNA fragments

[0069] pGRB and donor DNA fragments were simultaneously electroporated into electrocompetent cells containing pREDCas9. The revived cells after electroporation were plated on LB agar plates containing ampicillin and zithromycin and incubated overnight at 32°C. Colony PCR was performed using specially designed identification primers to verify the colonies, screen for positive recombinants, and maintain the cells.

[0070] 1.4 Plasmid Elimination

[0071] 1.4.1 Elimination of pGRB

[0072] Positive recombinants were incubated overnight in LB medium containing 0.2% arabinose. After appropriate dilution, they were spread onto LB plates containing zirconia-resistant bacteria and incubated overnight at 32°C. Single colonies that did not grow on ampicillin-resistant plates but grew on zirconia-resistant plates were selected and preserved.

[0073] 1.4.2 Elimination of pREDCas9 plasmid

[0074] The positive recombinant was transferred to antibiotic-free LB liquid medium and incubated overnight at 42°C. After appropriate dilution, it was spread onto antibiotic-free LB plates and incubated overnight at 37°C. Single colonies that did not grow on bilirubin-resistant plates and grew on antibiotic-free plates were selected for preservation.

[0075] The genetically engineered bacteria constructed in the 2 examples are described in Table 5.

[0076] Table 5

[0077]

[0078] The primer sequences used in the 3 examples are shown in Table 6.

[0079] Table 6

[0080]

[0081] In the 4 examples, the concentration of L-phenylalanine in the fermentation broth was determined by HPLC.

[0082] Collect the supernatant of the fermentation broth, dilute the supernatant to the concentration range of the determination (ensuring that the concentration of L-phenylalanine after dilution is within the range of the established standard curve), filter it through a 0.22 μm filter membrane, and detect the L-phenylalanine content by high performance liquid chromatography. The concentration of L-phenylalanine in the fermentation broth is calculated by comparing the peak area value with the standard curve.

[0083] The chromatographic conditions were as follows: Kromasil C18 column (250 mm × 460 mm, 5 μm), mobile phase was 10% acetonitrile solution, flow rate was set at 1.0 mL / min, column temperature was 40℃, retention time was 10 min, UV detection wavelength was 210 nm, injection volume was 20 μL, and peak elution time was approximately 2.612 min.

[0084] Construction of L-phenylalanine standard curve: L-phenylalanine solutions with concentrations of 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, and 0.5 g / L were prepared. Following the chromatographic conditions set above, the peak area values ​​of L-phenylalanine at the corresponding concentrations were measured by liquid chromatography. The linear relationship formula and standard curve between L-phenylalanine concentration and peak area were calculated using software.

[0085] Example 1:

[0086] This embodiment illustrates the construction of the genetically engineered bacterium for producing L-phenylalanine according to the present invention, specifically the genetically engineered bacterium PHE02.

[0087] Using the Escherichia coli genome as a template, based on its yghX upstream homologous arm primers (UP-yghX-S, UP-yghX-A) and downstream homologous arm primers (DN-yghX-S, DN-yghX-A) were designed based on the upstream and downstream sequences of the gene (NCBI GeneID: 2847694); according to aroG fbr Gene design primers (yghX::Ptrc-aroG-S, yghX::Ptrc-aroG-A), with the Ptrc promoter sequence designed in the upstream primer; the above fragments were fused using overlap PCR to obtain the target gene (upstream homologous arm -P trc - aroG fbr -Downstream homologous arm). Constructing pGRB- yghX The DNA fragment containing the target sequence was prepared by annealing with primers gRNA-yghX-S and gRNA-yghX-A. Using the *E. coli* genome as a template, based on its... tyrR Design upstream homologous arm primers (UP-tyrR-S, UP-tyrR-A) and downstream homologous arm primers (DN-tyrR-S, DN-tyrR-A) based on the upstream and downstream sequences of the gene (NCBIGeneID: 945879); according to pheA fbr Genes and tyrBPrimers (tyrR::Ptrc-pheA-tyrB-1, tyrR::Ptrc-pheA-tyrB-2, tyrR::Ptrc-pheA-tyrB-3, tyrR::Ptrc-pheA-tyrB-4) were designed for the gene (NCBI GeneID: 948563), with the Ptrc promoter sequence designed in the upstream primer of the gene. The above fragments were fused using overlap PCR to obtain the target gene (upstream homologous arm -P). trc - pheA fbr - tyrB- Downstream homologous arm). Constructing pGRB- tyrR The DNA fragment containing the target sequence was prepared by annealing with primers gRNA-tyrR-S and gRNA-tyrR-A. Competent cells of strain W3110 were prepared according to the methods shown in 1.3 and 1.4 to construct cells with enhanced... aroG fbr Gene expression intensity ( yghX ::P trc - aroG fbr )and pheA fbr -tyrB Gene expression intensity ( tyrR:: P trc -pheA fbr -tyrB The engineered strain PHE00 was successfully verified.

[0088] Among them, the aroG fbr The gene is a mutant gene that has been relieved of feedback inhibition, and its nucleotide sequence is shown in SEQ ID NO: 3; pheA fbr The gene is a mutant gene that has been relieved of feedback inhibition, and its nucleotide sequence is shown in SEQ ID NO: 4.

[0089] Using the Escherichia coli genome as a template, based on its yeeP Design upstream homologous arm primers (UP-yeeP-S, UP-yeeP-A) and downstream homologous arm primers (DN-yeeP-S, DN-yeeP-A) based on the upstream and downstream sequences of gene (NCBI GeneID: 946524); according to Gdy Primers (PyddG-S, yeeP-yddG-A) were designed based on the gene (NCBI GeneID: 945942); the above fragments were fused using overlap PCR to obtain the target gene (upstream homologous arm - yeeP::P yddG - Gdy-Downstream homologous arm). Constructing pGRB- yeeP The DNA fragment containing the target sequence was prepared by annealing with primers gRNA-yeeP-S and gRNA-yeeP-A. Competent cells of strain PHE00 were prepared according to the methods shown in 1.3 and 1.4 to construct cells with enhanced... Gdy Gene expression intensity ( yeeP::P yddG - Gdy The engineered strain PHE01 was successfully verified.

[0090] Using the Escherichia coli genome as a template, based on its yciQ upstream homologous arm primers (UP-yciQ-S, UP-yciQ-A) and downstream homologous arm primers (DN-yciQ-S, DN-yciQ-A) were designed based on the upstream and downstream sequences of the gene (NCBI GeneID: 945850); according to Gdy PCR primers (PyddG-S, PyddG-A) were designed based on the 200 bp upstream sequence of the gene (NCBI GeneID: 945942); PCR primers (ydeD-S, ydeD-A) were designed based on the gene sequence of the presumed metabolite transporter YdeD from Bacillus subtilis subsp. subtilis str. 168 (NCBI Gene ID: 939906); the above fragments were fused using overlap PCR to obtain the target gene (upstream homologous arm -P yddG - Dey- Downstream homologous arm). Constructing pGRB- yciQ The DNA fragment containing the target sequence was prepared by annealing with primers gRNA-yciQ-S and gRNA-yciQ-A. Competent cells of the engineered strain PHE01 were prepared according to the methods shown in 1.3 and 1.4 to construct cells with enhanced... Dey Gene expression intensity ( yciQ ::P yddG - Dey The engineered strain PHE02 was successfully verified.

[0091] Using the Escherichia coli genome as a template, based on its yciQ upstream homologous arm primers (UP-yciQ-S, UP-yciQ-A) and downstream homologous arm primers (DN-yciQ-S, DN-yciQ-A) were designed based on the upstream and downstream sequences of the gene (NCBI GeneID: 945850); according to GdyPrimers (PyddG-S, yciQ::yddG-A) were designed based on the gene (NCBI GeneID: 945942); the above fragments were fused using overlap PCR to obtain the target gene (upstream homologous arm -P). yddG - Gdy- Downstream homologous arm). Constructing pGRB- yciQ The DNA fragment containing the target sequence was prepared by annealing with primers gRNA-yciQ-S and gRNA-yciQ-A. Competent cells of the engineered strain PHE01 were prepared according to the methods shown in 1.3 and 1.4 to construct cells with enhanced... Gdy Gene expression intensity ( yciQ ::P yddG - Gdy The engineered strain PHE03 was successfully verified.

[0092] Example 2:

[0093] This embodiment illustrates the construction of the genetically engineered bacterium for producing L-phenylalanine according to the present invention, specifically the genetically engineered bacterium PHE04.

[0094] Using the Escherichia coli genome as a template, based on its yciQ Design upstream homologous arm primers (UP-yciQ-S, UP-yciQ-Ptrc-ydeD-A) and downstream homologous arm primers (DN-yciQ-S, DN-yciQ-A) based on the upstream and downstream sequences of the gene (NCBI GeneID: 945850); according to Dey PCR primers (yciQ-Ptrc-ydeD-S, ydeD-A) were designed based on the gene (NCBI Gene ID: 939906), with the Ptrc promoter incorporated into the upstream homologous arm primer. These fragments were then fused using overlap PCR to obtain the target gene (upstream homologous arm -P). trc - Dey -Downstream homologous arm). Constructing pGRB- yciQ The DNA fragment containing the target sequence was prepared by annealing with primers gRNA-yciQ-S and gRNA-yciQ-A. Competent cells of strain PHE00 were prepared according to the methods shown in 1.3 and 1.4 to construct cells with enhanced... Dey Gene expression intensity ( yciQ ::P trc -Dey The engineered strain PHE04 was successfully verified.

[0095] Example 3:

[0096] This embodiment is used to illustrate the construction of the genetically engineered bacteria for producing L-phenylalanine according to the present invention. Specifically, it can be genetically engineered bacteria PHE06, PHE07, PHE08, PHE09 or PHE10.

[0097] This embodiment will be provided by P yddG Starter-controlled Dey The gene was constructed in the pSTV28 plasmid, which was then electroporated into strains MG1655, W3110, BL21, DH5α, or BW25113 to obtain strains PHE06, PHE07, PHE08, PHE09, or PHE10, respectively. The empty pSTV28 plasmid was electroporated into strain MG1655 to obtain strain PHE05, which served as the control group. Positive strains were successfully verified by PCR.

[0098] Example 4:

[0099] This embodiment is used to illustrate the use of the overexpression method described in this invention. Dey A method for producing L-phenylalanine using genetically engineered bacteria.

[0100] (1) Activation culture: The strain was transferred to antibiotic-free LB medium for 12 h of resuscitation culture. The transfer volume ratio was about 5 μL / mL. The resuscitation culture conditions were 37 ℃ and 220 rpm.

[0101] (2) Seed culture: First, add 1 mL of 60% (m / v) glucose solution to the seed culture medium as the base sugar, and then add 3 mL of bacterial culture to 27 mL of seed culture medium. Seal the Erlenmeyer flask with sterile gauze. Then, culture at 37 ℃ and 220 rpm for 8-10 h.

[0102] (3) Fermentation culture: First, add 1 mL of 60% (m / v) glucose solution to the seed culture medium as the base sugar, and then transfer 3 mL of seed culture to 27 mL of fermentation culture medium. Seal the Erlenmeyer flask with sterile gauze. Then, carry out constant temperature and constant speed culture at 37 ℃ and 220 rpm for 24 h. During the fermentation process, the pH change of the bacterial solution needs to be judged according to the color development of phenol red in the fermentation broth, and the pH of the bacterial solution should be maintained at about 7.0 by manually adding ammonia. When the color of the fermentation broth does not change, it indicates that the bacteria are lacking glucose, and 1 mL of 60% (m / v) glucose needs to be added in time to maintain bacterial fermentation.

[0103] The preferred slant culture medium in this embodiment is prepared as follows: First, glucose, peptone, beef extract, yeast powder, sodium chloride, and agar powder are weighed according to the required concentration ratios of 5 g / L, 10 g / L, 10 g / L, 5 g / L, 5 g / L, and 20 g / L, respectively. The mixture is then diluted to volume with deionized water and thoroughly mixed. Subsequently, the pH of the culture medium is adjusted to approximately 7.0 with diluted ammonia water, and sterilization is performed at 121°C for 20 minutes.

[0104] The preferred shake-flask seed culture medium in this embodiment is as follows: First, yeast powder, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, sodium citrate, sodium chloride, calcium chloride, ferrous sulfate heptahydrate, and trace element mixture are weighed according to the required concentrations of 10 g / L, 4 g / L, 3 g / L, 2 g / L, 2 g / L, 1 g / L, 0.015 g / L, 0.005 g / L, and 1 mL / L, respectively. The solutions are then diluted to volume with deionized water and mixed thoroughly. Subsequently, the pH of the culture medium solution is adjusted to approximately 7.0 with diluted ammonia water, and sterilization is performed at 115 ℃ for 15 min.

[0105] The preferred shake-flask fermentation medium in this embodiment is as follows: First, according to the required quantities, yeast powder, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, sodium citrate, sodium chloride, calcium chloride, ferrous sulfate heptahydrate, and trace element mixture are weighed at concentration ratios of 5 g / L, 5 g / L, 3 g / L, 2 g / L, 2 g / L, 1 g / L, 0.015 g / L, 0.03 g / L, and 1.5 mL / L, respectively. The mixture is then diluted to volume with deionized water and thoroughly mixed. Subsequently, the pH of the medium is adjusted to approximately 7.0 with diluted ammonia water, and sterilization is performed at 115°C for 15 min. Note: The glucose in the shake-flask medium is sterilized separately at a concentration of 60% (m / v) at 115°C for 15 min.

[0106] Example 5:

[0107] This example is used to illustrate overexpression. Dey Differences in L-phenylalanine production performance between genetically modified PHE02, PHE01, and PHE03.

[0108] The PHE02 strain and control strains PHE01 and PHE03 were cultured in shake flasks using the culture method described in Example 4. The concentration of L-phenylalanine in the fermentation broth was measured (results are shown in Table 7).

[0109] Table 7

[0110]

[0111] Table 7 shows the results of overexpression GdyGiven that the gene has already enhanced the ability to efflux L-phenylalanine (PHE01), further enhancement is needed. Gdy Gene expression intensity significantly affected strain growth (PHE03), however, overexpression at this time... Dey The gene significantly improved the L-phenylalanine production capacity and enhanced the growth ability of the engineered strain (PHE02). Compared with PHE01, the L-phenylalanine accumulation in PHE02 increased from 10.8 g / L to 13.4 g / L, and the L-phenylalanine yield increased by 24.1%. The results showed that enhancing the L-phenylalanine production capacity in *E. coli* significantly improved the L-phenylalanine production capacity and growth ability of the strain. Dey Gene expression intensity can significantly improve the L-phenylalanine production performance of engineered strains.

[0112] Example 6:

[0113] This example is used to illustrate overexpression. Dey Differences in L-phenylalanine production performance between genetically modified PHE04 and PHE00.

[0114] The PHE04 strain and the control strain PHE00 were cultured in shake flasks using the culture method described in Example 4. The concentration of L-phenylalanine in the fermentation broth was measured (results are shown in Table 8).

[0115] Table 8

[0116]

[0117] Table 8 shows the results of overexpression Dey In the genetically modified PHE04, the accumulation of L-phenylalanine increased from 8.9 g / L to 10.9 g / L, and the L-phenylalanine yield increased by 22.5%. The results showed that this enhanced the L-phenylalanine content in *E. coli*. Dey Gene expression intensity can significantly improve the L-phenylalanine production performance of engineered strains.

[0118] Example 7:

[0119] This example is used to illustrate overexpression. Dey Differences in L-phenylalanine production performance among genetically modified PHE06, PHE07, PHE08, PHE09, or PHE10.

[0120] PHE05 to PHE10 were cultured in shake flasks using the cultivation method described in Example 4. The concentration of L-phenylalanine in the fermentation broth was measured (results are shown in Table 9).

[0121] Table 9

[0122]

[0123] Table 9 shows the results, enhancing... DeyGene expression intensity had a certain impact on the accumulation of L-phenylalanine in the strains. The L-phenylalanine production in PHE06, PHE07, PHE08, PHE09, or PHE10 reached 0.21 g / L, 0.31 g / L, 0.15 g / L, 0.13 g / L, or 0.25 g / L, respectively, while no L-phenylalanine accumulation was detected in the control group, PHE05. The results showed that enhancing L-phenylalanine accumulation in *E. coli*... Dey Gene expression intensity can significantly improve the production performance of L-phenylalanine in Escherichia coli.

[0124] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A genetically engineered bacterium for producing L-phenylalanine, characterized in that: The genetically engineered bacteria are Escherichia coli overexpressed with... ydeD The genes were modified in a specific way; Among them, the ydeD The nucleotide sequence of the gene is shown in SEQ ID NO: 1, or the nucleotide sequence encoding the protein shown in SEQ ID NO: 2; The genetically engineered bacteria also overexpressed aroG fbr Genes, and / or overexpression pheA fbr Gene; wherein, the described aroG fbr The gene nucleotide sequence is shown in SEQ ID NO:

3. pheA fbr The gene nucleotide sequence is shown in SEQ ID NO:

4.

2. The genetically engineered bacteria as described in claim 1, characterized in that: The genetically engineered bacteria also overexpressed yddG Gene.

3. The genetically engineered bacteria as described in claim 2, characterized in that: The overexpression ydeD Genetic pathways include: increasing gene size through autonomous replication plasmids. ydeD The copy number of a gene, and / or the increase through gene editing methods. ydeD Gene copy number, and / or modifications ydeD Gene expression regulatory sequences.

4. The genetically engineered bacteria as described in claim 3, characterized in that: The ydeD The gene is also linked to P yddG The promoter, the P yddG The nucleotide sequence of the promoter is shown in SEQ ID NO:

5.

5. The use of the genetically engineered bacteria according to any one of claims 1-4 in the fermentation production of L-phenylalanine.

6. A method for producing L-phenylalanine, characterized in that: include: The genetically engineered bacteria according to any one of claims 1-4 are cultured in a culture medium to produce L-phenylalanine; And, the L-phenylalanine is collected from the genetically engineered bacteria and / or the culture medium.

7. The method as described in claim 6, characterized in that: The method also includes: maintaining a constant culture temperature of 37°C and / or maintaining a pH of approximately 7.0.

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