Genetically engineered bacteria overexpressing ynal gene and method for producing l-ornithine

Abstract

This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to overexpression. ynal Genetically engineered bacteria and a method for producing L-ornithine. The genetically engineered bacteria are used to overexpress... ynal The genes were modified to make them comparable to unmodified E. coli. ynal Enhanced gene expression intensity or increased expression level results in modified genetically engineered bacteria having enhanced L-ornithine production capacity.

Description

Technical Field

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

[0002] L-ornithine has wide applications in medicine, industry, food, cosmetics, animal husbandry and other fields, and has important economic and social value.

[0003] Selecting high-efficiency strains with L-ornithine production capacity is key to the industrial application of microbial fermentation. Currently, there are two main methods for selecting L-amino acid production strains: (1) Irrational mutagenesis screening and adaptive evolution: Mutagenesis breeding mainly involves mutagenesis of wild-type chassis microorganisms using physical or chemical methods, combined with high-throughput quantitative screening methods for L-ornithine, to select strains with improved L-ornithine production performance. After multiple rounds of mutagenesis, superior production strains with better L-ornithine synthesis capacity are finally screened out. Adaptive evolution mainly involves gradually increasing the concentration of L-ornithine in the growth environment of L-ornithine production strains, enriching the population with genes related to tolerance to high concentrations of L-ornithine, and then combining high-throughput screening methods to obtain production strains that are tolerant and whose yield has not decreased, thereby improving the production activity of strains in the middle and late stages of fermentation. (2) Rational metabolic engineering modification: This method mainly utilizes efficient gene editing technology to systematically modify the L-ornithine synthesis network in chassis microorganisms to maximize the redirection of carbon metabolism to the L-ornithine synthesis pathway. This mainly includes blocking the L-ornithine degradation pathway, removing the feedback inhibition regulation mechanism of key enzymes in the synthesis pathway, enhancing the supply of the precursor L-glutamate, optimizing the balance of coenzyme supply in chassis cells, and modifying the L-ornithine transmembrane transport system.

[0004] Improving L-ornithine production performance in chassis microorganisms is a sustainable goal of rational metabolic engineering breeding. In the systematic reconstruction of the metabolic network of chassis microorganisms, enhancing or weakening the expression intensity of target genes is a common strategy for improving L-ornithine production performance. For example, weakening the expression of one or more genes involved in L-ornithine degradation, one or more genes involved in the competitive pathway of L-ornithine synthesis, or genes involved in carbon-nitrogen flow redistribution significantly promotes L-ornithine synthesis. Furthermore, increasing the expression of key enzymes in the L-ornithine synthesis pathway, increasing the expression of efflux membrane proteins of target products, and increasing the expression of key enzymes in the precursor synthesis pathway of target products also have significant beneficial effects on improving L-ornithine synthesis in chassis microorganisms. YnaI protein (composed of...) thenI (Gene-encoded) It belongs to the MScS family of membrane proteins and is a component of small-conductivity mechanosensitive ion channels in cells. There are currently no research reports on its relationship with the L-ornithine synthesis pathway. 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-ornithine.

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

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

[0008] As described in the first aspect of the present invention, the genetically engineered bacteria, Escherichia coli, has been modified to enable it to produce L-ornithine.

[0009] In a second aspect, the present invention provides a method for producing L-ornithine using genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-ornithine; and collecting the L-ornithine 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. thenI The genes were modified to make them comparable to unmodified E. coli. thenI The intensity of gene expression is increased or the expression level is raised.

[0011] As described in the second aspect of the present invention, the *Escherichia coli* has been modified to enable it to produce L-ornithine.

[0012] The beneficial effects of this invention are as follows:

[0013] thenI The relationship and role of the gene and its encoded protein in the L-ornithine synthesis pathway are still unclear. This invention confirms that overexpression... thenI The gene can significantly improve the L-ornithine production capacity of engineered strains. The overexpression... thenI A modified gene is one whose expression intensity is significantly increased compared to its original expression intensity. This invention uses the Orn7 strain as an example, after expression... thenI After gene modification, the L-ornithine yield in this strain increased by 10.94%; the embodiments of this invention use Orn10 strain as an example after expression... thenI After gene modification, the L-ornithine yield in this strain increased by 13.55%; the embodiments of this invention use Orn12 strain as an example after expression... thenI After gene modification, the L-ornithine yield in this strain increased by 11.32%. Attached Figure Description

[0014] Figure 1 and Figure 2 : Schematic diagram of the plasmids used in the gene editing method 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] Unless otherwise stated, the term "production" of L-ornithine (or other target products) in this invention refers to the process by which L-ornithine accumulates intracellularly or extracellularly in microbial cells, whether genetically modified or not, through transcription, translation, and metabolic catalysis, and can be separated and purified to obtain a detectable amount of L-ornithine final product.

[0017] Firstly, the present invention provides a genetically engineered bacterium for producing L-ornithine, specifically, belonging to *Escherichia coli*, and more specifically, the genetically engineered bacterium is an *Escherichia coli* strain overexpressing... thenI The genes were modified.

[0018] According to a first aspect of the present invention, the genetically engineered bacterium is an overexpression of *Escherichia coli*. thenI The genes were modified to make them comparable to unmodified E. coli. thenI Gene expression levels are increased, for example, by 150% or more, 200% or more, 300% or more.

[0019] According to a first aspect of the present invention, the *E. coli* is further modified to enable it to produce L-ornithine. For example, the *E. coli* has been modified to block or weaken the ornithine degradation pathway (e.g., to not express or weaken the expression of one or more genes among argF, argI, speC, speF, and proB); and / or the *E. coli* has been modified to remove key enzyme feedback inhibition (e.g., to introduce an argA mutant); and / or to enhance the flux of the ornithine synthesis pathway (e.g., to remove ArgR repression, to overexpress argCJBD, etc.); and / or to enhance the ornithine transport system; and / or to enhance the supply of the cofactor NADPH (e.g., to overexpress genes such as zwf, tkt, pPK, and gapC); and / or to strengthen central carbon metabolism (e.g., to overexpress genes such as pfkA, gap, pyk, gltA, and gdh); and / or to introduce heterologous argJ to achieve the acetyl cycle, etc.

[0020] According to a first aspect of the invention, overexpression thenI Genetic methods can be used by introducing and / or adding genes into the bacterial genome. thenI Gene copy number (e.g., increased by autonomously replicating plasmids such as pET28a, pTrc99a, and pSTV28) thenI The copy number of genes, or increasing the number of genes in bacterial chromosomes through gene editing or other methods. thenI (copy number of genes), or modifications thenI Gene expression regulatory sequences (e.g., promoters, ribosome binding sites, etc.), or a combination of the above methods.

[0021] In some implementations, overexpression thenI 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... mbhA、yeeP , yesP , yghX、ygaY , yjiT , etc , ycgH、ygaY、yeeL、ilvG Gene loci are all selectable. In other embodiments, the gene is also linked to a promoter, which can be selected based on conventional understanding among those skilled in the art to regulate gene expression according to production needs, such as P. trc Examples include BBa-J23100 and T7. In other embodiments, the promoter is... thenI The original promoter of a gene P native The nucleotide sequence is shown in SEQ ID NO: 4.

[0022] According to a first aspect of the invention, the thenI 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 YnaI protein. thenI Genes 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-ornithine using genetically engineered bacteria as described above, comprising: culturing the genetically engineered bacteria in a culture medium to produce L-ornithine; and collecting the L-ornithine from the genetically engineered bacteria and / or the culture medium.

[0024] According to a second aspect of the invention, the genetically engineered bacteria has been overexpressed thenI The genes were modified to make them comparable to unmodified E. coli. thenI The intensity of gene expression is increased or the expression level is raised.

[0025] According to a second aspect of the present invention, the *Escherichia coli* has been modified to enable it to produce L-ornithine, for example, by introducing gene mutations, gene knockouts, etc., to block the ornithine degradation pathway and / or to remove intracellular feedback repression and feedback inhibition to promote ornithine accumulation.

[0026] According to a second aspect of the invention, the L-ornithine includes not only L-ornithine in its free form, but may also include salts or hydrates of L-ornithine.

[0027] 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-ornithine 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.

[0028] 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.

[0029] 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; in still other embodiments, the method further includes: maintaining dissolved oxygen at approximately 35%.

[0030] 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-ornithine can then be recovered from the fermentation broth using any combination of conventional techniques (e.g., concentration, ion exchange chromatography, crystallization).

[0031] 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.

[0032] 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.

[0033] The present invention will be described in more detail below through specific embodiments.

[0034] 1. Methods of gene editing

[0035] The gene editing method used in this embodiment is based on 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 attached. Figure 1 and Figure 2 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℃.

[0036] The specific steps of this method are as follows:

[0037] 1.1 Construction of pGRB plasmid

[0038] 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.

[0039] 1.1.1 Target Sequence Design

[0040] The target sequence (PAM: 5'-NGG-3') was designed using CRISPR RGEN Tools.

[0041] 1.1.2 Preparation of DNA fragments containing the target sequence

[0042] Primer design: 5'-linearized vector terminal sequence (20 bp)-restriction site-target sequence (excluding PAM sequence)-linearized vector terminal sequence (20 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.

[0043] Table 1

[0044]

[0045] 1.1.3 Preparation of linear carriers

[0046] The vector was linearized using reverse PCR amplification.

[0047] 1.1.4 Recombination reaction

[0048] 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.

[0049] Table 2

[0050]

[0051] 1.1.5 Plasmid Transformation

[0052] 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.

[0053] 1.1.6 Cloning Identification

[0054] 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.

[0055] 1.2 Preparation of Recombinant DNA Fragments

[0056] For thenI Recombinant fragments of overexpressed genes are generated by yciQ Upstream and downstream homologous arms of genes and those derived from thenI Original promoter controlled thenI Genome composition (upstream homologous arm - P) native - thenI - Downstream homologous arm). Using primer design software Primer5, to... yciQ Using the upstream and downstream sequences of the gene as templates, primers for upstream and downstream homologous arms (approximately 400-500 bp in length) were designed. The upstream and downstream homologous arms and the target gene fragment were amplified separately by PCR, followed by overlap PCR to prepare the recombinant fragment. The PCR amplification system is shown in Table 3.

[0057] Table 3

[0058]

[0059] The systems for overlap PCR are shown in Table 4:

[0060] Table 4

[0061]

[0062] 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.

[0063] 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℃).

[0064] 1.3 Transformation of plasmids and recombinant DNA fragments

[0065] 1.3.1 Conversion of pREDCas9

[0066] 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.

[0067] 1.3.2 Preparation of electrotransformation competent cells of the target strain containing pREDCas9

[0068] 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.

[0069] 1.3.3 Transformation of pGRB and recombinant DNA fragments

[0070] 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.

[0071] 1.4 Plasmid Elimination

[0072] 1.4.1 Elimination of pGRB

[0073] 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.

[0074] 1.4.2 Elimination of pREDCas9 plasmid

[0075] 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.

[0076] 2. The primer sequences used in the embodiments are shown in Table 5:

[0077] Table 5

[0078]

[0079] 3. The culture medium used in the examples can be referred to as follows:

[0080] Slant culture medium: glucose 1-5 g / L, peptone 5-10 g / L, beef extract 5-10 g / L, yeast extract 1-5 g / L, NaCl 1-2.5 g / L, agar 15-20 g / L, the remainder being water, pH 7.0-7.2.

[0081] Seed culture medium: glucose 20-40 g / L, yeast extract 2-5 g / L, peptone 2-4 g / L, K₂HPO₄ 1-3 g / L, MgSO₄·7H₂O 1-2 g / L, FeSO₄·7H₂O 15-20 mg / L, MnSO₄·7H₂O 15-20 mg / L, V B1 V B3 V B5 V B12 V H Each 1-3 mg / L, L-arginine 2 g / L, the remainder water, pH 7.0-7.2.

[0082] Fermentation medium: glucose 20-40 g / L, yeast extract 1-3 g / L, peptone 2-3 g / L, K₂HPO₄ 3-6 g / L, MgSO₄·7H₂O 1-2 g / L, FeSO₄·7H₂O 15-20 mg / L, MnSO₄·7H₂O 15-20 mg / L, V B1 V B3 V B5 V B12 V H Each 1-3 mg / L, L-arginine 2 g / L, the remainder water, pH 7.0-7.2.

[0083] Example 1:

[0084] This embodiment illustrates the construction of the genetically engineered bacterium for producing L-ornithine according to the present invention, specifically the genetically engineered bacterium Orn16.

[0085] The genetically engineered bacterium Orn16 is an overexpression of Escherichia coli. thenI The genes were modified, and the *E. coli* strain was selected from the genetically engineered bacterium Orn7 (CN116162581A). Orn7 is a modified L-ornithine-producing bacterium that does not express the genes argF, argI, speC, and speF in the *E. coli* strain, but replaces the acetylornithine deacetylase gene argE with the glutamate acetyltransferase gene argJ from *Corynebacterium glutamicum* and overexpresses it. Furthermore, it overexpresses the argC, argJ, argB, and argD argine synthesis control genes from *Corynebacterium glutamicum*.

[0086] Using the Escherichia coli genome as a template, based on its yciQ Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI-GeneID: 945850). yciQ -S、UP- yciQ -A) and downstream homologous arm primer (DN- yciQ -S、DN- yciQ -A); according to thenI Design and amplification of upstream and downstream sequences of gene (NCBI GeneID: 945898) thenI Primers required for gene generation thenI -S and thenI -A. The above fragments were obtained by fusion using overlap PCR. yciQ::P native -thenI (Upstream homologous arm - P) native - thenI -Downstream homologous arm). Constructing pGRB- yciQ The DNA fragment containing the target sequence used is transmitted via primer gRNA- yciQ -S and gRNA- yciQ -A was obtained by annealing. Competent cells of the engineered strain Orn7 were prepared by following the methods shown in 1.3 and 1.4 to construct cells with enhanced... thenI The engineered strain Orn16 with high gene expression intensity.

[0087] Example 2:

[0088] This embodiment illustrates the construction of the genetically engineered bacterium for producing L-ornithine according to the present invention, specifically the genetically engineered bacterium Orn17.

[0089] The genetically engineered bacterium Orn17 is an overexpression of Escherichia coli. thenI The genes were modified, and the *E. coli* strain was selected from the genetically engineered bacterium Orn10 (CN116162581A). Orn10 is a modified L-ornithine-producing bacterium that does not express genes argF, argI, speC, and speF as *E. coli*, but overexpresses genes argO, pntA, and pntB. Furthermore, the acetylornithine deacetylase gene argE is replaced with the glutamate acetyltransferase gene argJ from *Corynebacterium glutamicum* and overexpressed. Additionally, the argC, argJ, argB, and argD args of *Corynebacterium glutamicum* are overexpressed.

[0090] Using the Escherichia coli genome as a template, based on its yciQ Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI-GeneID: 945850). yciQ -S、UP- yciQ -A) and downstream homologous arm primer (DN- yciQ -S、DN- yciQ -A); according to thenI Design and amplification of upstream and downstream sequences of gene (NCBI GeneID: 945898) thenI Primers required for gene generation thenI-S and thenI -A. The above fragments were obtained by fusion using overlap PCR. yciQ ::P native - thenI (Upstream homologous arm - P) native - thenI -Downstream homologous arm). Constructing pGRB- yciQ The DNA fragment containing the target sequence used is transmitted via primer gRNA- yciQ -S and gRNA- yciQ -A was obtained by annealing. Competent cells of the engineered strain Orn10 were prepared by following the methods described in 1.3 and 1.4 above, and a structure with enhanced... thenI The engineered strain Orn17 with high gene expression intensity.

[0091] Example 3:

[0092] This embodiment illustrates the construction of the genetically engineered bacterium for producing L-ornithine according to the present invention, specifically the genetically engineered bacterium Orn18.

[0093] The genetically engineered bacterium Orn18 is an overexpression of Escherichia coli. thenI The genes were modified, and the *E. coli* strain was selected from the genetically engineered bacterium Orn12 (CN116162581A). Orn12 is a modified L-ornithine-producing bacterium that does not express genes argF, argI, speC, and speF in addition to *E. coli*, but overexpresses genes argO, pntA, and pntB. Furthermore, the acetylornithine deacetylase gene argE is replaced with the glutamate acetyltransferase gene argJ from *Corynebacterium glutamicum* and overexpressed. Additionally, the argC, argJ, argB, and argD args of *Corynebacterium glutamicum* are overexpressed, and the promoter P is used with the nucleotide sequence shown in SEQ ID NO: 3. rpsL Control the expression of the gene sucA, which encodes the E1 subbody of α-ketoglutarate dehydrogenase.

[0094] Using the Escherichia coli genome as a template, based on its yciQ Design upstream homologous arm primers (UP-) based on the upstream and downstream sequences of the gene (NCBI-GeneID: 945850). yciQ -S、UP- yciQ -A) and downstream homologous arm primer (DN- yciQ -S、DN- yciQ -A); according to thenI Design and amplification of upstream and downstream sequences of gene (NCBI GeneID: 945898) thenI Primers required for gene generation thenI -S and thenI-A. The above fragments were obtained by fusion using overlap PCR. yciQ ::P native - thenI (Upstream homologous arm - P) native - thenI -Downstream homologous arm). Constructing pGRB- yciQ The DNA fragment containing the target sequence used is transmitted via primer gRNA- yciQ -S and gRNA- yciQ -A was obtained by annealing. Competent cells of the engineered strain Orn12 were prepared by following the methods shown in 1.3 and 1.4 to construct cells with enhanced... thenI The engineered strain Orn18 with high gene expression intensity.

[0095] Example 4:

[0096] This embodiment is used to illustrate the use of the overexpression method described in this invention. thenI A method for producing L-ornithine using genetically engineered bacteria.

[0097] (1) Slant culture: Take the -80℃ preserved strain and streak it onto the activated slant, incubate at 37℃ for 12 h, and subculture once;

[0098] (2) Shake flask seed culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. Seal the flask with nine layers of gauze and culture at 37℃ and 200 rpm for 7-10 h.

[0099] (2) Shake-flask fermentation: Inoculate 10% of the fermentation culture volume into a 500 mL Erlenmeyer flask containing fermentation medium (final volume 30 mL), seal with nine layers of gauze, and incubate at 37℃ with shaking at 200 r / min. During fermentation, maintain the pH at 7.0 by adding ammonia water; add 60% (m / v) Fermentation was maintained by glucose solution; the fermentation cycle was 12 hours.

[0100] The preferred composition of the slant culture medium in this embodiment is: 1 g / L glucose, 5 g / L peptone, 5 g / L beef extract, 1 g / L yeast extract, 1 g / L NaCl, 15 g / L agar, and the remainder is water, pH 7.0.

[0101] The preferred seed culture medium composition in this embodiment is: glucose 20 g / L, yeast extract 2-5 g / L, peptone 2 g / L, K2HPO4 1 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 15 mg / L, MnSO4·7H2O 15 mg / L, V B1 V B3V B5 V B12 V H Each 1 mg / L, L-arginine 2 g / L, the remainder water, pH 7.0.

[0102] The preferred fermentation medium composition in this embodiment is: glucose 20 g / L, yeast extract 1 g / L, peptone 2 g / L, K2HPO4 3 g / L, MgSO4·7H2O 1 g / L, FeSO4·7H2O 15 mg / L, MnSO4·7H2O 15 mg / L, V B1 V B3 V B5 V B12 V H Each 1 mg / L, L-arginine 2 g / L, the remainder being water, pH 7.0.

[0103] Example 5:

[0104] This example is used to illustrate overexpression. thenI Differences in L-ornithine production performance between genetically modified Orn16 and the original strain Orn7.

[0105] Orn16 and Orn7 were cultured in shake flasks using the culture method described in Example 4 to evaluate overexpression. thenI The effect of genes on L-ornithine synthesis. The OD of the strain was detected using a UV spectrophotometer. 600 The concentration of L-ornithine in the fermentation supernatant was detected by HPLC, and the results are shown in Table 6.

[0106] Table 6

[0107]

[0108] Table 6 shows the results, enhancing... thenI Gene expression intensity did not significantly affect the growth of the engineered strain; meanwhile, the L-ornithine concentration in strain Orn16 was 6.59 g / L. Compared with the L-ornithine production in the control strain Orn7, the L-ornithine yield in strain Orn16 increased by 10.94% (from 5.94 g / L to 6.59 g / L). The results show that enhancing L-ornithine production in *E. coli*... thenI Gene expression intensity can significantly improve the L-ornithine production performance of engineered strains.

[0109] Example 6:

[0110] This example is used to illustrate overexpression. thenI Differences in L-ornithine production performance between genetically modified Orn17 and the original strain Orn10.

[0111] Orn17 and Orn10 were cultured in shake flasks using the culture method described in Example 4 to evaluate overexpression. thenI The effect of genes on L-ornithine synthesis. The OD of the strain was detected using a UV spectrophotometer. 600 The concentration of L-ornithine in the fermentation supernatant was detected by HPLC, and the results are shown in Table 7.

[0112] Table 7

[0113]

[0114] Table 7 shows the results, enhancing... thenI Gene expression intensity did not significantly affect the growth of the engineered strain; meanwhile, the L-ornithine concentration in strain Orn17 was 8.46 g / L. Compared with the L-ornithine production in the control strain Orn10, the L-ornithine yield in strain Orn17 increased by 13.55% (from 7.45 g / L to 8.46 g / L). The results show that enhancing L-ornithine production in *E. coli*... thenI Gene expression intensity can significantly improve the L-ornithine production performance of engineered strains.

[0115] Example 7:

[0116] This example is used to illustrate overexpression. thenI Differences in L-ornithine production performance between genetically modified Orn18 and the original strain Orn12.

[0117] Orn18 and Orn12 were cultured in shake flasks using the culture method described in Example 4 to evaluate overexpression. thenI The effect of genes on L-ornithine synthesis. The OD of the strain was detected using a UV spectrophotometer. 600 The concentration of L-ornithine in the fermentation supernatant was detected by HPLC, and the results are shown in Table 8.

[0118] Table 8

[0119]

[0120] Table 8 shows the results, enhancing... thenI Gene expression intensity did not significantly affect the growth of the engineered strain; meanwhile, the L-ornithine concentration in strain Orn18 was 9.05 g / L. Compared with the L-ornithine production in the control strain Orn10, the L-ornithine yield in strain Orn18 increased by 11.32% (from 8.13 g / L to 9.05 g / L). The results show that enhancing the L-ornithine production in *E. coli*... thenI Gene expression intensity can significantly improve the L-ornithine production performance of engineered strains.

[0121] 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-ornithine, characterized in that: The genetically engineered bacteria are Escherichia coli overexpressed with... ynaI The genes were modified in a specific way; The genetically engineered bacteria do not express genes argF, argI, speC, and speF, and replace the ornithine deacetylase gene argE with the glutamate acetyltransferase gene argJ from Corynebacterium glutamicum and overexpress it, and also overexpress the argine synthesis operator genes argC, argJ, argB, and argD from Corynebacterium glutamicum. The ynaI 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.

2. The genetically engineered bacteria as described in claim 1, characterized in that: The genetically engineered bacteria also overexpress E. coli's own genes argO, pntA, and pntB; and / or use the promoter P with the nucleotide sequence shown in SEQ ID NO:

3. rpsL Control the expression of the gene sucA, which encodes the E1 subbody of α-ketoglutarate dehydrogenase.

3. The genetically engineered bacteria as described in claim 1, characterized in that: The overexpression ynaI Genetic pathways include: increasing gene size through autonomous replication plasmids. ynaI The copy number of a gene, and / or the increase through gene editing methods. ynaI Gene copy number, and / or modifications ynaI Gene expression regulatory sequences.

4. The use of the genetically engineered bacteria according to any one of claims 1-3 in the fermentation production of L-ornithine.

5. A method for producing L-ornithine, characterized in that: include: The genetically engineered bacteria according to any one of claims 1-3 are cultured in a culture medium to produce L-ornithine; And, the L-ornithine is collected from the genetically engineered bacteria and / or the culture medium.

6. The method as described in claim 5, characterized in that: The method further includes: maintaining a constant culture temperature of 37°C and / or maintaining the pH. 7.0, and / or maintain dissolved oxygen at 35%.

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