A method for modifying an l-lysine high-yield production strain and application thereof

Abstract

The application discloses a kind of L-lysine high-yield production strain modification method and its application, belong to the field of bioengineering technology.The application obtains recombinant bacteria by knocking out CbrB gene and / or CbrC gene on the genome of escherichia coli, and the yield and conversion rate of L-lysine can be improved by fermenting the recombinant bacteria, and the mortality in the fermentation process of strain can be reduced.The application has important application value.The application finds through experiment that the influence of the two genes on escherichia coli has synergistic effect, L-lysine hydrochloride has osmotic pressure stress effect on strain, and silencing related gene protein can change membrane lipid composition (increase the proportion of unsaturated fatty acid), maintain membrane fluidity.Improve the tolerance to lysine hydrochloride, reduce the mortality in later fermentation stage.

Description

Technical Field

[0001] This invention specifically relates to a method for modifying a high-yield L-lysine-producing strain and its application, belonging to the field of biotechnology. Background Technology

[0002] Lysine, chemically known as 2,6-diaminohexanoic acid, is a basic essential amino acid that the body cannot synthesize and must obtain from food. Lysine is mainly found in animal-based foods and legumes; cereal grains contain very little lysine, and it is easily destroyed during processing, thus it is considered the first limiting amino acid.

[0003] Lysine plays a vital role in promoting human growth and development, enhancing immunity, fighting viruses, promoting fat oxidation, and alleviating anxiety. It also promotes the absorption of certain nutrients and works synergistically with others to enhance their physiological functions. Lysine regulates metabolic balance, provides structural components for carnitine synthesis, and carnitine promotes fatty acid synthesis in cells. Adding small amounts of lysine to food can stimulate the secretion of pepsin and gastric acid, improving gastric juice secretion and thus increasing appetite and promoting growth and development in children. Lysine also enhances calcium absorption and accumulation, accelerating bone growth. A deficiency in lysine can lead to insufficient gastric juice secretion, resulting in anorexia, nutritional anemia, and even central nervous system dysfunction and developmental delays. Lysine is used in medicine as an adjunct to diuretics, to treat lead poisoning caused by decreased blood chloride levels, and to form salts with acidic drugs (such as salicylic acid) to reduce adverse reactions. Combined with methionine, it can inhibit severe hypertension. Studies have also shown that lysine supplementation can accelerate the recovery from herpes infections and inhibit their recurrence. Only the L-form of lysine is absorbed by the body; the CAS number for L-lysine is 56-87-1. Due to the wide application of L-lysine in various fields, its demand is gradually increasing each year. Therefore, various studies are underway to develop efficient microbial strains and fermentation technologies for L-lysine production. For example, overexpressing key genes involved in L-lysine synthesis in microbial strains to increase their activity; or deleting genes that do not need to be expressed (byproducts or toxins affecting cell growth, etc.) from microbial strains. However, with the increasing demand for L-lysine year by year, further research is needed to effectively increase L-lysine production capacity. Summary of the Invention

[0004] The main objective of this invention is to increase the yield of L-amino acids. However, the objectives of this invention are not limited to the subject matter described herein. Those skilled in the art will clearly understand other objectives not mentioned herein through the following description.

[0005] This invention first protects a method for modifying a strain to increase L-lysine production, which can be used for microorganisms that inhibit or downregulate the expression and / or activity of endometrial proteins in vivo. Using three high-L-lysine-producing *Escherichia coli* strains—lys-1 (CCTCC NO: M2019435), lys-2 (E. coli NRRLB-12185), and lys-3 (E. coli GDMCC NO. 1.318)—as starting strains, an empty vector fragment was introduced into the gene fragments encoding cbrB and cbrC using a gene modification system and plasmids purchased from Professor Yang Sheng. The *E. coli* NRRLB-12185 strain is a low-L-lysine-producing strain and can be purchased from the Agricultural Research Service Culture Collection (NRRL).

[0006] This invention provides a high-yielding L-lysine-producing *Escherichia coli* strain FMME-lys2.0. This *E. coli* strain is based on the chassis strain FMME-lys (*E. coli* CCTCC NO: M2019435). The strain is formed by inserting a 32bp target site from the first 200bp segment of the host strain's cbrB sequence into an empty vector sequence as shown in SEQ ID NO.3, thereby disrupting cbrB and reducing the expression of the inner membrane protein in *E. coli*, thus increasing the efficiency of L-lysine export; thus forming the FMME-lys1.0 strain.

[0007] Furthermore, the host strain of Escherichia coli is FMME-lys1.0. Based on this, a 32bp target site is inserted into the fragment before the beginning of cbrC in the first 200bp of the fragment, thereby disrupting cbrC and improving the tolerance of Escherichia coli to lysine hydrochloride, thus forming the FMME-lys2.0 strain.

[0008] The present invention also provides an engineered Escherichia coli strain, which is obtained by silencing the cbrB and / or cbrC proteins on the genome of Escherichia coli CCTCCNO:M2019435 as a chassis cell.

[0009] In one embodiment, the amino acid sequence of the cbrB protein is shown in SEQ ID NO.1; SEQ ID NO.1: MSVSRRVIHHGLYFAVLGPLIGVLFLVLYIFFAKEPLVLWVIIHPIFLLLSITTGAIPALLTGVMVACLPEKIGSQKRYRCLAGGIGGVVITEIYCAVIVHIKGMASSELFENILSGDSLVVRIIPALLAGVVMSRIITRLPGLDISCPETDSLS

[0010] The amino acid sequence of the cbrC protein is shown in SEQ ID NO.2. SEQ ID NO.2:

[0011] MTQNIRPLPQFKYHPKPLETGAFEQDKTVECDCCEQQTSVYYSGPFYCVDEVEHLCPWCIADGSAAEKF

[0012] AGSFQDDASIEGVEFEYDEEDEFAGIKNTYPDEMLKELVERTPGYHGWQQEFWLAHCGDFCVFIGYVGW

[0013] NDIKDRLDEFANLEEEDCENFGIRNSDLAKCLQKGGHCQGYLFRCLHCGKLRLWGDFS

[0014] The present invention also provides the application of cbrB protein and / or cbrC protein in improving the L-lysine stress resistance of Escherichia coli, wherein the application is to silence cbrB protein and / or cbrC protein on the Escherichia coli genome.

[0015] In one embodiment, the *E. coli* includes, but is not limited to, *E. coli* CCTCC NO: M2019435, *E. coli* NRRLB-12185, and *E. coli* GDMCC NO. 1.318. In one embodiment, the amino acid sequence of the cbrB protein is shown in SEQ ID NO. 1, and the amino acid sequence of the cbrC protein is shown in SEQ ID NO. 2. In one embodiment, the silencing method involves replacing the gene fragment in SEQ ID NO. 4 of the upstream sequence of the cbrB gene with the target gene fragment shown in SEQ ID NO. 3; and replacing the gene fragment in SEQ ID NO. 5 of the upstream sequence of the cbrC gene with the target gene fragment shown in SEQ ID NO. 3.

[0016] This invention also provides a method for increasing the biomass at the fermentation endpoint of a high-L-lysine-producing *Escherichia coli*, wherein the method involves silencing the cbrB and / or cbrC proteins on the *E. coli* genome, wherein the high-L-lysine-producing *E. coli* is *E. coli* CCTCC NO: M2019435; in one embodiment, the amino acid sequence of the cbrB protein is shown in SEQ ID NO.1, and the amino acid sequence of the cbrC protein is shown in SEQ ID NO.2. In one embodiment, the silencing method involves replacing the gene fragment of SEQ ID NO.4 in the upstream sequence of the cbrB gene with the target gene fragment shown in SEQ ID NO.3; and replacing the gene fragment of SEQ ID NO.5 in the upstream sequence of the cbrC gene with the target gene fragment shown in SEQ ID NO.3.

[0017] This invention provides a method for constructing the above-mentioned engineered Escherichia coli, wherein the method comprises: silencing the cbrB gene and / or cbrC gene on the genome using gene editing techniques.

[0018] In one implementation, the method for silencing the cbrB and cbrC genes can be any of the following:

[0019] (i) Knockout of the cbrB and cbrC genes using gene editing techniques; cbrB base fragment:

[0020] Ttcttcacactcccttcacttaccccgcttaaattggcgctcaaaggtaagtaaagggagtttgatatgtctgtttcacgtcgggtaatacatcacggactttattttgcagtt

[0021] ttaggaccgttaattggtgttctgtttcttgtcctctacatattcttcgcaaaagaaccgctggttct ttgggtgataatacatccaatttttctcttattgtcgataactacggga

[0022] gctattcctgcgttgttaaccggtgtaatggttgcgttatcgttgtctggctggtggcataggtggcg tcgttatcaccgagatctattgtgcagttattgtacatattaaggg

[0023] catggcttcctcggagttgtttgaaaacattctttctggtgacagtctcgttgtccgcatcattcctg cattgctggcaggtgtggtgatgagcagaatcattacccgtctac

[0024] ccggattggatatttcatgtcctgaaacagactctttaagttaa gcgggatactttatctttgggctactcaaaagcagacaggatgtttct

[0025] The underlined part represents the cbrB base sequence that needs to be knocked out.

[0026] cbrC base fragment:

[0027] gcgggatactttatctttgggctactcaaaagcagacaggatgtttct atgactcaaaatatcaggcc gttaccccaattcaaatatcatcccaagccactggaaacagg

[0028] cgcatttgaacaggataaaaccgtagagtgcgattgctgtgaacaacagacgtcagtttattactcgg gtcccttttattgcgttgatgaagttgaacatctctgtccgtgg

[0029] tgtattgcggacggttctgctgctgagcatagaaggtgttgaatttgagtatgatgaagaggacgaat ttgccggtattaagaacacatatcctgatgaaatgctgaaag

[0030] agttggttgaacgcacgccaggttatcatggatggcagcaggaattctggctcgcgcattgtggcgat ttctgtgtttttatcggctatgtgggctggaatgatataaaag

[0031] atcgcctcgatgaatttgccaaccttgaagaagattgtgagaatttcggtattagaaattctgatcta gctaaatgcctgcaaaagggtggtcattgtcagggttatctcttc

[0032] cgctgtctccactgcggcaagctgagactgtggggtgatttttcgtag ttatttaaataatgagaacaggccg

[0033] The underlined part represents the cbrC base sequence that needs to be knocked out.

[0034] (ii) Replace the gene fragment in SEQ ID NO.4 of the upstream sequence of the cbrB gene with the target gene fragment shown in SEQ ID NO.3;

[0035] Replace the gene fragment in SEQ ID NO.5 of the upstream sequence of the cbrC gene with the target gene fragment shown in SEQ ID NO.3.

[0036] In one embodiment, the method includes:

[0037] (i) Construction of pDONOR-empty vector plasmid: Using the genome of Escherichia coli as a template, the stop codon as the target fragment, and 20 bp from each end of the target gene fragment as homologous arms, PCR was performed using primers to construct the target gene fragment with the nucleotide sequence shown in SEQ ID NO.3; then, reverse PCR was performed on the pDONOR plasmid using primers to form the vector of the pDONOR plasmid; the two fragments were homologously recombinated using homologous recombination enzyme to form the pDONOR-empty vector plasmid.

[0038] (ii) Construction of pQCascade-cbrB and pQCascade-cbrC plasmids: 32bp target sequences were found 200bp upstream of the cbrB and cbrC fragments, as shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; the 32bp sequence shown in SEQ ID NO.10 of the original pQCascade plasmid was replaced with the target sequence using primers, and reverse PCR was performed to construct pQCascade-cbrB and pQCascade-cbrC plasmids, respectively.

[0039] (iii) Construction of engineered Escherichia coli strain with silenced cbrB gene: pDONOR-empty vector plasmid and pQCascade-cbrB were electroporated into Escherichia coli CCTCC NO:M2019435 chassis strain, respectively. After induction with rhamnose, the empty vector plasmid was successfully inserted 200bp upstream of the cbrB gene. After removing the plasmid, engineered Escherichia coli strain with silenced cbrB gene was obtained.

[0040] (iV) Construction of engineered E. coli strain with silenced cbrC gene: pDONOR-empty vector plasmid and pQCascade-cbrC were electroporated into E. coli CCTCC NO:M2019435 chassis strain, respectively. After induction with rhamnose, the empty vector plasmid was successfully inserted 200bp upstream of the cbrC gene. After removing the plasmid, engineered E. coli strain with silenced cbrB gene was obtained.

[0041] (Vi) Construction of engineered E. coli strains with silenced cbrB and cbrC genes:

[0042] The pDONOR-empty vector plasmid and pQCascade-cbrC were electroporated into the E. coli engineered bacteria with the cbrB gene silenced in step (iii). The empty vector plasmid was successfully inserted into the cbrC gene 200 bp upstream by rhamnose induction. After removing the plasmid, the cbrB and cbrC genes of the E. coli engineered bacteria were silenced.

[0043] The base sequence after silencing the cbrB gene is as follows:

[0044] atgtctgtttcacgtcgggtaatacatcacggactttattttgcagttttaggaccgttaattggtgttctgtttcttgtcctctacatattcttcgcaa aagaaccgctggttctttgggtgataatacatccaatttttctcttattgtcgataactacgggagctattcctgcgttgttaaccggtgtaatggttgc ttgggtgttgtttgaagtat aagttgacatatctgtactaaaagatggcataaattggaagtgtaaggtggcatagtctagtatttaaccaaatgg ttaaatggttgactcaccgggaattcgagataaattgcactgaaatctagaggtcgaaattcacctcgaaagcaag ctgataaaccgatacaattaaaggctccttttggagccttttttttggagattttcaacgtgaaaaaattattatt cgcaattccaagctaattcacctcgaaagcaagctgataaaccgatacaattaaaggctccttttggagccttttt tttggagattttcaacgtgaaaaaattattattcgcaattccaagctctgcctcgcgcgtttcggtgatgacggtg aaaacctctgacacatgcagctcccggagacggtcagcttgtctgtaagcggatgccgggagcagacaagcccgtc agggcgcgtcagcgggtttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatggg ctcgatcccctcggatcgatccccatgtaatgaataaaagcagtaattaatacatctgtttcatttgaagcgcgaa agctaaagttttcgcatttatcggatccggctgctaatcgagttaattaaactagtgagctcggtacccggggatc ctctagaggtcgaaattcacctcgaaagcaagctgataaaccgatacaattaaaggctccttttggagcctttttt tttggagattttcaacgtgaaaaaattattattcgcaattccaagctaattcacctcgaaagcaagctgataaacc gatacaattaaaggctccttttggagcctttttttttggagattttcaacgtgaaaaaattattattcgcaattcc aagctctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagctt gtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagc catgacccagtcacgtagcgatagcggagtgtatgggctcgatcccctcggatccaattggtcttaattttcctta attatttttaaagttagactgatttagacttggaaaagcttaatgattggagagctaaattgactaatagtattca gtcaagtttaattagttttaagcgatccatgcataactatttctgtacaatgctatattttcaccaattaataatt tttaatcaagcctacattatgaaatatactatacccatttgaactcttctatttgtaccattgtcggtagcaaaaa cttatggggttttgaacgtcacttaaattgtaagcatttgcgatggaggcgcgtttagagtcaaccttgattctga tatgctccgaatttttggtaagaatataagtgtgagagtagctaatgtggatacgcctgagttaagggaaaaatgt gaaaatgaaataactcgttatcatgcaaagtgactaaggttataatcttccgtttatggcacatagcagccaacta aacttgacagtatttttatgtggttggctttataaaaccagcatttggtaacattatgccaatttttacttcaata ttatgccaacatacactacactaacggagctgtagcacaataagctcgtttgtacttatgccaacttatacttcaa acaacattgg gttatcgttgtctggctggtggcataggtggcgtcgttatcaccgagatctattgtgcagttattgtacatattaagggcatggcttcctcggagttgtttgaaaacattctttctgg tgacagtctcgttgtccgcatcattcctgcattgctggcaggtgtggtgatgagcagaatcattacccgtctacccggattggatatttcatgtcctgaaacagactctttaagttaa

[0045] (Note: Underlined segments represent inserted empty vector fragments, while ununderlined segments represent cbrB gene fragments.)

[0046] The base sequence following silencing the cbrC gene is as follows:

[0047] atgactcaaaatatcaggccgttaccccaattcaaatatcatcccaagccactggaaacaggcgcatttgaacaggataaaaccgtagagtgcgattgctgtgaacaacagacgtcagtttattactcgggtcccttttattgcgttgatgaagttgaacatctctgtccgtggtgtattgcggacggttctgctgctga ttgggtgttgtttgaagtat aagttgacatatctgtactaaaagatggcataaattggaagtgtaaggtggcatagtctagtatttaaccaaatgg ttaaatggttgactcaccgggaattcgagataaattgcactgaaatctagaggtcgaaattcacctcgaaagcaag ctgataaaccgatacaattaaaggctccttttggagccttttttttggagattttcaacgtgaaaaaattattatt cgcaattccaagctaattcacctcgaaagcaagctgataaaccgatacaattaaaggctccttttggagccttttt tttggagattttcaacgtgaaaaaattattattcgcaattccaagctctgcctcgcgcgtttcggtgatgacggtg aaaacctctgacacatgcagctcccggagacggtcagcttgtctgtaagcggatgccgggagcagacaagcccgtc agggcgcgtcagcgggtttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatggg ctcgatcccctcggatcgatccccatgtaatgaataaaagcagtaattaatacatctgtttcatttgaagcgcgaa agctaaagttttcgcatttatcggatccggctgctaatcgagttaattaaactagtgagctcggtacccggggatc ctctagaggtcgaaattcacctcgaaagcaagctgataaaccgatacaattaaaggctccttttggagcctttttt tttggagattttcaacgtgaaaaaattattattcgcaattccaagctaattcacctcgaaagcaagctgataaacc gatacaattaaaggctccttttggagcctttttttttggagattttcaacgtgaaaaaattattattcgcaattcc aagctctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagctt gtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagc catgacccagtcacgtagcgatagcggagtgtatgggctcgatcccctcggatccaattggtcttaattttcctta attatttttaaagttagactgatttagacttggaaaagcttaatgattggagagctaaattgactaatagtattca gtcaagtttaattagttttaagcgatccatgcataactatttctgtacaatgctatattttcaccaattaataatt tttaatcaagcctacattatgaaatatactatacccatttgaactcttctatttgtaccattgtcggtagcaaaaa cttatggggttttgaacgtcacttaaattgtaagcatttgcgatggaggcgcgtttagagtcaaccttgattctga tatgctccgaatttttggtaagaatataagtgtgagagtagctaatgtggatacgcctgagttaagggaaaaatgt gaaaatgaaataactcgttatcatgcaaagtgactaaggttataatcttccgtttatggcacatagcagccaacta aacttgacagtatttttatgtggttggctttataaaaccagcatttggtaacattatgccaatttttacttcaata ttatgccaacatacactacactaacggagctgtagcacaataagctcgtttgtacttatgccaacttatacttcaa acaacattgg gcatagaaggtgttgaatttgagtatgatgaagaggacgaatttgccggtattaagaacacatatcctgatgaaatgctgaaagagttggttgaacgcacgccaggttatcatggatggcagcaggaattctggctcgcgcattgtggcgatttctgtgtttttatcggctatgtgggctggaatgatataaaagatcgcctcgatgaatttgccaaccttgaagaagattgtgagaatttcggtattagaaattctgatctagctaaatgcctgcaaaagggtggtcattgtcagggttatctcttccgctgtctccactgcggcaagctgagactgtggggtgatttttcgtag

[0048] (Note: The underlined part is the inserted empty fragment, and the non-underlined part is the gene fragment of cbrC)

[0049] The present invention also provides a method for producing L-lysine by fermentation, wherein the method utilizes engineered Escherichia coli strains with silenced cbrB and / or cbrC genes to produce L-lysine by fermentation.

[0050] In one embodiment, the fermentation temperature is 30-37°C, and the fermentation time is 30-42 seconds. In another embodiment, the method involves inoculating the engineered Escherichia coli seed culture into a fermentation medium containing glucose and then fermenting it. Further, the seed culture is added at a volume ratio of 15-20%.

[0051] In one embodiment, the seed culture is prepared by streaking an engineered strain of *Escherichia coli* onto a slant culture medium and culturing it at 33–37°C for 12–16 hours. The culture is then inoculated into a primary seed culture medium at a volume ratio of 5–15% and cultured at 33–37°C until the OD (Organic Demand) reaches zero. 600 The OD value was 4.0–5.0. A primary seed culture was prepared and inoculated into a secondary culture medium at a volume ratio of 5–15%, and cultured at 33–37°C until the OD value reached 4.0–5.0. 600 It ranges from 15.0 to 18.0.

[0052] The method for producing L-lysine using engineered *E. coli* strains with silenced cbrB and / or cbrC genes via fermentation includes: streaking the engineered *E. coli* strains with silenced cbrB and / or cbrC genes onto a slant culture medium and culturing at 33–37°C for 12–16 hours; inoculating at 5–15% onto a primary seed culture medium at 33–37°C with shaking for 5–8 hours; inoculating at mid-logarithmic growth (OD) of approximately 4–5; inoculating at 5–15% onto a secondary culture medium with shaking for 5–8 hours; inoculating at mid-logarithmic growth (OD) of approximately 15–18; and inoculating at 15–20% onto the fermentation medium, controlling the aeration rate at 3–7 vvm and the temperature at 33–37°C, continuing stirring at 600–800 rpm, and immediately stopping the fermentation once the glucose is exhausted.

[0053] The slant culture medium comprises: yeast extract (Oxoid) 5 g / L, tryptone (Oxoid) 10 g / L, sodium chloride 5 g / L, agar powder 20 g / L, and sodium pyruvate 0.5 g / L. It is sterilized at 121°C for 15 min.

[0054] The primary seed culture medium comprises: 3.0 g / L sucrose, 5 g / L yeast extract (Oxoid), 8 g / L tryptone (Oxoid), 4.5 g / L (NH4)2SO4, 4 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, 22 mg / L FeSO4·7H2O, 13 mg / L MnSO4·H2O, 5 g / L monosodium glutamate, 0.3 g / L L-threonine, 0.3 g / L L-methionine, 0.55 g / L sodium pyruvate, and 5 mg / L biotin.

[0055] The secondary seed culture medium comprises: glucose 50 g / L, (NH4)2SO4 14.4 g / L, KH2PO4 1.44 g / L, MgSO4·7H2O 1.2 g / L, corn steep liquor powder 5.92 g / L, hair hydrolysate 3.12 g / L, FeSO4·7H2O 24.9 mg / L, MnSO4·H2O 15.2 mg / L, ZnSO4·7H2O 102.4 mg / L, CuSO4·5H2O 80.6 mg / L, L-threonine 368 mg / L, L-methionine 368 mg / L, betaine hydrochloride 1.6 g / L, PABA 2 mg / L, VB1 1 mg / L, nicotinamide 1 mg / L, biotin 5 mg / L, and light calcium carbonate 1250 mesh 10 g / L.

[0056] The fermentation medium comprises: glucose 30 g / L, 85% H3PO4 0.2 mL / L, potassium chloride 0.5 g / L, beet molasses 18 mL / L, betaine hydrochloride 1.5 g / L, MgSO4·7H2O 3.29 g / L, FeSO4·7H2O 49.4 mg / L, MnSO4·H2O 35.8 mg / L, ZnSO4·7H2O 152.9 mg / L, CuSO4·5H2O 120 mg / L, L-threonine 300 mg / L, corn steep liquor powder 7.4 g / L, antifoaming agent 2 g / L, vitamin B1 60 mg / L, nicotinamide 10 mg / L, and biotin 0.6 mg / L.

[0057] A fourth objective of this invention is to provide a microbial preparation of engineered Escherichia coli containing the silenced cbrB gene and / or cbrC gene.

[0058] The present invention also provides the use of the microbial preparation of the engineered Escherichia coli containing the silenced cbrB gene and / or cbrC gene in food or pharmaceuticals.

[0059] Optionally, the application involves producing L-lysine using a microbial preparation of engineered Escherichia coli containing the silenced cbrB gene and / or cbrC gene.

[0060] The present invention also provides the above-described method for constructing engineered Escherichia coli or the use of the above-described engineered Escherichia coli in any of the following:

[0061] (a) Use in the preparation of L-lysine or in food, pharmaceuticals, chemicals and feed containing L-lysine;

[0062] (b) Application in improving the resistance of Escherichia coli to L-lysine stress;

[0063] (c) Application in increasing the biomass of high-L-lysine-producing Escherichia coli at the fermentation endpoint or reducing the mortality rate of high-L-lysine-producing Escherichia coli during fermentation.

[0064] (d) Application in regulating the production of L-lysine in Escherichia coli.

[0065] In one embodiment, the drug further contains pharmaceutically acceptable pharmaceutical excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0066] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0067] In one embodiment, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0068] In one embodiment, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0069] In one embodiment, the injectable dosage form includes, but is not limited to, an injection solution or an injection powder for injection;

[0070] In one embodiment, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0071] The present invention also provides the application of the cbrB protein with the above-mentioned amino acid sequence as shown in SEQ ID NO.1 and / or the cbrC protein with the amino acid sequence shown in SEQ ID NO.2 in improving the production of L-lysine by Escherichia coli, wherein the application is to silence the cbrB protein and / or cbrC protein on the genome of Escherichia coli.

[0072] In one embodiment, the Escherichia coli includes, but is not limited to, Escherichia coli CCTCC NO:M2019435, E. coli NRRLB-12185, and Escherichia coli GDMCC NO.1.318.

[0073] Beneficial effects

[0074] Escherichia coli is the most commonly used cell factory with a clear genetic background. Further increasing L-lysine production and the beneficial effects of producing strains in silencing cbrB and / or cbrC proteins mainly focus on the following two aspects:

[0075] First, by silencing cbrB and / or cbrC proteins using three different L-lysine-producing strains, with each strain performing triplicate experiments, the conversion rate and yield of L-lysine were significantly improved. The lys-1 strain showed an approximately 18.37% increase in lysine yield per shake flask, the lys-2 strain approximately 38.9%, and the lys-3 strain approximately 21.32%. Figure 1 As shown.

[0076] Furthermore, these two genes have a synergistic effect: L-lysine hydrochloride exerts osmotic stress on the strain, and silencing the related gene protein can alter membrane lipid composition (increasing the proportion of unsaturated fatty acids) and maintain membrane fluidity. This improves tolerance to lysine hydrochloride and reduces mortality in the later stages of fermentation. Attached Figure Description

[0077] Figure 1 Production comparison chart.

[0078] Figure 2 Mortality rate during fermentation.

[0079] Figure 3 Comparison of osmotic pressure of the final fermentation samples.

[0080] Figure 4 : Schematic diagram of CRISPR-related transposase-mediated site-directed transposition. Detailed Implementation

[0081] In the following examples, conventional experimental methods were used, and all materials were commercially available. The original plasmids of pQCascade (abbreviated PQ) and pDonor-GFP (abbreviated PD) and the Pcut plasmid involved in the following examples were purchased from Professor Yang Sheng; the purchase channels were: addgene website PDONNOR-GFP(175578)PQ-IS186(175581).

[0082] pDonor-GFP is a commercially available plasmid. The pQ plasmid carries the Kana resistance gene, and the PD plasmid carries the Cm resistance gene. The JM109 strain was used to carry the relevant plasmids.

[0083] Through extensive experimentation, the inventors of this invention modified the L-lysine-producing bacterium FMME-lys (Escherichia coli FMME-lys) to obtain the engineered bacterium FMME-lys1.0, whose genotype is FMME-lys-Δ. c brB. Using FMME-lys1.0 as the chassis strain, FMME-lys2.0 strain was iteratively developed, and the genotype of the engineered strain was FMME-lys-Δ. c brBΔ c brC.

[0084] The Escherichia coli FMME-lys is Escherichia coli CCTCC NO: M2019435, which is described in Chinese invention patent with publication number CN 110964670B.

[0085] The detection methods involved in the following embodiments are as follows:

[0086] Amino acid detection

[0087] The conventional ninhydrin colorimetric method was used for detection, as detailed in Liu Feifei's article "Study on conditions for quantitative detection of lysine by ninhydrin colorimetric method" published in the journal "Chinese Food Additives".

[0088] Glucose assay method:

[0089] Analysis was performed using the SBA-40 biosensor analyzer, as seen in Li Xianmin's article "Study on Influencing Factors of Glucose and L-Lactic Acid Determination by Biosensor Analyzer" published in the journal Asia-Pacific Traditional Medicine.

[0090] Calculation of glucose yield:

[0091] Glucose yield (%) = Maximum L-lysine yield (g / L) / Total glucose added.

[0092] The mortality rate of the strain was tested.

[0093] The instrument used for testing was a flow cytometer (model: Attune CytPix).

[0094] Sample processing:

[0095] Cell collection: Adherent cells: Digest with trypsin without EDTA (EDTA may induce apoptosis). Digestion time should not be too long, and gently pipette to avoid mechanical damage. Suspension cells: Collect the cell suspension directly, avoiding violent shaking or high-speed centrifugation (recommended centrifugation speed: 300-400g, 5 minutes).

[0096] Wash cells: Wash cells 1-2 times with pre-cooled PBS (containing 1% BSA or fetal bovine serum) to remove dead cell debris and serum interference from the culture medium. After centrifugation, discard the supernatant and gently resuspend the cells.

[0097] Staining (choose an appropriate dye): Nucleic acid dye method (suitable for distinguishing between dead and live cells): Propidium iodide (PI): final concentration 1-5 μg / mL, incubate in the dark for 15-30 minutes (4℃ or room temperature). 7-AAD: final concentration 5-20 μg / mL, incubate in the dark for 20 minutes (room temperature). DAPI: final concentration 0.1-1 μg / mL (requires permeabilization, used only for cell fixation). Membrane integrity detection methods (e.g., trypan blue, but flow cytometry is not commonly used and requires microscopy).

[0098] The principle of the bacterial mortality test: PI labels dead cells with membrane damage (late apoptosis / necrosis). Annexin V labels early apoptotic cells (membrane phospholipid eversion but intact membrane).

[0099] Mortality rate calculation:

[0100] Mortality rate (%) = (Number of late-stage apoptotic cells + Number of necrotic cells + Total number of cells) × 100% Mortality rate (%) = (Total number of cells + Number of late-stage apoptotic cells + Number of necrotic cells) × 100%

[0101] Note: Depending on the experimental objective, only PI-positive cells (necrosis) may be counted, or Annexin V may be used in conjunction to distinguish the apoptosis stage.

[0102] How to calculate conversion rate (%):

[0103] Definition: Conversion rate (%) = Mass of substrate consumed (g) / Mass of product (g) × 100%

[0104] The substrate involved in the fermentation process is glucose, and the product is L-lysine. Therefore, the formula for calculating the conversion rate is as follows:

[0105] Conversion rate calculation formula:

[0106]

[0107] The culture media involved in the following examples are as follows:

[0108] The components of slant culture medium include:

[0109] Yeast extract (Oxoid) 5 g / L, tryptone (Oxoid) 10 g / L, sodium chloride 5 g / L, agar powder 20 g / L, sodium pyruvate 0.5 g / L, sterilized at 121℃ for 15 min.

[0110] The primary seed culture medium consists of:

[0111] Sucrose 3.0 g / L, yeast extract (Oxoid) 5 g / L, tryptone (Oxoid) 8 g / L, (NH4)2SO4 4.5 g / L, K2HPO4 4 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 22 mg / L, MnSO4·H2O 13 mg / L, monosodium glutamate 5 g / L, L-threonine 0.3 g / L, L-methionine 0.3 g / L, sodium pyruvate 0.55 g / L, biotin 5 mg / L. Sterilize at 121℃ for 15 min.

[0112] The components of the secondary seed culture medium include:

[0113] Glucose 50 g / L, (NH4)2SO4 14.4 g / L, KH2PO4 1.44 g / L, MgSO4·7H2O 1.2 g / L, corn steep liquor powder 5.92 g / L, hair hydrolysate 3.12 g / L, FeSO4·7H2O 24.9 mg / L, MnSO4·H2O 15.2 mg / L, ZnSO4·7H2O 102.4 mg / L, CuSO4·5H2O 80.6 mg / L, L-threonine 368 mg / L, L-methionine 368 mg / L, betaine hydrochloride 1.6 g / L, PABA 2 mg / L, VB1 1 mg / L, nicotinamide 1 mg / L, biotin 5 mg / L, light calcium carbonate 1250 mesh 10 g / L. Adjust the pH to 7.6-7.7 with ammonia water, then sterilize at 121℃ for 15 minutes.

[0114] The fermentation medium consists of:

[0115] Ingredients: Glucose 30 g / L, 85% H3PO4 0.2 mL / L, Potassium chloride 0.5 g / L, Beet molasses 18 mL / L, Betaine hydrochloride 1.5 g / L, MgSO4·7H2O 3.29 g / L, FeSO4·7H2O 49.4 mg / L, MnSO4·H2O 35.8 mg / L, ZnSO4·7H2O 152.9 mg / L, CuSO4·5H2O 120 mg / L, L-Threonine 300 mg / L, Corn steep liquor powder 7.4 g / L, Antifoaming agent 2 g / L, Vitamin B1 60 mg / L, Nicotinamide 10 mg / L, Biotin 0.6 mg / L. Adjust the pH to 6.6-6.7 with ammonia.

[0116] The primers used in the following examples are shown in Table 1:

[0117] Table 1: Primer Table

[0118]

[0119]

[0120] Example 1: Construction of a strain that silences the cbrB gene in the genome of an L-lysine-producing strain

[0121] Three L-lysine-producing strains (lys-1: *E. coli* CCTCC NO: M2019435, lys-2: *E. coli* NRRLB-12185, lys-3: *E. coli* GDMCC NO. 1.318) were used as chassis strains and genetically modified. Using the MUCICAT gene editing method, the target sequence (terminator) nucleotide sequence (SEQ ID NO. 3) was used to replace the 32 bp upstream target sequence (ctgtctgccggaaaagatcgggtcacagaaac, SEQ ID NO. 4) of the cbrB gene in the L-lysine-producing strains. The specific steps are as follows:

[0122] 1. Construct expression vectors using the MUCICAT gene editing method

[0123] (1) Obtaining different segments:

[0124] Using Escherichia coli type strain MG1655 (NCBI number GCA_000005845.2), PCR amplification was performed using primers P1 and P2. The amplification product was recovered to obtain the target gene (terminator) fragment with the nucleotide sequence as shown in SEQ ID NO.3.

[0125]

[0126] Using the original PD-GFP plasmid as a template, reverse PCR amplification of the plasmid was performed using primers P3 and P4; the vector fragment for forming the pDONOR plasmid was: PD-vector.

[0127] Using the original PQ plasmid as a template, PCR amplification was performed using primers P5, P6, P7, and P8 to obtain the PQ-CbrB-1 fragment (SEQ ID No. 6) and the PQ-CbrB-2 fragment (SEQ ID No. 7).

[0128] The glue from the four segments mentioned above was recycled.

[0129] (2) Eliminate template plasmid:

[0130] The four fragments obtained in step (1) – the target gene fragment, the PD-vector, PQ-CbrB-1, and PQ-CbrB-2 – were digested using the Qcut-DPN1 enzyme to eliminate the template plasmid.

[0131] (3) Strains containing PD-empty vector plasmid and PQ-CbrB plasmid

[0132] Using ABclone's homologous recombination enzyme, the fragments obtained in step (2) that have eliminated the template plasmid, the target gene fragment and the PD-vector, PQ-CbrB-1 and PQ-CbrB-2, were homologously recombinated.

[0133] Because the PQ plasmid is large, it was split into two fragments for PCR. Both fragments, PQ-CbrB-1 and PQ-CbrB-2, were products of inverse PCR, each approximately 5000 bp in size. Then, the target gene fragment and the PD-vector, along with the PQ-CbrB-1 and PQ-CbrB-2 fragments, underwent homologous recombination to form the PD-empty vector plasmid and the PQ-CbrB plasmid.

[0134] Two plasmids were electroporated into competent JM109 cells to form strains JM109-PD and JM109-PQ.

[0135] (4) Culture of strains

[0136] After culturing the strains from step (3) for 1 hour, centrifuge at 5000 rpm for 2 minutes. Spread the JM109-PD strain onto resistant solid LB agar plates with a final concentration of 33 μg / mL Cm (chloramphenicol), while spread the JM109-PQ strain onto resistant solid LB agar plates with a final concentration of 50 μg / mL Kana. After culturing for 12 hours, single colonies grew.

[0137] (5) Sequencing verification

[0138] The single colony of the JM109-PD strain in (4) was subjected to colony PCR using PD-YZ-s and PD-YZ-x. After gel verification, the band size of the empty vector terminator sequence was 1784bp. Then the correct JM109-PD strain was sent for sequencing.

[0139] JM109-PQ cannot be verified by colony PCR and can only be verified by sequencing. The sequencing primer for JM109-PQ is PQ-YZ. After verifying the correct strain, the bacteria were preserved, cultured, and the correct plasmid was extracted. The plasmid was extracted using the Novizan plasmid extraction kit.

[0140] PD-empty vector plasmid and PQ-CbrB plasmid were prepared respectively.

[0141] (6) The two plasmids extracted in step (5) are simultaneously electroporated into the three competent strains lys-1, lys-2, and lys-3 to form strains lys-1-PD-PQ, lys-2-PD-PQ, and lys-3-PD-PQ.

[0142] The three strains lys-1-PD-PQ, lys-2-PD-PQ, and lys-3-PD-PQ were plated onto solid LB plates with dual resistance at a final concentration of 33 μg / mL Cm (chloramphenicol) and a final concentration of 50 μg / mL Kana, respectively, and then cultured.

[0143] The three strains lys-1-PD-PQ, lys-2-PD-PQ, and lys-3-PD-PQ are collectively referred to as the lys-PD-PQ series strains.

[0144] 2. Induced expression of plasmids in lys-PD-PQ series strains

[0145] Next, the two plasmids contained in the lys-PD-PQ series strains were used to induce the strains. The target gene (terminator) was inserted 32 bp upstream of the cbrB gene of the chassis strains lys-1, lys-2, and lys-3 (SEQ ID NO.4), respectively, forming lys1.1 (CCTCC NO: M2019435Δ). c brB), lys-2.1(NRRLB-12185Δ c brB), lys-3.1(GDMCC NO.1.318Δ c brB) strain.

[0146] The specific steps are as follows:

[0147] (1) The lys-PD-PQ series strains (lys-1-PD-PQ, lys-2-PD-PQ, lys-3-PD-PQ) obtained in step 1 were streaked on solid LB plates with three kinds of resistance to induce the strains. The resistances were 33 μg / mL Cm, 50 μg / mL Kana, and 1 mg / L dehydrotetracycline hydrochloride.

[0148] (2) Perform colony PCR on the single colonies grown in step (1). The verification primers used are PJY-YZ-S-1 and PJY-YZ-X-2 to verify that the target gene enters the target sequence.

[0149] If the gel electrophoresis shows a band size of 1784 bp, it proves that the empty target gene has been inserted into the target site. If the sequencing confirms that the lys-PD-PQ series strain is correct, the next step of modification will be carried out.

[0150] (3) The lys-PD-PQ series strains (lys-1-PD-PQ, lys-2-PD-PQ, lys-3-PD-PQ) were prepared as electrotransfer competent cells. After electrotransferring the Pcut plasmid (purchased by Professor Yang Sheng to eliminate PD and PQ plasmids), the cells were cultured for 3 h and then plated on a solution of 100 μg / mL Amp and 10 Mmol / L rhamnose to induce the lys-PD-PQ series strains. The cells were cultured for 24 h.

[0151] (4) The single colonies induced in (3) were streaked on 50 μg / mL Kana plates, 33 μg / mL Cm plates, and 100 μg / mL Amp plates for 24 h. If the strains did not grow on Kana and Cm plates but grew on Amp plates, it proved that the PQ and PD plasmids were successfully eliminated. The correct strains were then cultured into LB.

[0152] (5) The strain obtained in (4) was streaked onto a solid LB plate containing 10 g / L sucrose for secondary induction to eliminate the Pcut plasmid and cultured for 24 h.

[0153] (6) Incubate the single colonies from (5) on 100 μg / mL Amp plates and antibiotic-free LB solid medium for 24 h. If no growth occurs on the Amp plate but growth occurs on the antibiotic-free plate, it proves that the Pcut plasmid elimination is complete. Incubate the plasmid-eliminated strains in liquid LB medium for 24 h. This yields strains lys1.1, lys-2.1, and lys-3.1. The gene modification principle diagram is shown below. Figure 1 As shown.

[0154] We will use lys1.1(CCTCC NO:M2019435Δ) c brB), lys2.1(NRRLB-12185Δc brB), lys3.1(GDMCCNO.1.318Δ c brB), collectively referred to as lys generation 1 bacteria.

[0155] Example 2: Iterative construction of second-generation lys strains from the first generation of lys strains

[0156] Using lys1.1(CCTCC NO:M2019435Δ) c brB), lys2.1(NRRLB-12185Δ c brB), lys3.1(GDMCCNO.1.318Δ c Using brB as the chassis strain, genetic modification was performed. The MUCICAT gene editing method was employed to replace the 32bp upstream target sequence (aaagtttgcaggtagttttcaggatgatgcca, SEQ ID NO.5) of the cbrC gene in the L-lysine-producing strain with the target gene (terminator) in SEQ ID NO.3. The specific steps are as follows:

[0157] 1. Strains obtained by silencing the CbrC gene in the genome of the first generation of *Lys* bacteria.

[0158] (1) Obtaining different segments:

[0159] Using the genome of Escherichia coli strain MG1655 as a template, PCR amplification was performed using primers P1 and P2. The amplification products were recovered, and the target gene fragment with the nucleotide sequence shown in SEQ ID NO.3 was obtained. Using the original PD-GFP plasmid as a template, inverse PCR amplification of the plasmid was performed using primers P3 and P4; the vector fragment for forming the pDONOR plasmid was: PD-vector.

[0160] Using the original PQ plasmid as a template, PCR amplification was performed using primers P9, P10, P11, and P12 to obtain the PQ-CbrC-1 fragment (SEQ ID No. 8) and the PQ-CbrC-2 fragment (SEQ ID No. 9).

[0161] The glue from the four segments mentioned above was recycled.

[0162] (2) Eliminate template plasmid:

[0163] The four fragments obtained in step (1) – the target gene fragment, the PD-vector, the PQ-CbrC-1 fragment, and the PQ-CbrC-2 fragment – ​​were digested using the Qcut-DPN1 enzyme to eliminate the template plasmid.

[0164] (3) Strains containing PD-empty vector plasmid and PQ-CbrC plasmid

[0165] Using ABclone's homologous recombination enzyme, the fragments obtained in step (2) that eliminated the template plasmid—the target gene fragment and the PD-vector, and the two sets of fragments, PQ-CbrC-1 and PQ-CbrC-2—were subjected to homologous recombination. Since the PQ plasmid was large, it was divided into two parts for PCR. Both fragments, PQ-CbrC-1 and PQ-CbrC-2, were products of reverse PCR, and each fragment was approximately 5000 bp in size. Then, the target gene fragment and the PD-vector, and the two sets of fragments, PQ-CbrC-1 and PQ-CbrC-2, were subjected to homologous recombination to form the PD-empty vector plasmid and the PQ-CbrC plasmid.

[0166] Two plasmids were electroporated into competent JM109 cells, resulting in strains JM109-PD and JM109-PQ.

[0167] (4) Culture of strains

[0168] After culturing the strains from step (3) for 1 hour, centrifuge at 5000 rpm for 2 minutes. Spread the JM109-PD strain onto resistant solid LB agar plates with a final concentration of 33 μg / mL Cm (chloramphenicol), while spread the JM109-PQ strain onto resistant solid LB agar plates with a final concentration of 50 μg / mL Kana. After culturing for 12 hours, single colonies grew.

[0169] (5) Sequencing verification

[0170] The single colony of the JM109-PD strain in (4) was subjected to colony PCR using PD-YZ-s and PD-YZ-x. The colony was verified by running a gel. If the band size was 1784bp, the correct JM109-PD strain was sent for sequencing.

[0171] JM109-PQ cannot be verified by colony PCR and can only be verified by sequencing. The sequencing primer for JM109-PQ is PQ-YZ. After verifying that the strain is correct, it is preserved, cultured, and the correct plasmid is extracted. The plasmid is extracted using the Novizan plasmid extraction kit.

[0172] PD-empty vector plasmid and PQ-CbrC plasmid were prepared respectively.

[0173] (6) Based on the first generation of lys strains, the coliform E2 tolerance protein CbrC was knocked out to complete the iterative construction of the second generation of lys strains: the two plasmids extracted in step (5) were simultaneously electroporated into the competent strains of the first generation of lys strains to form strain lys-x.1-PD-PQ. The recipient strain of electroporation changed from the lys chassis strain to the first generation of lys strains, and an iterative operation was performed.

[0174] Specifically, the two plasmids extracted in step (5) were simultaneously electroporated into the first generation of lys bacteria (lys1.1 (CCTCCNO:M2019435Δ). c brB), lys2.1(NRRLB-12185Δ c brB), lys3.1(GDMCC NO.1.318Δ c Among the competent strains of brB), the lys-x.1-PD-PQ series of strains were formed.

[0175] The lys-x.1-PD-PQ series strains were plated on dual-resistance solid LB agar plates with a final concentration of 33 μg / mL Cm (chloramphenicol) and a final concentration of 50 μg / mL Kana. Single colonies were grown, and the best-growing strains were cultured.

[0176] (7) Following the method in Example 1, PJY-YZ-S-3 and PJY-YZ-X-4 were used to verify the process of induction and plasmid elimination in Example 2 when the empty vector fragment entered the cbrC gene. After eliminating the plasmid, strains were obtained that silenced the CbrB and CbrC genes on the genome of the first generation of lys strains and named them as the second generation of lys strains.

[0177] Lys-1.2 (CCTCC NO: M2019435Δ) was prepared separately. c brBΔ c brC), lys-2.2(NRRLB-12185Δ c brBΔ c brC), lys-3.2(GDMCC NO.1.318Δ c brBΔ c brC)

[0178] 2. The specific implementation method is the same as in Example 1 and step 1, except that lys-1: Escherichia coli CCTCC NO: M2019435, lys-2: E. coli NRRLB-12185, and lys-3: Escherichia coli GDMCC NO.1.318 are used as chassis strains for gene modification. The MUCICAT gene editing method is used to replace the 32bp upstream target sequence (SEQ ID NO.5) of the cbrC gene of the L-lysine producing strain with the target gene (terminator) of SEQ ID NO.3.

[0179] lys-1.1-1 (CCTCC NO: M2019435Δ) was prepared separately. c brC), lys-2.2-1(NRRLB-12185Δ c brC), lys-3.2-1(GDMCC NO.1.318Δ c brC).

[0180] Example 3: Comparative fermentation experiment of three chassis strains (original strains) and LYS Generation I strains (cbrB knockout strains), LYS Generation I BC strains (cbrBC knockout strains), and LYS Generation II strains (cbrB and cbrBC knockout strains).

[0181] 1. In addition to the three generations of strains mentioned above, we also supplemented the series of genes with single-gene silencing of the cbrC gene in the three chassis strains lys-1, lys-2, and lys-3. We named them lys-1.1-1 (CCTCC NO: M2019435ΔcbrC), lys-2.2-1 (NRRLB-12185ΔcbrC), and lys-3.2-1 (GDMCC NO.1.318ΔcbrC), collectively referred to as lys first-generation strain-1. Shake-flask experiments were conducted on the three chassis strains, the lys first-generation series, lys first-generation strain-1, and the lys second-generation series to obtain fermentation broths. The L-lysine content in the fermentation broth was then tested.

[0182] The specific steps are as follows:

[0183] (1) Preparation of seed liquid

[0184] First, 100 μL of the bacterial strain in the glycerol tube was inoculated into 30 mL of LB medium and incubated at 37°C for 24 h. Then, it was streaked onto a slant tube and incubated at 37°C for 16-24 h. Next, the slant was washed with 10 mL of sterile water to obtain the slant wash solution.

[0185] Take 2.5 mL (2.5% inoculum) of the slant bacterial solution and inoculate it into 100 mL of primary seed culture medium. Incubate at 37°C and 180 rpm for 7-8 hours until the primary seed culture reaches OD. 600 If the OD value is 4-5, inoculate 5 mL of bacterial suspension (inoculation volume, 10% by volume) into 50 mL of secondary culture medium, incubate at 37°C and 180 rpm for 7-8 hours, and wait for the OD value to be determined. 600 Once the seed culture reaches 16-17 cm, inoculate with fermentation medium at a rate of 20% (v / v). The formulations for each seed culture medium are as described above.

[0186] (2) Fermentation preparation of lysine

[0187] 1) Composition of the fermentation medium used during fermentation (g / L):

[0188] Glucose 30g / L, 85% H3PO4 0.2mL, potassium chloride 0.5g / L, beet molasses 18mL, betaine hydrochloride 1.5g / L, MgSO4·7H2O 3.29g / L, FeSO4·7H2O 49.4mg, MnSO4·H2O 35.8mg, ZnSO4·7H2O 152.9mg, CuSO4·5H2O 120mg, L-threonine 300mg, corn steep liquor powder 7.4g / L, vitamin B1 60mg, nicotinamide 10mg, biotin 0.6mg;

[0189] 2) The control process for the shake flask is as follows: Inoculum size: 20% (v / v); Culture temperature: 37℃; Shaker speed: 180 rpm; pH control: No pH adjustment is needed for 0-10 hours, after 10 hours, the pH should be controlled between 6 and 7; Shaker manufacturer configuration: HYL-C 3 Combined shaking table; fermentation cycle: 30-42 hours.

[0190] 2. After fermentation, the L-lysine content in the fermentation broth was determined using the ninhydrin colorimetric method. The detection results for the fermentation of the three chassis strains and the first and second generation lysine strains are shown in Table 2.

[0191] Table 2: OD, pH before fermentation and experimental results after fermentation

[0192]

[0193] The results showed that compared with L-lysine-producing chassis strains lys-1, lys-2, and lys-3, strains lys-1.1, lys-2.1, and lys-3.1 significantly increased lysine production. Furthermore, lys-1.2, lys-2.2, and lys-3.2 significantly increased L-lysine production compared to the other three strains. In other words, knocking out CbrB and CbrC from the *E. coli* chassis strains can improve L-lysine production and conversion rate.

[0194] Example 4: Effects of silencing other genes on the strain

[0195] The specific implementation method is the same as in Example 1, except that the chassis strain is adjusted to lys-1 (Escherichia coli CCTCC NO: M2019435). Following the method in Example 1, the MUCICAT gene editing method is used to replace the target sequence (terminator) upstream of the sthA gene, AmtB gene, pck gene, or sucCD gene of the L-lysine-producing strain with the target gene (terminator) whose nucleotide sequence is shown in SEQ ID NO.3 (Table 3).

[0196] The process of obtaining strains with different genes on the genome of the silencing lys-1 strain is the same as in step 1, except that the genes to be silenced are sthA, AmtB, pck, and sucCD. That is, the 32bp sequence shown in SEQ ID NO.10 of the original pQCascade plasmid is replaced with the 32bp target sequences of sthA, AmtB, pck, and sucCD, respectively, to knock out these genes. The target gene (terminator) shown in SEQ ID NO.3 is used to replace the 32bp target sequence upstream of the sthA, AmtB, pck, or sucCD gene of the L-lysine producing strain. The specific target sequences are shown in Table 3.

[0197] New strains lys-1-ΔsthA, lys-1-ΔAmtB, lys-1-Δpck, and lys-1-ΔsucCD were formed.

[0198] Table 3: Target sequences involved

[0199] Gene name 32bp target sequence sthA tcatccaacagatgttgatttcacccatccac AmtB ttctcagctgtgttgattttcgtggtggtatg pck aaaagataagtatatcgtccgtgacgatacca sucCD tgaagaagtggttattgcgcgttgccccctgg

[0200] Fermentation was carried out according to the method in Example 3, and the test results are shown in Table 4.

[0201] Table 4: Results of the strain in the shake flask

[0202] strain number genotype <![CDATA[OD 562 ]]> Lysine production (g / L) Sugar consumption (g) Conversion rate % Lys-1 Original strain 16.8 22.53 2.219 50.78 Lys-1-ΔsthA (gene ID: 948461) ΔsthA 18.9 21.3 2.175 49.0 lys-1-ΔsucCD (ID: 945312, 945314) △sucC / D 11.3 2.4 1.05 11.4 lys-1-Δpck(ID: 945667) Δpck 6.9 2.8 0.85 16.4 lys-1-ΔAmtB(ID: 945084) ΔAmtB 17.9 20 2.15 46.5

[0203] Although the yield of the strain did not increase significantly, this modification method is universal and applicable to the silencing of various genes.

[0204] Example 5: Comparison of mortality rates of three chassis strains and LYS first-generation and second-generation strains.

[0205] Next, the mortality rates of the three chassis strains and the first and second generation of lys strains were tested using a flow cytometer (model: Attune CytPix). The specific steps are as follows:

[0206] 1. First, inoculate 100 μL of the bacterial strain in the glycerol tube into 30 mL of LB medium and incubate at 37°C for 24 h. Then, streak it onto a slant tube and incubate at 37°C for 16-24 h. Next, wash the slant with 10 mL of sterile water to obtain the slant wash solution.

[0207] Take 2.5 mL (2.5% inoculum) of the slant bacterial solution and inoculate it into 100 mL of primary seed culture medium. Incubate at 37°C and 180 rpm for 7-8 hours until the primary seed culture reaches OD. 600 If the OD value is 4-5, inoculate 5 mL of bacterial suspension (inoculation volume, 10% by volume) into 50 mL of secondary culture medium, incubate at 37°C and 180 rpm for 7-8 hours, and wait for the OD value to be determined. 600 When the cells reach 16-17, they are inoculated with fermentation medium (step (2) of Example 4), with an inoculation amount of 20% (v / v), a culture temperature of 37°C, a shaking speed of 180 rpm, and fermentation is carried out. Samples are taken every 4 hours to detect the mortality rate.

[0208] 2. Mortality rate detection

[0209] Filter the sample using a 40 μm cell sieve to prevent cell clumps from clogging the flow cytometer nozzles. Adjust the cell suspension concentration to 1 × 10⁻⁶. 6 ~1×10 7 cells / mL (too high a concentration can cause clogging, too low a concentration will result in unstable data). Perform immediate analysis (if short-term storage is required, store at 4°C in the dark and complete the analysis within 1 hour). The results are shown in Table 5 below:

[0210] Table 5: Mortality Rate (%)

[0211]

[0212] Therefore, we can see that after knocking out the CbrB and CbrC genes, the cell mortality rate, especially in the later stages, decreased progressively, which has a significant effect on extending fermentation time and increasing yield.

[0213] 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 modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An engineered strain of *Escherichia coli*, characterized in that, The engineered Escherichia coli strain is an L-lysine-producing strain, obtained by silencing the cbrB and cbrC genes on the genome of Escherichia coli using E. coli as the chassis cell.

2. The engineered Escherichia coli strain according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the cbrB gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the cbrC gene is shown in SEQ ID NO.

2.

3. The engineered Escherichia coli strain according to claim 2, characterized in that, The silencing method involves replacing the target gene fragment shown in SEQ ID NO.3 with... cbrB The gene fragment in SEQ ID NO.4 of the upstream sequence of the gene; replace it with the target gene fragment shown in SEQ ID NO.

3. cbrC The gene fragment of SEQ ID NO.5 in the upstream sequence of the gene.

4. A method for improving the L-lysine stress resistance of Escherichia coli, characterized in that, The method involves silencing the cbrB and cbrC genes on the Escherichia coli genome.

5. The method according to claim 4, characterized in that, The amino acid sequence of the protein encoded by the cbrB gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the cbrC gene is shown in SEQ ID NO.

2.

6. The method according to claim 5, characterized in that, The silencing method involves replacing the target gene fragment shown in SEQ ID NO.3 with... cbrB The gene fragment in SEQ ID NO.4 of the upstream sequence of the gene; replace it with the target gene fragment shown in SEQ ID NO.

3. cbrC The gene fragment of SEQ ID NO.5 in the upstream sequence of the gene.

7. A method for increasing the biomass at the fermentation endpoint of an L-lysine-producing strain, characterized in that, The method involves silencing the cbrB and cbrC genes on the Escherichia coli genome.

8. The method according to claim 7, characterized in that, The amino acid sequence of the cbrB protein is shown in SEQ ID NO.1, and the amino acid sequence of the cbrC protein is shown in SEQ ID NO.

2.

9. The method according to claim 8, characterized in that, The silencing method involves replacing the target gene fragment shown in SEQ ID NO.3 with... cbrB The gene fragment in SEQ ID NO.4 of the upstream sequence of the gene; replace it with the target gene fragment shown in SEQ ID NO.

3. cbrC The gene fragment of SEQ ID NO.5 in the upstream sequence of the gene.

10. A method for preparing L-lysine, characterized in that, The method involves fermentation using the engineered Escherichia coli strain described in any one of claims 1 to 3.

11. The preparation method according to claim 10, characterized in that, The fermentation temperature is 30~37 ℃, and the fermentation time is 30~42 h.

12. The preparation method according to claim 11, characterized in that, The method involves inoculating the engineered Escherichia coli seed culture into a fermentation medium containing glucose and then fermenting it to obtain the product.

13. The preparation method according to claim 12, characterized in that, The seed solution was added at a volume ratio of 15-20%.

14. The preparation method according to claim 13, characterized in that, The seed culture is prepared by streaking the engineered Escherichia coli strain onto an agar slant culture medium and culturing it at 33-37°C for 12-16 h. Then, it is inoculated into a primary seed culture medium at a volume ratio of 5-15% and cultured at 33-37°C until the OD reaches [value missing]. 600 The value is 4.0~5.0; The primary seed culture was prepared and inoculated into the secondary culture medium at a volume ratio of 5-15%, and cultured at 33-37℃ until OD reached. 600 The value is between 15.0 and 18.

0.

15. The use of the engineered Escherichia coli according to any one of claims 1 to 3 in any of the following: (a) Its use in the preparation of L-lysine or in foods and pharmaceuticals containing L-lysine; (b) Application in improving the resistance of Escherichia coli to L-lysine stress; (c) Application in increasing the biomass of Escherichia coli producing L-lysine at the fermentation endpoint or reducing the lethality of Escherichia coli producing L-lysine during fermentation.

16. The application according to claim 15, characterized in that, The medicine also contains pharmaceutically acceptable excipients.

17. The application according to claim 16, characterized in that, The excipients include one or more of the following: adhesives, diluents, wetting agents, disintegrants, absorption promoters, surfactants, colorants, lubricants, and coating materials.

18. The application according to claim 17, characterized in that, The binder is: cellulose derivative, alginate, gelatin, and polyvinylpyrrolidone; the diluent is: pregelatinized starch, dextrin, sucrose, lactose, and mannitol; the humectant is: glycerol; the disintegrant is: sodium carboxymethyl starch, croscarmellose, and dry starch; the absorption promoter is: quaternary ammonium compound; the surfactant is: polysorbate, fatty acid sorbitan, and fatty acid glycerides; the colorant is: titanium dioxide, sunset yellow, methylene blue, and pharmaceutical grade iron oxide red; the lubricant is: hydrogenated vegetable oil, talc, and polyethylene glycol; and the coating material is: acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate.

19. The application according to claim 18, characterized in that, The dosage forms of the drug are oral, injectable, and inhaled.

20. The application according to claim 19, characterized in that, The oral dosage forms are tablets, capsules, granules, and oral liquids.

21. The application according to claim 19, characterized in that, The injectable dosage forms are injection solutions and injection powders for injection.

22. The application according to claim 19, characterized in that, The inhalation formulation is an aerosol.

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