CN118147035A - Recombinant microorganism and application thereof in preparation of L-lysine - Google Patents
Recombinant microorganism and application thereof in preparation of L-lysine Download PDFInfo
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- CN118147035A CN118147035A CN202410470432.2A CN202410470432A CN118147035A CN 118147035 A CN118147035 A CN 118147035A CN 202410470432 A CN202410470432 A CN 202410470432A CN 118147035 A CN118147035 A CN 118147035A
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a recombinant microorganism and application thereof in preparation of L-lysine. The present invention provides recombinant microorganisms: introducing the Ncgl1531 gene or a biological material related to the Ncgl1531 gene into a host microorganism to obtain a recombinant microorganism; introducing the mutant gene or a biological material related to the mutant gene into a host microorganism to obtain a recombinant microorganism; and (3) replacing the Ncgl1531 gene in the host microorganism genome with a mutant gene to obtain the recombinant microorganism. The invention also provides application of the Ncgl1531 protein or the mutant protein in the positive regulation of L-lysine production by microorganisms. The inventors found that the mutation of Ncg and 11531 gene is advantageous for increasing the yield of L-lysine, and that the increase of the expression activity (point mutation or overexpression) of Ncg and 11531 gene in microorganisms is advantageous for accumulation of L-lysine.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a recombinant microorganism and application thereof in preparation of L-lysine.
Background
L-lysine (CAS No. 56-87-1) is widely used in various fields, and its global demand is increasing every year, so that various researches are being conducted to develop efficient microbial strains and fermentation process technologies for producing L-lysine. For example, a key gene involved in L-lysine synthesis is overexpressed in a microbial strain, so that its activity is increased; or deleting genes (by-products or toxins affecting cell growth, etc.) that do not need to be expressed in the microbial strain. However, as the demand for L-lysine increases year by year, research is still required to effectively increase L-lysine productivity.
Lysine (Lysine), chemical name 2, 6-diaminocaproic acid. Lysine is an essential basic amino acid, which the body cannot synthesize itself and must be supplemented from food. Lysine is mainly present in animal foods and beans, and the cereal foods have a very low lysine content and are easily damaged during processing and are thus lacking, and thus are called first limiting amino acids.
Lysine has positive nutritional significance in the aspects of promoting the growth and development of human bodies, enhancing the immunity of organisms, resisting viruses, promoting fat oxidation, relieving anxiety and emotion and the like, can promote the absorption of certain nutrients, can cooperate with certain nutrients, and can better exert the physiological functions of various nutrients. Lysine can regulate the metabolic balance of the human body, lysine provides a structural component for the synthesis of carnitine, which promotes the synthesis of fatty acids in cells. The food is added with a small amount of lysine, can stimulate the secretion of pepsin and gastric acid, improves the gastric secretion efficacy, and plays roles of stimulating appetite and promoting the growth and development of infants. Lysine also increases calcium absorption and accumulation in the body, and accelerates bone growth. If lysine is lacking, anorexia and nutritional anemia can occur due to insufficient gastric secretion, which results in central nerve obstruction and dysplasia. Lysine can be used as an auxiliary drug of diuretics in medicine to treat lead poisoning caused by chloride reduction in blood, and can also be used for generating salt with acidic drugs (such as salicylic acid and the like) to relieve adverse reactions, and can be used for inhibiting severe hypertension when being used with methionine.
Disclosure of Invention
The object of the present invention is to provide recombinant microorganisms and their use for the preparation of L-lysine.
The present invention provides a recombinant microorganism which is (a 1) or (a 2) or (a 3) as follows:
(a1) Introducing the Ncgl1531 gene or a biological material related to the Ncgl1531 gene into a host microorganism to obtain a recombinant microorganism;
(a2) Introducing the mutant gene or a biological material related to the mutant gene into a host microorganism to obtain a recombinant microorganism;
(a3) And (3) replacing the Ncgl1531 gene in the host microorganism genome with a mutant gene to obtain the recombinant microorganism.
The invention also provides application of the recombinant microorganism in preparation of L-lysine.
The invention also provides application of the Ncgl1531 protein or the mutant protein in the positive regulation of L-lysine production by microorganisms.
The invention also provides application of the Ncgl1531 gene or the mutant gene in the positive regulation of L-lysine production by microorganisms.
The invention also provides an application of the Ncgl1531 gene or the Ncgl1531 gene related biological material or the mutant gene related biological material in preparing products for improving the L-lysine production capacity of microorganisms.
Specifically, the Ncgl1531 protein comprises an amino acid sequence shown in SEQ ID NO. 2.
Illustratively, the Ncgl1531 protein is shown in SEQ ID NO. 2.
Illustratively, the Ncgl1531 protein consists of the protein shown in SEQ ID NO. 2 and a tag protein.
The Ncgl1531 gene is a gene encoding an Ncgl1531 protein.
Specifically, the Ncgl1531 gene comprises a nucleotide sequence as shown in SEQ ID NO. 1.
Illustratively, the Ncgl1531 gene is set forth in SEQ ID NO. 1.
Illustratively, the Ncgl1531 gene consists of the segment shown in SEQ ID NO. 1 and a segment encoding a tag protein.
The Ncgl1531 gene related biomaterial is an expression cassette or a recombinant vector with the Ncgl1531 gene.
Illustratively, the recombinant vector having the Ncgl1531 gene is a recombinant expression vector obtained by inserting the Ncgl1531 gene into an expression vector.
Illustratively, the expression vector is a pK18mobsacB plasmid or pXMJ plasmid.
Illustratively, the recombinant expression vector may be recombinant plasmid pK18-Ncgl1531OE or recombinant plasmid pXMJ-Ncgl 1531.
The recombinant plasmid pK18-Ncgl1531OE differs from the pK18mobsacB plasmid only in that "AGGATCCCC" in the pK18mobsacB plasmid has been replaced by a specific DNA molecule consisting of, in sequence from upstream to downstream, the double-stranded DNA molecule shown in SEQ ID NO:7, the double-stranded DNA molecule shown in SEQ ID NO: 542-1581 in SEQ ID NO:5, and the double-stranded DNA molecule shown in SEQ ID NO: 8.
The recombinant plasmid pXMJ-Ncgl 1531 differs from the pXMJ plasmid only in that the double stranded DNA molecule shown at positions 542-1581 in SEQ ID NO. 5 is substituted for "TCTAGAGGATCCCCG" in the pXMJ plasmid.
The mutant protein is obtained by mutating one or more amino acid residues of the Ncgl1531 protein.
Specifically, the mutant protein is obtained by mutating the 35 th amino acid residue of the amino acid sequence shown in SEQ ID NO. 2 in the Ncgl1531 protein from glutamic acid to other amino acids.
Specifically, the mutant protein is obtained by mutating the 35 th amino acid residue of the amino acid sequence shown in SEQ ID NO. 2 in the Ncgl1531 protein from glutamic acid (E) to lysine (K).
Specifically, the mutant protein comprises an amino acid sequence shown as SEQ ID NO. 4.
Illustratively, the mutant protein is shown in SEQ ID NO. 4.
Illustratively, the mutant protein consists of the protein shown in SEQ ID NO. 4 and a tag protein.
The mutant gene is a gene encoding a mutant protein.
Specifically, the mutant gene is obtained by mutating one or more nucleotides of the Ncgl1531 gene.
Specifically, the mutant gene is obtained by mutating one or more codons of the Ncgl1531 gene.
Specifically, the mutant gene is obtained by mutating the codon of the 35 th amino acid residue of the amino acid sequence shown in SEQ ID NO.2 in the Ncgl1531 gene from the codon of the glutamic acid to the codon of other amino acids.
Specifically, the mutant gene is obtained by mutating the codon of the 35 th amino acid residue of the amino acid sequence shown in SEQ ID NO. 2 in the Ncgl1531 gene from the codon of the glutamic acid to the codon of the lysine.
Specifically, the mutant gene comprises a nucleotide sequence shown as SEQ ID NO. 3.
Illustratively, the mutant gene is shown in SEQ ID NO. 3.
Illustratively, the mutant gene consists of the segment shown in SEQ ID NO. 3 and a segment encoding a tag protein.
The mutant gene related biological material is an expression cassette or a recombinant vector with the mutant gene.
Illustratively, the recombinant vector having the mutant gene is a recombinant expression vector obtained by inserting the mutant gene into an expression vector.
Illustratively, the expression vector is a pK18mobsacB plasmid or pXMJ plasmid.
Illustratively, the recombinant expression vector may be recombinant plasmid pK18-Ncgl1531 E35K, recombinant plasmid pK18-Ncgl1531 E35K OE, or recombinant plasmid pXMJ19-Ncgl1531 E35K.
The recombinant plasmid pK18-Ncgl1531 E35K differs from the pK18mobsacB plasmid only in that the double stranded DNA molecule shown in SEQ ID NO. 6 is substituted for "AGGATCCCC" in the pK18mobsacB plasmid.
The recombinant plasmid pK18-Ncgl1531 E35K OE differs from the pK18mobsacB plasmid only in that "AGGATCCCC" in the pK18mobsacB plasmid is replaced by a specific DNA molecule consisting of the double-stranded DNA molecule shown in SEQ ID NO. 7, the double-stranded DNA molecule shown in SEQ ID NO. 9, and the double-stranded DNA molecule shown in SEQ ID NO.8 in this order from upstream to downstream.
The recombinant plasmid pXMJ-Ncgl 1531 E35K differs from the pXMJ plasmid only in that the double stranded DNA molecule shown in SEQ ID NO. 9 is substituted for "TCTAGAGGATCCCCG" in the pXMJ plasmid.
The recombinant microorganism may be exemplified by recombinant bacteria Y-Ncgl1531-1, recombinant bacteria Y-Ncgl1531-2, recombinant bacteria Y-Ncgl1531-3, recombinant bacteria Y-Ncgl1531-4, recombinant bacteria Y-Ncgl1531-5, recombinant bacteria L-Ncgl1531-1, recombinant bacteria L-Ncgl1531-2, recombinant bacteria L-Ncgl1531-3, recombinant bacteria L-Ncgl1531-4 or recombinant bacteria L-Ncgl1531-5.
Compared with the genomic DNA of Corynebacterium glutamicum YP097158, the genomic DNA of recombinant bacterium L-Ncgl1531-1 differs only in that: the coding region of the wild type Ncgl1531 gene shown in SEQ ID No. 1 was replaced with the coding region of the Ncgl1531 E35K gene shown in SEQ ID No. 3.
Compared with the genomic DNA of Corynebacterium glutamicum YP097158, the genomic DNA of recombinant bacterium L-Ncgl1531-3 differs only in that: the region of genomic DNA intermediate the upstream homology arm shown as SEQ ID NO. 7 and the downstream homology arm shown as SEQ ID NO. 8 is replaced with a double-stranded DNA molecule shown as SEQ ID NO. 9. The recombinant bacterium L-Ncgl1531-3 has 1 copy of the wild-type Ncgl1531 gene and 1 copy of the Ncgl1531 E35K gene in the genomic DNA.
Compared with the genomic DNA of recombinant bacterium L-Ncgl1531-3, the genomic DNA of recombinant bacterium L-Ncgl1531-2 differs only in that: the exogenous fragment integrated into the genome DNA replaces the coding region of the Ncgl1531 E35K gene shown in SEQ ID NO. 3 with the coding region of the wild type Ncgl1531 gene shown in SEQ ID NO. 1. The recombinant strain L-Ncgl1531-2 has two copies of the wild type Ncgl1531 gene in the genomic DNA.
Recombinant bacterium L-Ncgl1531-4: recombinant plasmid pXMJ-Ncgl 1531 was introduced into Corynebacterium glutamicum YP097158 to obtain recombinant strain L-Ncgl1531-4.
Recombinant bacterium L-Ncgl1531-5: recombinant plasmid pXMJ-Ncgl 1531- E35K was introduced into Corynebacterium glutamicum YP097158 to obtain recombinant strain L-Ncgl1531-5.
Compared with the genomic DNA of Corynebacterium glutamicum ATCC13032, the genomic DNA of recombinant bacterium Y-Ncgl1531-1 differs only in that: the coding region of the wild type Ncgl1531 gene shown in SEQ ID No. 1 was replaced with the coding region of the Ncgl1531 E35K gene shown in SEQ ID No. 3.
Compared with the genomic DNA of Corynebacterium glutamicum ATCC13032, the genomic DNA of recombinant bacteria Y-Ncgl1531-3 differs only in that: the region of genomic DNA intermediate the upstream homology arm shown as SEQ ID NO. 7 and the downstream homology arm shown as SEQ ID NO. 8 is replaced with a double-stranded DNA molecule shown as SEQ ID NO. 9. The genomic DNA of recombinant strain Y-Ncgl1531-3 has 1 copy of the wild-type Ncgl1531 gene and 1 copy of the Ncgl1531 E35K gene.
Compared with the genomic DNA of recombinant strain Y-Ncgl1531-3, the genomic DNA of recombinant strain Y-Ncgl1531-2 differs only in that: the exogenous fragment integrated into the genome DNA replaces the coding region of the Ncgl1531 E35K gene shown in SEQ ID NO. 3 with the coding region of the wild type Ncgl1531 gene shown in SEQ ID NO. 1. The recombinant strain Y-Ncgl1531-2 has two copies of the wild-type Ncgl1531 gene in the genomic DNA.
Recombinant strain Y-Ncgl1531-4: recombinant plasmid pXMJ-Ncgl 1531 was introduced into Corynebacterium glutamicum ATCC13032 to obtain recombinant strain Y-Ncgl1531-4.
Recombinant strain Y-Ncgl1531-5: recombinant plasmid pXMJ-Ncgl 1531 and E35K was introduced into Corynebacterium glutamicum ATCC13032 to obtain recombinant strain Y-Ncgl1531-5.
The method for preparing L-lysine by using the recombinant microorganism comprises the following steps: fermenting the recombinant microorganism.
The person skilled in the art can carry out the fermentation using fermentation methods known in the art. Optimization and improvement of the fermentation process can also be carried out by routine experimentation. The fermentation of the bacteria may be performed in a suitable medium under fermentation conditions known in the art. The medium may comprise: carbon source, nitrogen source, trace elements, and combinations thereof. During the culture, the pH of the culture may be adjusted. In addition, the culture may include prevention of bubble generation, for example, by using an antifoaming agent. In addition, the culturing may include injecting a gas into the culture. The gas may comprise any gas capable of maintaining aerobic conditions of the culture. In the cultivation, the temperature of the culture may be 20 to 45 ℃.
The method may further comprise the steps of: lysine was obtained from the culture. Obtaining lysine from a culture may be accomplished in a variety of ways, including but not limited to: the culture is treated with sulfuric acid or hydrochloric acid or the like, followed by a combination of methods such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation.
In the fermentation, the formula of an exemplary fermentation medium is shown in Table 3, and the balance is water.
An exemplary fermentation control process in the fermentation is shown in table 4.
Illustratively, in the fermentation, the system OD 600nm value may be 0.3-0.5 at the initial time of completion of inoculation.
Specifically, any of the above microorganisms may be a bacterium.
Any of the above bacteria include, but are not limited to, the following: corynebacterium genus bacteria, preferably Corynebacterium acetoacidophilus (Corynebacterium acetoacidophilum), corynebacterium aceti (Corynebacterium acetoglutamicum), corynebacterium meyenii (Corynebacterium callunae), corynebacterium glutamicum (Corynebacterium glutamicum), brevibacterium flavum (Brevibacterium flavum), brevibacterium lactofermentum (Brevibacterium lactofermentum), corynebacterium ammoniagenes (Corynebacterium ammoniagenes), corynebacterium beijing (Corynebacterium pekinense), brevibacterium saccharolyticum (Brevibacterium saccharolyticum), brevibacterium roseum (Brevibacterium roseum), brevibacterium thiogenum (Brevibacterium thiogenitalis).
Any of the above bacteria is a bacterium having an ability to produce lysine.
"Bacterium having an ability to produce lysine" means that the bacterium has the following ability: ability to produce and accumulate lysine in the medium and/or cells of the bacteria. Thus, lysine can be collected when bacteria are cultured in the medium.
The bacteria may be naturally harvested wild-type bacteria or modified bacteria.
"Modified bacteria" refers to engineered bacteria obtained by artificial mutation and/or mutagenesis of naturally acquired wild-type bacteria.
Specifically, the corynebacterium glutamicum may be Corynebacterium glutamicum ATCC13032 or Corynebacterium glutamicum YP097158.
Lysine as defined above is meant to be lysine in the broad sense and includes lysine in free form, salts of lysine or mixtures of both.
Specifically, the lysine is L-lysine.
Any of the above methods or applications may also be used for the preparation of downstream products of lysine.
The L-lysine may be replaced with other products.
The recombinant microorganism may be replaced with a recombinant biological cell.
That is, the recombinant biological cells can be used to produce a variety of products, including but not limited to lysine in the examples. The products produced may also be glutamic acid, valine, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, alpha ketoglutaric acid, citric acid, ornithine, citrulline and the like.
Ncg11531 encodes a membrane protein (membrane protein), which is one of the components of cell membranes and may be related to cell membrane permeability. Although no report has been found in the literature on this gene, the specific function and mechanism of action are not clear. The inventor finds that the mutation of the gene is favorable for improving the yield of L-lysine in the mutagenesis process of corynebacterium glutamicum by accident, and meanwhile, the continuous research shows that the improvement of the expression activity (point mutation or over-expression) of the gene in microorganisms is favorable for accumulation of L-lysine.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged. pK18mobsacB plasmid, double-stranded circular DNA (shown as SEQ ID NO: 13). pXMJ19 plasmid: bioVector plasmid vector cell Gene preservation center. NEBuilder enzyme: NEB corporation. In the primers, the underlined regions are used for homologous recombination with the same sequences on the plasmid backbone.
Corynebacterium glutamicum ATCC13032, corynebacterium glutamicum accession No. 13032 in ATCC (Corynebacterium glutamicum).
Corynebacterium glutamicum (Corynebacterium glutamicum) YP097158 was deposited at China general microbiological culture Collection center (CGMCC) with accession number CGMCC No.12856 in the Ministry of China including Kogyo-Chamber North Star West Lu No.1 and 3 of Beijing, and was deposited at the center of China including 16 th month.
Sequencing confirmed that the genomic DNA of Corynebacterium glutamicum ATCC13032 and Corynebacterium glutamicum YP097158 each had the following sections: the segment shown in SEQ ID No. 5 (template strand) (wherein the 1236-1581 positions are promoters and the 654-1235 positions are wild-type Ncgl1531 gene), the segment shown in SEQ ID No. 7 (corresponding to the NCgl1740 gene part-coding region and the NCgl1741 gene promoter region and part-coding region), and the segment shown in SEQ ID No. 8 (corresponding to the NCgl1742 gene part-coding region).
The coding region of the wild type Ncgl1531 gene is shown as SEQ ID NO. 1, and the wild type Ncgl1531 protein shown as SEQ ID NO. 2 is coded. The coding region of the Ncgl1531 E35K gene is shown as SEQ ID NO. 3, and the Ncgl1531 E35K protein shown as SEQ ID NO. 4 is encoded. The difference between the Ncgl1531 and the wild-type Ncgl1531 proteins, E35K, is only the mutation of the amino acid residue at position 35 from glutamic acid (E) to lysine (K). The difference between the Ncgl1531 gene and the wild type Ncgl1531 gene, the Ncgl1531 E35K gene, was only that the 103 th nucleotide of the coding region was mutated from G to A.
Example 1L-lysine fermentation experiment
Test strain: corynebacterium glutamicum ATCC13032, recombinant bacterium Y-Ncgl1531-1, recombinant bacterium Y-Ncgl1531-2, recombinant bacterium Y-Ncgl1531-3, recombinant bacterium Y-Ncgl1531-4, recombinant bacterium Y-Ncgl1531-5, and recombinant bacterium Y-Ncgl1531-6; corynebacterium glutamicum YP097158, recombinant bacterium L-Ncgl1531-1, recombinant bacterium L-Ncgl1531-2, recombinant bacterium L-Ncgl1531-3, recombinant bacterium L-Ncgl1531-4, recombinant bacterium L-Ncgl1531-5, and recombinant bacterium L-Ncgl1531-6. Recombinant bacteria were prepared from examples 3, 4, 5 and 6, respectively.
Fermentation was performed using a BLBIO-5GC-4-H model fermenter (Shanghai Bai Biotechnology Co., ltd.). The formulation of the fermentation medium is shown in Table 1. The fermentation control process is shown in Table 2. At the initial time of completing inoculation, the OD 600nm value of the system is 0.3-0.5. After the fermentation is finished, detecting the yield of the L-lysine by adopting an ninhydrin colorimetric method. At least 3 biological replicates were set and the results averaged ± standard deviation.
The method for detecting the lysine yield of the fermentation liquor by ninhydrin staining is as follows:
① After fermentation, the mixture was centrifuged at 5000rpm/min for 5min, and the supernatant was collected.
② Taking a 96-well plate, adding 5 mu L of the supernatant obtained in the step ①, 0.066mL of the solution I and 0.037mL of the solution II into each well, fully and uniformly mixing, and tightly sealing the cover by using a colloid 96-well sealing cover.
Solution I:630mL buffer+6 g ferric trichloride+373 mL methyl cellosolve. Solution II:1g of ninhydrin was dissolved in 100mL of buffer. Buffer (ph 2.2): 1.43g of Na 2HPO4 and 18.8g of anhydrous citric acid were weighed and water was added to 1L.
③ After completion of step ②, the 96-well plate was kept at a constant temperature of 100℃for 40min, and then rapidly cooled to room temperature with water.
④ After completion of step ③, 200 μl DMSO was added to each well and mixed well.
⑤ After completion of step ④, 200. Mu.L to 96-well ELISA plates were sampled and the absorbance peak at 480nm was measured using an ELISA reader.
A standard curve was made using L-lysine standard, and then L-lysine production was calculated using the absorbance peak.
The results are shown in tables 3 and 4. Overexpression of the wild-type Ncgl1531 gene or the Ncgl1531 E35K gene in Corynebacterium glutamicum contributes to increased L-lysine production. The site-directed mutagenesis of the wild-type Ncgl1531 gene into the Ncgl1531 E35K gene in Corynebacterium glutamicum contributes to an improved L-lysine yield. The wild type Ncgl1531 gene in Corynebacterium glutamicum is knocked out, reducing L-lysine production. Over-expression of the Ncgl1531 E35K gene in C.glutamicum increased L-lysine production more significantly than over-expression of the wild-type Ncgl1531 gene.
TABLE 1 fermentation Medium formulation
| Composition of the components | Concentration in the Medium |
| Starch hydrolyzing sugar | 30g/L |
| Ammonium sulfate | 12g/L |
| Magnesium sulfate | 0.87g/L |
| Molasses | 20g/L |
| Acidified corn steep liquor | 3mL/L |
| Phosphoric acid | 0.4mL/L |
| Potassium chloride | 0.53g/L |
| Defoaming agent (2% bubble enemy) | 4mL/L |
| Ferrous sulfate | 120mg/L |
| Manganese sulfate | 120mg/L |
| Nicotinamide | 42mg/L |
| Calcium pantothenate | 6.3mg/L |
| Vitamin B1 | 6.3mg/L |
| Copper sulfate | 0.9mg/L |
| Zinc sulfate | 1.0mg/L |
| Biotin | 0.88mg/L |
TABLE 2 fermentation control process
TABLE 3 fermentation yield of modified bacteria starting from Corynebacterium glutamicum ATCC13032
TABLE 4 fermentation yield of modified bacteria starting from Corynebacterium glutamicum YP097158
EXAMPLE 2 construction of recombinant vector expressing Ncgl1531 E35K Gene
1. The genomic DNA of Corynebacterium glutamicum ATCC13032 is used as a template, and a primer pair consisting of P1 and P2 is used for PCR amplification to recover an amplified product.
2. The genomic DNA of Corynebacterium glutamicum ATCC13032 is used as a template, and a primer pair consisting of P3 and P4 is used for PCR amplification to recover an amplified product.
3. The pK18mobsacB plasmid was digested with restriction enzymes Xbal I and BamH I to recover a linearized plasmid backbone of about 6 kb.
4. The amplification product obtained in step 1, the amplification product obtained in step 2 and the linearized plasmid backbone obtained in step 3 were ligated with NEBuilder enzyme (50 ℃ C., 30 min) to obtain recombinant plasmid pK18-Ncgl1531 E35K. The recombinant plasmid pK18-Ncgl1531 E35K has been subjected to sequencing verification. The recombinant plasmid pK18-Ncgl1531 E35K differs from the pK18mobsacB plasmid only in that the double stranded DNA molecule shown in SEQ ID NO. 6 is substituted for "AGGATCCCC" in the pK18mobsacB plasmid.
P1：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGGGTGACATCCTGGAAATAG-3'；
P2：5'-CAGCGATGCGGAAATTCAGAAATACACCGCAGCTTTC-3'；
P3：5'-GAAAGCTGCGGTGTATTTCTGAATTTCCGCATCGCTG-3'；
P4：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCACAAGCACGCAGTAGATG-3'。
EXAMPLE 3 construction of recombinant bacterium in which wild-type Ncgl1531 Gene was replaced with homologous Ncgl1531 E35K Gene
1. Preparation of recombinant L-Ncgl1531-1
1. The recombinant plasmid pK18-Ncgl1531 E35K was introduced into Corynebacterium glutamicum YP097158 by electric shock, and then cultured in a solid medium containing 50mg/L kanamycin at 30℃for 40h. And (3) picking a single colony, carrying out PCR amplification by adopting a primer pair consisting of P1 and P4, recovering an amplification product, and carrying out sequencing verification, wherein the strain with the sequencing result shown in SEQ ID No.6 is a target strain.
Solid medium A (pH 7.0): comprises 10g/L of sucrose, 10g/L of polypeptone, 10g/L of beef extract, 5g/L of yeast powder, 2g/L of urea, 2.5g/L of sodium chloride, 18g/L of agar powder and the balance of water. The following is the same.
2. And (3) streaking and inoculating the target strain obtained in the step (1) onto a solid culture medium B plate, and culturing at 30 ℃ for 40h.
Solid medium b (ph 7.0): comprises 150g/L sucrose, 10g/L polypeptone, 10g/L beef extract, 5g/L yeast powder, 2g/L urea, 2.5g/L sodium chloride, 18g/L agar powder and the balance of water. The following is the same.
3. After the step 2 is completed, single colonies are picked from the flat plate and respectively inoculated to a non-antibacterial flat plate and an antibacterial flat plate, and strains which do not grow on the antibacterial flat plate and grow on the non-antibacterial flat plate are selected to be target strains.
Non-antibacterial plates: solid medium formazan plate. The following is the same.
Antimicrobial plates: a solid medium plate containing 50mg/L kanamycin. The following is the same.
4. And (3) taking the target strain obtained in the step (3), carrying out PCR amplification by adopting a primer pair consisting of P1 and P4, recovering an amplification product, and carrying out sequencing verification, wherein if the amplification product has an Ncgl1531 E35K gene coding region and does not have a wild Ncgl1531 gene coding region, the strain is recombinant bacterium L-Ncgl1531-1.
Sequencing shows that the genomic DNA of recombinant bacterium L-Ncgl1531-1 differs from the genomic DNA of Corynebacterium glutamicum YP097158 only in that: the coding region of the wild type Ncgl1531 gene shown in SEQ ID No. 1 was replaced with the coding region of the Ncgl1531 E35K gene shown in SEQ ID No. 3.
2. Preparation of recombinant Y-Ncgl1531-1
Corynebacterium glutamicum ATCC13032 was used in place of Corynebacterium glutamicum YP097158, in the same manner as in step one.
Recombinant strain Y-Ncgl1531-1 was obtained.
Sequencing shows that the genomic DNA of recombinant strain Y-Ncgl1531-1 differs from the genomic DNA of Corynebacterium glutamicum ATCC13032 only in that: the coding region of the wild type Ncgl1531 gene shown in SEQ ID No.1 was replaced with the coding region of the Ncgl1531 E35K gene shown in SEQ ID No. 3.
EXAMPLE 4 construction of recombinant bacteria overexpressing the wild-type Ncgl1531 Gene or the Ncgl1531 E35K Gene on the genome
1. Construction of recombinant plasmid pK18-Ncgl1531OE
1. The genomic DNA of Corynebacterium glutamicum ATCC13032 is used as a template, and a primer pair consisting of P5 and P6 is used for PCR amplification to recover an amplified product. Sequencing shows that the amplified product has upstream homology arm shown as SEQ ID No. 7.
2. The genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and the amplification product was recovered by PCR amplification using a primer set composed of P7 and P8. Sequencing shows that the amplified product is shown in 542-1581 bits of SEQ ID No. 5.
3. The genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and the amplification product was recovered by PCR amplification using a primer set composed of P9 and P10. Sequencing shows that the amplified product has the downstream homology arm shown as SEQ ID NO. 8.
4. The pK18mobsacB plasmid was digested with restriction enzymes Xbal I and BamH I to recover a linearized plasmid backbone of about 6 kb.
5. And (3) connecting the amplification product obtained in the step (1), the amplification product obtained in the step (2), the amplification product obtained in the step (3) and the linearized plasmid skeleton obtained in the step (4) by using NEBuilder enzyme (50 ℃ for 30 min) to obtain the recombinant plasmid pK18-Ncgl1531OE. The recombinant plasmid pK18-Ncgl1531OE has been sequenced. The recombinant plasmid pK18-Ncgl1531OE differs from the pK18mobsacB plasmid only in that "AGGATCCCC" in the pK18mobsacB plasmid has been replaced by a specific DNA molecule consisting of, in sequence from upstream to downstream, the double-stranded DNA molecule shown in SEQ ID NO:7, the double-stranded DNA molecule shown in SEQ ID NO: 542-1581 in SEQ ID NO:5, and the double-stranded DNA molecule shown in SEQ ID NO: 8.
2. Construction of recombinant plasmid pK18-Ncgl1531 E35K OE
1. The genome DNA of recombinant bacterium L-Ncgl1531-1 is used as a template, and a primer pair consisting of P5 and P6 is used for PCR amplification, and an amplification product is recovered.
2. The genome DNA of recombinant bacterium L-Ncgl1531-1 is used as a template, and a primer pair consisting of P7 and P8 is used for PCR amplification, and an amplification product is recovered.
3. The genome DNA of recombinant bacterium L-Ncgl1531-1 is used as a template, and a primer pair consisting of P9 and P10 is used for PCR amplification, and an amplification product is recovered.
4. The pK18mobsacB plasmid was digested with restriction enzymes Xbal I and BamH I to recover a linearized plasmid backbone of about 6 kb.
5. The amplification product obtained in step 1, the amplification product obtained in step 2, the amplification product obtained in step 3 and the linearized plasmid backbone obtained in step 4 are ligated by NEBuilder enzyme (50 ℃ C., 30 min) to obtain recombinant plasmid pK18-Ncgl1531 E35K OE. The recombinant plasmid pK18-Ncgl1531 E35K OE was sequenced. The recombinant plasmid pK18-Ncgl1531 E35K OE differs from the pK18mobsacB plasmid only in that "AGGATCCCC" in the pK18mobsacB plasmid is replaced by a specific DNA molecule consisting of the double-stranded DNA molecule shown in SEQ ID NO. 7, the double-stranded DNA molecule shown in SEQ ID NO. 9, and the double-stranded DNA molecule shown in SEQ ID NO. 8 in this order from upstream to downstream.
3. Preparation of recombinant L-Ncgl1531-3
1. The recombinant plasmid pK18-Ncgl1531 E35K OE was introduced into Corynebacterium glutamicum YP097158 by electric shock and then cultured in solid medium methanol at 30℃for 40h. And (3) picking a single colony, carrying out PCR amplification by adopting a primer pair consisting of P11 and P12, recovering an amplified product, and carrying out sequencing verification, wherein if the amplified product is 1629bp, the strain is a target strain. The sequencing result of the amplified product is shown as SEQ ID NO. 10.
2. And (3) streaking and inoculating the target bacteria obtained in the step (1) onto a solid culture medium B plate, and culturing at 30 ℃ for 40h. And selecting a single colony, carrying out PCR amplification by adopting a primer pair consisting of P13 and P14, recovering an amplified product, and carrying out sequencing verification, wherein if the amplified product is 1359bp, the bacterium is a target bacterium, namely recombinant bacterium L-Ncgl1531-3. The sequencing result of the amplified product is shown as SEQ ID NO. 11.
Sequencing shows that the genomic DNA of recombinant bacteria L-Ncgl1531-3 differs from the genomic DNA of Corynebacterium glutamicum YP097158 only in that: the region of genomic DNA intermediate the upstream homology arm shown as SEQ ID NO. 7 and the downstream homology arm shown as SEQ ID NO. 8 is replaced with a double-stranded DNA molecule shown as SEQ ID NO. 9. The recombinant bacterium L-Ncgl1531-3 has 1 copy of the wild-type Ncgl1531 gene and 1 copy of the Ncgl1531 E35K gene in the genomic DNA.
4. Preparation of recombinant L-Ncgl1531-2
1. The recombinant plasmid pK18-Ncgl1531OE was introduced into Corynebacterium glutamicum YP097158 by electric shock and then cultured in solid medium methanol at 30℃for 40h. And (3) picking a single colony, carrying out PCR amplification by adopting a primer pair consisting of P11 and P12, recovering an amplified product, and carrying out sequencing verification, wherein if the amplified product is 1629bp, the strain is a target strain.
2. And (3) streaking and inoculating the target bacteria obtained in the step (1) onto a solid culture medium B plate, and culturing at 30 ℃ for 40h. And selecting a single colony, carrying out PCR amplification by adopting a primer pair consisting of P13 and P14, recovering an amplified product, and carrying out sequencing verification, wherein if the amplified product is 1359bp, the bacterium is a target bacterium, namely recombinant bacterium L-Ncgl1531-2.
Sequencing shows that compared with the genomic DNA of recombinant bacterium L-Ncgl1531-3, the genomic DNA of recombinant bacterium L-Ncgl1531-2 only differs in that: the exogenous fragment integrated into the genome DNA replaces the coding region of the Ncgl1531 E35K gene shown in SEQ ID NO. 3 with the coding region of the wild type Ncgl1531 gene shown in SEQ ID NO. 1. The recombinant strain L-Ncgl1531-2 has two copies of the wild type Ncgl1531 gene in the genomic DNA.
5. Preparation of recombinant Y-Ncgl1531-3
Corynebacterium glutamicum ATCC13032 was used instead of Corynebacterium glutamicum YP097158, and the procedure was the same as in step three.
Recombinant strain Y-Ncgl1531-3 was obtained.
Sequencing shows that the genomic DNA of recombinant strain Y-Ncgl1531-3 differs from the genomic DNA of Corynebacterium glutamicum ATCC13032 only in that: the region of genomic DNA intermediate the upstream homology arm shown as SEQ ID NO. 7 and the downstream homology arm shown as SEQ ID NO. 8 is replaced with a double-stranded DNA molecule shown as SEQ ID NO. 9. The genomic DNA of recombinant strain Y-Ncgl1531-3 has 1 copy of the wild-type Ncgl1531 gene and 1 copy of the Ncgl1531 E35K gene.
6. Preparation of recombinant Y-Ncgl1531-2
Corynebacterium glutamicum ATCC13032 was used in place of Corynebacterium glutamicum YP097158, and the procedure was the same as in the fourth step.
Recombinant strain Y-Ncgl1531-2 was obtained.
Sequencing shows that compared with the genomic DNA of the recombinant bacteria Y-Ncgl1531-3, the genomic DNA of the recombinant bacteria Y-Ncgl1531-2 only has the following differences: the exogenous fragment integrated into the genome DNA replaces the coding region of the Ncgl1531 E35K gene shown in SEQ ID NO. 3 with the coding region of the wild type Ncgl1531 gene shown in SEQ ID NO. 1. The recombinant strain Y-Ncgl1531-2 has two copies of the wild-type Ncgl1531 gene in the genomic DNA.
P5：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAATGCGTTCTGGACTGAGG-3'；
P6：5'-GACACAGCCCAAGGCAACTAGTGCACCGAGAACAGATG-3'；
P7：5'-CATCTGTTCTCGGTGCACTAGTTGCCTTGGGCTGTGTC-3'；
P8：5'-GATTTAATTGCGCCATCTGCTTGCCCACGTAGATCAC-3'；
P9：5'-GTGATCTACGTGGGCAAGCAGATGGCGCAATTAAATC-3'；
P10：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCTATGACACCTTCAACGGATC-3'。
P11:5'-TCCAAGGAAGATACACGCC-3' (corresponding to the outer side of the upper homology arm NCgl 1740);
P12:5'-GAAGTTACGCACCTCAACG-3' (corresponding to the inside of the Ncgl1531 gene);
p13:5'-GAGCAACGAACAACATCAAC-3' (corresponding to the inside of the Ncgl1531 gene);
p14:5'-TGGTCGTTGGAATCTTGC-3' (corresponding to the outer side of the lower homology arm NCgl 1742).
EXAMPLE 5 construction of recombinant bacteria overexpressing the wild-type Ncgl1531 Gene or the Ncgl1531 E35K Gene on plasmids
1. Construction of recombinant plasmid pXMJ-Ncgl 1531 E35K
1. The genome DNA of recombinant bacterium L-Ncgl1531-1 is used as a template, and a primer pair consisting of P15 and P16 is used for PCR amplification, and an amplification product is recovered.
2. The pXMJ plasmid was double digested with restriction enzymes Xbal I and BamH I, and the linearized plasmid backbone of about 6kb was recovered.
3. The amplification product obtained in step 1 and the linearized plasmid backbone obtained in step 2 were ligated with NEBuilder enzyme (50 ℃ C., 30 min) to obtain recombinant plasmid pXMJ-Ncgl 1531 E35K. The recombinant plasmid pXMJ-Ncgl 1531 E35K has been subjected to sequencing verification. The recombinant plasmid pXMJ-Ncgl 1531 E35K differs from the pXMJ plasmid only in that the double stranded DNA molecule shown in SEQ ID NO. 9 is substituted for "TCTAGAGGATCCCCG" in the pXMJ plasmid.
2. Construction of recombinant plasmid pXMJ-Ncgl 1531
1. The genomic DNA of Corynebacterium glutamicum ATCC13032 is used as a template, and a primer pair consisting of P15 and P16 is used for PCR amplification to recover an amplified product.
2. The pXMJ plasmid was double digested with restriction enzymes Xbal I and BamH I, and the linearized plasmid backbone of about 6kb was recovered.
3. The amplification product obtained in step 1 and the linearized plasmid backbone obtained in step 2 were ligated with NEBuilder enzyme (50 ℃ C., 30 min) to obtain recombinant plasmid pXMJ-Ncgl 1531. The recombinant plasmid pXMJ-Ncgl 1531 has been sequenced. The recombinant plasmid pXMJ-Ncgl 1531 differs from the pXMJ plasmid only in that the double stranded DNA molecule shown at positions 542-1581 in SEQ ID NO. 5 is substituted for "TCTAGAGGATCCCCG" in the pXMJ plasmid.
3. Preparation of recombinant bacteria
Recombinant plasmid pXMJ-Ncgl 1531 was introduced into Corynebacterium glutamicum YP097158 to obtain recombinant strain L-Ncgl1531-4.
Recombinant plasmid pXMJ-Ncgl 1531- E35K was introduced into Corynebacterium glutamicum YP097158 to obtain recombinant strain L-Ncgl1531-5.
Recombinant plasmid pXMJ-Ncgl 1531 was introduced into Corynebacterium glutamicum ATCC13032 to obtain recombinant strain Y-Ncgl1531-4.
Recombinant plasmid pXMJ-Ncgl 1531 and E35K was introduced into Corynebacterium glutamicum ATCC13032 to obtain recombinant strain Y-Ncgl1531-5.
P15：5'-CAGAATAATTAAGCTTGCATGCCTGCAGGTCGACTAGTTGCCTTGGGCTGTGTC-3'；
P16：5'-CCAAAACAGCCAAGCTGAATTCGAGCTCGGTACCCTTGCCCACGTAGATCAC-3'。
EXAMPLE 6 construction of recombinant bacterium having wild-type Ncgl1531 Gene deleted from genome
1. Preparation of recombinant plasmid pK 18-. DELTA.Ncgl 1531
1. The genomic DNA of Corynebacterium glutamicum ATCC13032 is used as a template, and a primer pair consisting of P17 and P18 is used for PCR amplification to recover an amplified product.
2. The genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and the amplification product was recovered by PCR amplification using a primer set composed of P19 and P20.
3. The pK18mobsacB plasmid was digested with restriction enzymes Xbal I and BamH I to recover a linearized plasmid backbone of about 6 kb.
4. And (3) connecting the amplification product obtained in the step (1), the amplification product obtained in the step (2) and the linearized plasmid skeleton obtained in the step (3) by using NEBuilder enzyme (50 ℃ for 30 min) to obtain the recombinant plasmid pK 18-delta Ncgl1531. The recombinant plasmid pK 18-. DELTA.Ncgl 1531 was verified by sequencing. The recombinant plasmid pK 18-. DELTA.Ncgl 1531 differs from the pK18mobsacB plasmid only in that "AGGATCCCC" in the pK18mobsacB plasmid was replaced with the double stranded DNA molecule shown in SEQ ID NO. 12.
2. Preparation of recombinant L-Ncgl1531-6
1. The recombinant plasmid pK 18-. DELTA.Ncgl 1531 was introduced into Corynebacterium glutamicum YP097158 by electric shock, and then cultured in a solid medium plate at 30℃for 40h. Single colony is selected, and PCR amplification is carried out by adopting a primer pair consisting of P17 and P20, so that the strains with the sizes of 1540bp and 1979bp bands can be obtained by simultaneous amplification as positive strains.
2. And (3) streaking and inoculating the positive strain obtained in the step (1) onto a solid culture medium B plate, and culturing at 30 ℃ for 40h.
3. After the step 2 is completed, single colonies are picked from the flat plate and respectively inoculated to a non-antibacterial flat plate and an antibacterial flat plate, and strains which do not grow on the antibacterial flat plate and grow on the non-antibacterial flat plate are selected to be positive strains.
4. And (3) taking the positive strain obtained in the step (3), and carrying out PCR (polymerase chain reaction) amplification by adopting a primer pair consisting of P17 and P20, wherein only the strain with 1540bp band obtained by amplification is the positive strain, namely the recombinant strain L-Ncgl1531-6.
Sequencing shows that the genomic DNA of recombinant bacteria L-Ncgl1531-6 differs from the genomic DNA of Corynebacterium glutamicum YP097158 only in that: the DNA molecule shown in SEQ ID NO. 5 was replaced with the DNA molecule shown in SEQ ID NO. 12.
3. Preparation of recombinant Y-Ncgl1531-6
Corynebacterium glutamicum ATCC13032 was used instead of Corynebacterium glutamicum YP097158, and the procedure was the same as in the second step.
Recombinant strain Y-Ncgl1531-6 was obtained.
Sequencing shows that the genomic DNA of recombinant strain Y-Ncgl1531-6 differs from the genomic DNA of Corynebacterium glutamicum ATCC13032 only in that: the DNA molecule shown in SEQ ID NO. 5 was replaced with the DNA molecule shown in SEQ ID NO. 12.
P17：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGTTGAGTTTGCGAGTGAC-3'；
P18：5'-GTTTGACGCCTCGGTTTACTTCATCGGAACTCGTTTC-3'；
P19：5'-GAAACGAGTTCCGATGAAGTAAACCGAGGCGTCAAAC-3'；
P20：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAGAAGGTGGGACTTGAAGG-3'。
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. Recombinant microorganism, which is (a 1) or (a 2) or (a 3) as follows:
(a1) Introducing the Ncgl1531 gene or a biological material related to the Ncgl1531 gene into a host microorganism to obtain a recombinant microorganism;
(a2) Introducing the mutant gene or a biological material related to the mutant gene into a host microorganism to obtain a recombinant microorganism;
(a3) Replacing the Ncgl1531 gene in the host microorganism genome with a mutant gene to obtain a recombinant microorganism;
The Ncgl1531 gene is a gene encoding Ncgl1531 protein;
the Ncgl1531 gene related biological material is an expression cassette or a recombinant vector with the Ncgl1531 gene;
the mutant gene is a gene encoding mutant protein;
the mutant protein is obtained by mutating one or more amino acid residues of the Ncgl1531 protein;
the mutant gene related biological material is an expression cassette or a recombinant vector with the mutant gene.
2. The recombinant microorganism of claim 1, wherein: the Ncgl1531 protein comprises an amino acid sequence shown in SEQ ID NO. 2.
3. The recombinant microorganism of claim 2, wherein: the mutant protein is obtained by mutating the 35 th amino acid residue of the amino acid sequence shown in SEQ ID NO. 2 in the Ncgl1531 protein from glutamic acid to other amino acids.
4. The recombinant microorganism of claim 3, wherein: the mutant protein is obtained by mutating the 35 th amino acid residue of the amino acid sequence shown in SEQ ID NO. 2 in the Ncgl1531 protein from glutamic acid to lysine.
5. Use of a recombinant microorganism according to any one of claims 1 to 4 for the preparation of L-lysine.
Application of Ncgl1531 protein or mutant protein in positive regulation of L-lysine production by microorganisms;
the mutant protein is obtained by mutating one or more amino acid residues of the Ncgl1531 protein.
Application of Ncgl1531 gene or mutant gene in positive regulation of L-lysine production by microorganisms;
The Ncgl1531 gene is a gene encoding Ncgl1531 protein;
the mutant gene is a gene encoding mutant protein;
the mutant protein is obtained by mutating one or more amino acid residues of the Ncgl1531 protein.
Use of the Ncgl1531 gene or the biological material associated with the mutant gene or the mutant gene for the preparation of a product for improving the ability of a microorganism to produce L-lysine;
The Ncgl1531 gene is a gene encoding Ncgl1531 protein;
the Ncgl1531 gene related biological material is an expression cassette or a recombinant vector with the Ncgl1531 gene;
the mutant gene is a gene encoding mutant protein;
the mutant protein is obtained by mutating one or more amino acid residues of the Ncgl1531 protein;
the mutant gene related biological material is an expression cassette or a recombinant vector with the mutant gene.
9. Use according to any one of claims 6 to 8, wherein: the Ncgl1531 protein comprises an amino acid sequence shown in SEQ ID NO. 2.
10. The use according to claim 9, wherein:
The mutant protein is obtained by mutating the 35 th amino acid residue of the amino acid sequence shown in SEQ ID NO. 2 in the Ncgl1531 protein from glutamic acid to other amino acids;
Preferably, the mutant protein is obtained by mutating the 35 th amino acid residue of the amino acid sequence shown in SEQ ID NO. 2 in the Ncgl1531 protein from glutamic acid to lysine.
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