CN111909944A - Recombinant strain modified by lysC gene and construction method and application thereof - Google Patents
Recombinant strain modified by lysC gene and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a nucleotide sequence and a recombinant strain comprising the same, which is formed by carrying out point mutation on a promoter region sequence of lysC gene in Corynebacterium glutamicum, wherein the mutated promoter region gene sequence is shown as SEQ ID NO. 2. Compared with the non-mutated strain, the recombinant strain has the advantages that the yield of the L-lysine is improved, the stability of the strain is good, and the production cost can be further reduced when the recombinant strain is used as an L-lysine production strain.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and microorganisms, and particularly relates to a recombinant strain modified by lysC (lysogeny C) gene and a construction method and application thereof.
Background
L-lysine is the most important of the eight essential amino acids that cannot be synthesized in humans and animals. At present, lysine is the amino acid feed additive with the largest global use amount at present, about 90% of L-lysine products are used for the feed additive, about 5% are used as food additives, and the rest 5% are used as medical intermediates. For example, the addition of a proper amount of L-lysine in the soybean meal can greatly improve the utilization rate of protein and promote the growth of livestock. Due to the remarkable rate of development of the domestic feed industry, the market demand for L-lysine is increasing. But the fermentation acid production level is low, the production scale is small, so that the production cost is high, and the method is difficult to compete with the same imported product. Therefore, it is necessary to breed L-lysine high-producing strain, and the lysine industry in China has international competitiveness.
The method for producing L-lysine is a protein hydrolysis method at the earliest, and a chemical synthesis method developed later uses a highly toxic raw material, so that the method has a serious environmental problem, an enzyme method is slowly eliminated due to unstable enzyme activity, small scale, high cost and the like, and the direct fermentation method is a lysine production method widely adopted at present until the success of producing lysine by adopting direct fermentation of microorganisms in Japan in 1960 is achieved, so that the research and development of lysine production are really promoted.
However, with the national advocation of conservation-oriented society, how to achieve energy conservation and emission reduction in the production process of L-lysine and reduce energy consumption and environmental pollution become serious problems in each L-lysine production enterprise. Therefore, the breeding of the production strain with high L-lysine synthesis and low byproduct accumulation, which has independent intellectual property rights, has important significance for realizing the sustainable development of enterprises.
The L-lysine is produced by microbial fermentation, and a plurality of key enzymes exist in the pathway to participate in the synthesis of the L-lysine. Examples thereof include sugar-1, 6-bisphosphatase (FBPase), glucose-6-phosphate dehydrogenase (G6PDH), glucose-6-phosphate dehydrogenase (6PGDH), glyceraldehyde-3-phosphate dehydrogenase (GADPH), phosphoenolpyruvate carboxylase (PEPCx), pyruvate carboxylase (PCx), Aspartokinase (AK), aspartate semialdehyde dehydrogenase (ASADH), and the like. Wherein AK is encoded by lysC gene, which is one of the key enzymes in the synthesis of L-lysine. Corynebacteria, which are the most important species used in amino acid production, include Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium flavum (c. flavum), Corynebacterium crenatum (c. crenatum), Corynebacterium beijing, etc., among which studies on aspartokinase of some strains have been reported.
Disclosure of Invention
The invention provides a promoter nucleotide sequence which comprises a nucleotide sequence formed by mutating base groups at the 45bp position and the 47bp position of a promoter region shown in SEQ ID NO. 1.
According to the invention, the nucleotide guanine (G) at the-45 bp position of the promoter region shown in SEQ ID NO.1 is mutated into adenine (A), and the nucleotide guanine (G) at the-47 bp position is mutated into thymine (T).
According to the invention, the promoter nucleotide sequence is as follows:
(a) a nucleotide sequence shown as SEQ ID NO. 2; in the alternative, the first and second sets of the first,
(b) a nucleotide sequence having 90% or more, preferably 95%, 98% or more identity to the nucleotide sequence shown in SEQ ID NO. 2, and which retains the enhanced activity of the promoter of (a) and maintains adenine (A) at the-45 bp position and thymine (T) at the-47 bp position.
The invention also provides an expression cassette comprising the promoter, comprising the promoter and a coding sequence operably linked to the promoter. In one embodiment of the present invention, the coding sequence is the coding sequence of the lysC gene.
The present invention also provides a recombinant vector comprising the promoter nucleotide sequence of the present invention.
According to the present invention, the recombinant vector is constructed by linking the promoter nucleotide sequence of the present invention to a shuttle plasmid; as an embodiment of the present invention, the shuttle plasmid is a pK18mobsacB plasmid.
The invention also provides a recombinant strain containing the promoter nucleotide sequence or the recombinant vector.
The recombinant strain comprises a nucleotide sequence shown as SEQ ID NO. 2. The nucleotide sequence shown in SEQ ID NO. 2 is a promoter region of lysC gene. Further, the nucleotide sequence shown in SEQ ID NO. 2 is linked to a lysC gene coding sequence. In particular, the recombinant strain may comprise an expression cassette or a recombinant vector of the present invention as described above. Specifically, the recombinant strain of the present invention is transformed with an expression cassette or a recombinant vector. According to the recombinant strain, the mutant promoter nucleotide sequence is introduced into a host strain to be recombined; the host strain may be selected from L-lysine producing strains known in the art, for example, at least one selected from Corynebacterium, which may be Corynebacterium glutamicum, Corynebacterium flavum, Corynebacterium crenatum, Corynebacterium pekinense; corynebacterium glutamicum is preferred. As an embodiment of the invention, the host strain is YP97158 (preservation number: CGMCC No.12856, preservation date: 2016, 8 and 16 days, preservation unit: institute of microbiology, national academy of sciences).
The recombinant strain of the invention takes pK18mobsacB plasmid as a vector.
The recombinant strain according to the present invention may further comprise other modifications.
The invention also provides a construction method of the recombinant strain for producing the L-lysine, which comprises the following steps:
(1) the promoter region shown as SEQ ID NO.1 is modified to mutate the-45 bp position and the-47 bp position to obtain the nucleotide sequence of the promoter region containing the mutation.
According to the invention, the mutation is that the-45 bp nucleotide guanine (G) of the promoter region shown in SEQ ID NO.1 is mutated into adenine (A), and the-47 bp nucleotide guanine (G) is mutated into thymine (T). Specifically, the nucleotide sequence of the promoter region after mutation is shown as SEQ ID NO. 2. Further, the construction method further comprises the following steps:
(2) connecting the nucleotide sequence of the mutant promoter region with a plasmid to construct a recombinant vector;
(3) and (3) introducing the recombinant vector into a host strain to obtain the L-lysine-producing recombinant strain containing the mutant promoter region.
According to the present invention, in the step (1), the method for mutating comprises mutagenesis, PCR site-directed mutagenesis or homologous recombination, preferably PCR site-directed mutagenesis.
According to the invention, said step (1) comprises:
two pairs of primers for amplifying the promoter region of lysC gene were designed, and the nucleotide sequence of the mutant promoter region was obtained by PCR technique.
In one embodiment of the present invention, the primers in step (1) are:
P1:5'CCGGAATTCG ACCAAGGATG AGGGCTTTG 3'(EcoR I)(SEQ ID NO:3)
P2:5'AGTTACCCGC TCAATTATAC CTTTATAAAC 3'(SEQ ID NO:4)
P3:5'GTTTATAAAG GTATAATTGA GCGGGTAACT 3'(SEQ ID NO:5)
P4:5'ACATGCATGC GCGTACGCGA AGTGGCACAT 3'(Sph I)(SEQ ID NO:6)。
in one embodiment of the present invention, the step (1) comprises: carrying out PCR amplification by using Corynebacterium glutamicum ATCC13032 as a template and primers P1 and P2, and P3 and P4 respectively to obtain two DNA-containing fragments; the two DNA fragments were used as templates, and P1 and P4 were used as primers, and Overlap PCR amplification (Overlap PCR) was performed to obtain a DNA fragment containing the nucleotide sequence (SEQ ID NO:2) of the promoter region of the present invention.
According to the present invention, in step (1), the two ends of the obtained DNA fragment contain EcoR I and Sph I cleavage sites by Overlap PCR amplification (Overlap PCR).
According to the invention, said step (2) comprises: and (3) carrying out agarose gel electrophoresis and separation and purification on the product amplified by the overlapping PCR reaction, and connecting the fragment subjected to double enzyme digestion (EcoR I/Sph I) with the shuttle plasmid subjected to the same double enzyme digestion (EcoR I/Sph I) to obtain the allelic replacement recombinant vector.
According to the invention, the shuttle plasmid is pK18mobsacB plasmid; the constructed recombinant vector is pK18-Plys C(G(-45)A,G(-47)T)。
In one embodiment of the invention, the recombinant plasmid has a kanamycin resistance marker.
In one embodiment of the present invention, the conversion of step (3) is an electrical conversion process; exemplarily, in the step (3), the recombinant vector is transformed into the strain YP 97158.
The present invention also provides the use of the recombinant strain according to the fourth aspect for the production of L-lysine; or a method for increasing the fermentation amount of L-lysine; or a method for producing L-lysine.
According to the application and the method, the recombinant strain is adopted for fermentation, and the L-lysine is prepared. According to the application and method of the present invention, the recombinant strain of the present invention can be used alone, or can be used in combination with other L-lysine producing bacteria.
Advantageous effects
The recombinant strain is obtained by introducing point mutation into the promoter region of the lysC gene, and compared with the strain without mutation, the obtained strain greatly improves the yield of L-lysine, further improves the generation efficiency, reduces the generation cost and is convenient to popularize and apply.
Detailed Description
The present invention will be described in further detail with reference to examples. However, those skilled in the art will appreciate that the scope of the present invention is not limited to the following examples. In light of the present disclosure, those skilled in the art will recognize that many variations and modifications may be made to the embodiments described above without departing from the spirit and scope of the present invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples are all commercially available products or can be prepared by known methods; the manipulations performed are all known in the art or performed according to the user's manual of commercially available products.
Example 1 construction of transformation vector pK18-PlysC containing promoter region of point-mutated lysC Gene(G (-45)A,G(-47)T)
Two pairs of primers for amplifying sequences of promoter regions of lysC genes are designed and synthesized according to a genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, and point mutation is introduced in the promoter region (SEQ ID NO:1) of the lysC gene in the background of strain YP97158 (preservation number: CGMCC No.12856, preservation date: 2016, 8, 16, 2016, institute of microbiology, institute of China academy of sciences, No. 3 of Western No.1, telephone: 010-64807355) in Beijing, and-45 bp G and-47 bp G of the nucleotide sequence of the promoter region of the lysC gene are changed into A and-47 bp G and T (SEQ ID NO:2) in an allelic replacement manner.
The primers were designed as follows (synthesized by Shanghai Invitrogen corporation):
P1:5'CCGGAATTCG ACCAAGGATG AGGGCTTTG 3'(EcoR I)(SEQ ID NO:3)
P2:5'AGTTACCCGC TCAATTATAC CTTTATAAAC 3'(SEQ ID NO:4)
P3:5'GTTTATAAAG GTATAATTGAGCGGGTAACT 3'(SEQ ID NO:5)
P4:5'ACATGCATGCGCGTACGCGAAGTGGCACAT 3'(Sph I)(SEQ ID NO:6)。
the construction method comprises the following steps: using Corynebacterium glutamicum ATCC13032 as a template, and primers P1 and P2, and P3 and P4 respectively, and performing PCR amplification, wherein the PCR system comprises the following components: 10 XEx Taq Buffer 5. mu.L, dNTP mix (2.5 mM each) 4. mu.L, Mg2+4 μ L (25mM), 2 μ L each of primers (10pM), 0.25 μ L of Ex Taq (5U/. mu.L) in total volume of 50 μ L, and the PCR amplification was performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 40s, 30 cycles, and over-extension at 72 ℃ for 10min to obtain two DNA fragments (lysC promoter Up and lysC promoter Down fragments) containing point mutations and having lengths of 729bp and 760bp, respectively. The two DNA fragments were separated and purified by agarose gel electrophoresis, and then a fragment (Up-Down fragment) of about 1459bp in length was amplified by Overlap PCR using the two purified DNA fragments as templates and P1 and P4 as primers. And (3) PCR system: 10 XEx Taq Buffer 5. mu.L, dNTP mix (each2.5mM)4μL， Mg2+4 μ L (25mM), 2 μ L each of primers (10pM), 0.25 μ L of Ex Taq (5U/. mu.L) in total volume of 50 μ L, and the PCR amplification was performed as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 90s, 30 cycles, and over-extension at 72 ℃ for 10 min. And separating and purifying the Up-Down fragment by agarose gel electrophoresis, wherein the fragment comprises a lysC gene promoter region and upstream and downstream sequences thereof, and both ends of the fragment respectively contain EcoR I and Sph I enzyme cutting sites. This DNA fragment resulted in the change of the-45 bp nucleotide guanine (G) to adenine (A) and the-47 bp nucleotide guanine (G) to thymine (T) in the promoter region of the YP97158 lysC gene. The fragment was purified and recovered after double digestion (EcoR I/Sph I), and ligated with the shuttle plasmid pK18mobsacB (available from Addgene) after the same double digestion (EcoR I/Sph I) to obtain the allele substitution plasmid pK18-PlysC(G(-45)A,G(-47)T)The plasmid contains a kanamycin resistance marker. And the vector pK18-PlysC(G(-45)A,G(-47)T)Sending to a sequencing company for sequencing identification, and carrying out sequencing identification on the vector pK18-PlysC containing the correct point mutation(G(-45)A,G(-47)T)And (5) storing for later use.
Example 2 construction of PlysC comprising Point mutations(G(-45)A,G(-47)T)Of (4) an engineered strain
Substitution of the allele for the plasmid pK18-PlysC(G(-45)A,G(-47)T)Transformed into an L-lysine-producing bacterium patent strain YP97158 by electric shock (the construction method can be seen in WO2014121669A 1; the wild lysC gene promoter is reserved on the chromosome of the strain through sequencing confirmation), and the single colony generated by culture is identified by a primer P1 and a universal primer M13F respectively, so that the strain with a band of 1500bp is a positive strain. The positive strain was cultured on a medium containing 15% sucrose, and the single colonies resulting from the culture were cultured on a medium containing kanamycin and a medium not containing kanamycin, respectively, and the strains that grew on the medium not containing kanamycin were further identified by PCR using the following primers (synthesized by Shanghai Invitrogen Co.):
P5:5'ATCAATATATGGTCTGTTTA 3'(SEQ ID NO:7)
P6:5'CTTGGTGGCAACGATCCGTT 3'(SEQ ID NO:8)
the PCR amplification products were subjected to sscp electrophoresis (plasmid pK 18-PlysC) after denaturation at high temperature and ice-bath(G (-45)A,G(-47)T)The amplified fragment is a positive control, the YP97158 amplified fragment is a negative control, and water is used as a blank control), and the electrophoretic positions of the fragments are different due to different fragment structures, so that the strains of which the electrophoretic positions are inconsistent with the positions of the negative control fragments and the positive control fragments are strains of which allelic replacement is successful. The target fragment of the positive strain was amplified again by PCR and ligated to PMD19-T vector for sequencing, and the sequence in which the base sequence was mutated by sequence alignment confirmed that the strain was successfully substituted by allele, and was designated YPL-4-009.
The basic culture medium used for the above-mentioned culture has the same composition, and on the basic culture medium composition sucrose is added to 15%, or kanamycin is added, and the described basic culture medium composition is as follows:
| composition (I) | Formulation of |
| Sucrose | 10g/L |
| Polypeptone | 10g/L |
| Beef extract | 10g/L |
| Yeast powder | 5g/L |
| Urea | 2g/L |
| Sodium chloride | 2.5g/L |
| Agar powder | 20g/L |
| Ph | 7.0 |
| Culture conditions | 32 degree |
Preparation of PAGE for sscp electrophoresis
EXAMPLE 3L-lysine fermentation experiment
The strain YPL-4-009 and the original strain YP97158 constructed in example 2 were subjected to fermentation experiments in a BLBIO-5GC-4-H model fermenter (purchased from Bailan Biotech Co., Ltd., Shanghai) with the media shown in Table 1 and the control process shown in Table 2. Each strain was replicated three times, and the results are shown in Table 3.
TABLE 1 fermentation Medium formulation
| Composition (I) | Formulation of |
| Starch hydrolysis sugar | 30g/L |
| Ammonium sulfate | 12g/L |
| Magnesium sulfate | 0.87g/L |
| Molasses for health protection | 20g/L |
| Acidified corn steep liquor | 3mL/L |
| Phosphoric acid | 0.4mL/L |
| Potassium chloride | 0.53g/L |
| Defoaming agent (2% foam) | 4mL/L |
| Ferrous sulfate | 120mg/L |
| Manganese sulfate | 120mg/L |
| Nicotinamide | 42mg/L |
| Calcium pantothenate | 6.3mg/L |
| Vitamin B1 | 6.3mg/L |
| Solution of copper or zinc salt | 0.6g/L |
| Biotin | 0.88mg/L |
TABLE 2 fermentation control Process
TABLE 3 results of L-lysine fermentation experiments
The conversion rate is 100% of the total mass of lysine/total consumption of glucose
As a result, as shown in Table 3, point mutation of the lysC gene promoter in Corynebacterium glutamicum, PlysC(G (-45)A,G(-47)T)And contributes to the improvement of the yield of the L-lysine.
SEQUENCE LISTING
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Claims (10)
1. A promoter nucleotide sequence comprises a nucleotide sequence formed by mutating the base of the 45bp position and the 47bp position of a promoter region shown in SEQ ID NO. 1.
Preferably, the nucleotide guanine (G) at the-45 bp position of the promoter region shown in SEQ ID NO.1 is mutated to adenine (A), and the nucleotide guanine (G) at the-47 bp position is mutated to thymine (T).
2. The promoter nucleotide sequence according to claim 1, comprising:
(a) a nucleotide sequence shown as SEQ ID NO. 2; in the alternative, the first and second sets of the first,
(b) a nucleotide sequence having 90% or more, preferably 95%, 98% or more identity to the nucleotide sequence shown in SEQ ID NO. 2, and which retains the enhanced activity of the promoter of (a) and maintains adenine (A) at the-45 bp position and thymine (T) at the-47 bp position.
3. An expression cassette comprising the promoter nucleotide sequence of claim 1 or 2 and a coding sequence operably linked to the promoter nucleotide sequence. Preferably, the coding sequence is the coding sequence of the lysC gene.
4. A recombinant vector comprising the promoter nucleotide sequence of claim 1.
Preferably, the promoter nucleotide sequence is linked to a shuttle plasmid to construct the recombinant vector; preferably, the shuttle plasmid is the pK18mobsacB plasmid.
5. A recombinant strain comprising the promoter nucleotide sequence of claim 1 or the recombinant vector of claim 4. Preferably, it comprises the nucleotide sequence shown in SEQ ID NO. 2. Preferably, the recombinant strain comprises the nucleotide sequence shown in SEQ ID NO. 2 linked to the lysC gene coding sequence.
6. The recombinant strain according to claim 5, which is formed by introducing the promoter nucleotide sequence into a host strain; preferably, the host strain is corynebacterium glutamicum, for example YP 97158. Preferably, the recombinant strain is a pK18mobsacB plasmid as a vector.
7. A method for constructing a recombinant strain for producing L-lysine comprises the following steps:
(1) the promoter region shown as SEQ ID NO.1 is modified to mutate the-45 bp position and the-47 bp position to obtain the nucleotide sequence of the promoter region containing the mutation.
Preferably, the mutation is that the nucleotide guanine (G) at the-45 bp position of the promoter region shown in SEQ ID NO.1 is mutated into adenine (A), and the nucleotide guanine (G) at the-47 bp position is mutated into thymine (T). Specifically, the nucleotide sequence of the promoter region after mutation is shown as SEQ ID NO. 2. Preferably, the construction method further comprises the steps of:
(2) connecting the nucleotide sequence of the mutant promoter region with a plasmid to construct a recombinant vector;
(3) and (3) introducing the recombinant vector into a host strain to obtain the L-lysine-producing recombinant strain containing the mutant promoter region.
8. The building method according to claim 7, the step (1) comprising:
two pairs of primers for amplifying the promoter region of lysC gene were designed, and the nucleotide sequence of the mutant promoter region was obtained by PCR technique.
Preferably, the primers in step (1) are:
P1:5'CCGGAATTCG ACCAAGGATG AGGGCTTTG 3'(EcoR I)(SEQ ID NO:3)
P2:5'AGTTACCCGC TCAATTATAC CTTTATAAAC 3'(SEQ ID NO:4)
P3:5'GTTTATAAAG GTATAATTGA GCGGGTAACT 3'(SEQ ID NO:5)
P4:5'ACATGCATGC GCGTACGCGA AGTGGCACAT 3'(Sph I)(SEQ ID NO:6)。
preferably, the shuttle plasmid is a pK18mobsacB plasmid;
preferably, the introduction of step (3) is an electrical conversion method; exemplarily, in the step (3), the recombinant vector is transformed into the strain YP 97158.
9. A method for producing L-lysine comprising using the recombinant strain of claim 5.
10. Use of the promoter nucleotide sequence of claim 1, the expression cassette of claim 3, the recombinant vector of claim 4, or the recombinant strain of claim 5 for the preparation of L-lysine.
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| CN202010514037.1A CN111909944A (en) | 2020-06-08 | 2020-06-08 | Recombinant strain modified by lysC gene and construction method and application thereof |
| JP2022574296A JP2023528619A (en) | 2020-06-08 | 2020-12-31 | Recombinant strain producing L-amino acid, and construction method and use thereof |
| EP20939601.9A EP4163377A1 (en) | 2020-06-08 | 2020-12-31 | Recombinant strain for producing l-amino acid, construction method therefor, and application thereof |
| PCT/CN2020/142133 WO2021248902A1 (en) | 2020-06-08 | 2020-12-31 | Recombinant strain for producing l-amino acid, construction method therefor, and application thereof |
| KR1020227045499A KR20230042224A (en) | 2020-06-08 | 2020-12-31 | Recombinant strains producing L-amino acids and their construction methods and applications |
| US18/001,218 US20230313122A1 (en) | 2020-06-08 | 2020-12-31 | Recombinant strain for producing l-amino acid, construction method therefor, and application thereof |
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| WO2021248902A1 (en) * | 2020-06-08 | 2021-12-16 | 内蒙古伊品生物科技有限公司 | Recombinant strain for producing l-amino acid, construction method therefor, and application thereof |
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| CN117106783A (en) * | 2023-10-24 | 2023-11-24 | 齐齐哈尔龙江阜丰生物科技有限公司 | Recombinant strain with high lysine yield, construction method and application thereof |
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