CN116640791A - Construction method and application of recombinant microorganism - Google Patents
Construction method and application of recombinant microorganism Download PDFInfo
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- CN116640791A CN116640791A CN202310463896.6A CN202310463896A CN116640791A CN 116640791 A CN116640791 A CN 116640791A CN 202310463896 A CN202310463896 A CN 202310463896A CN 116640791 A CN116640791 A CN 116640791A
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- 244000005700 microbiome Species 0.000 title claims abstract description 25
- 238000010276 construction Methods 0.000 title claims abstract description 12
- 101150000475 pntAB gene Proteins 0.000 claims abstract description 49
- 239000004472 Lysine Substances 0.000 claims abstract description 36
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims abstract description 29
- 230000001105 regulatory effect Effects 0.000 claims abstract description 17
- 238000013518 transcription Methods 0.000 claims abstract description 17
- 230000035897 transcription Effects 0.000 claims abstract description 17
- 230000014509 gene expression Effects 0.000 claims abstract description 15
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 22
- 150000001413 amino acids Chemical group 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 229940024606 amino acid Drugs 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
- 241000186254 coryneform bacterium Species 0.000 claims description 4
- 238000003209 gene knockout Methods 0.000 claims description 4
- 230000035772 mutation Effects 0.000 claims description 4
- 101150073820 pntA gene Proteins 0.000 claims description 4
- 101150011666 pntB gene Proteins 0.000 claims description 4
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 claims description 4
- 241000186216 Corynebacterium Species 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 3
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- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 108091034117 Oligonucleotide Proteins 0.000 claims description 2
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 claims description 2
- 239000000074 antisense oligonucleotide Substances 0.000 claims description 2
- 238000012230 antisense oligonucleotides Methods 0.000 claims description 2
- 229940009098 aspartate Drugs 0.000 claims description 2
- 230000009368 gene silencing by RNA Effects 0.000 claims description 2
- 230000006801 homologous recombination Effects 0.000 claims description 2
- 238000002744 homologous recombination Methods 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 238000004904 shortening Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 12
- 230000002779 inactivation Effects 0.000 abstract description 8
- 235000003704 aspartic acid Nutrition 0.000 abstract description 5
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 241000186031 Corynebacteriaceae Species 0.000 abstract 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 39
- 235000018977 lysine Nutrition 0.000 description 25
- 238000000855 fermentation Methods 0.000 description 14
- 230000004151 fermentation Effects 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 13
- 239000012634 fragment Substances 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 238000012408 PCR amplification Methods 0.000 description 10
- 241000186226 Corynebacterium glutamicum Species 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 8
- 241001485655 Corynebacterium glutamicum ATCC 13032 Species 0.000 description 7
- 235000019766 L-Lysine Nutrition 0.000 description 7
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- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 3
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- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
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- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 2
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- 229920001817 Agar Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
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- 235000019764 Soybean Meal Nutrition 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000019730 animal feed additive Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 150000001510 aspartic acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
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- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
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- 239000008103 glucose Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
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- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
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- 239000004455 soybean meal Substances 0.000 description 1
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- 230000009466 transformation Effects 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- C12Y—ENZYMES
- C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
- C12Y106/01—Oxidoreductases acting on NADH or NADPH (1.6) with NAD+ or NADP+ as acceptor (1.6.1)
- C12Y106/01002—NAD(P)+ Transhydrogenase (AB-specific) (1.6.1.2)
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/265—Micrococcus
- C12R2001/28—Micrococcus glutamicus ; Corynebacterium glutamicum
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a construction method of recombinant microorganisms and application thereof. The construction method comprises the following steps: the expression level of a transcription regulatory factor Cg12680 in the microorganism is reduced, and the expression level of a transhydrogenase gene pntAB is improved. According to the research of the invention, the inactivation of the transcription regulatory factor Cg12680 can improve the lysine production capacity of the coryneform bacteria to a certain extent, and when the expression level of the transhydrogenase gene pntAB is improved, the lysine production capacity of the coryneform bacteria is further greatly improved, which has great significance in the production field of lysine or other aspartic acid families.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a construction method of recombinant microorganisms and application thereof.
Background
Aspartic acid and amino acids of the aspartic acid family have wide application in the fields of feed, food, medicine, chemical industry and the like. Among them, L-lysine is an essential basic amino acid and is widely used in animal feed, medicine and food industries. The L-lysine can promote the animal body to absorb other amino acids when being used as an animal feed additive, thereby improving the quality of the feed. Therefore, it is important to improve the productivity of L-lysine.
At present, the most commonly used production method of L-lysine is a microbial fermentation method. Corynebacterium glutamicum (Corynebacterium glutamicum) is a gram-positive microorganism with the characteristics of fast growth rate, non-pathogenic, and weak ability to degrade self-metabolites. As a conventional industrial microorganism, corynebacterium glutamicum is widely used for the production of various amino acids, nucleotides and other organic acids.
From a biochemical point of view, lysine belongs to the amino acid family of aspartic acids. Corynebacterium glutamicum undergoes a multi-step enzymatic reaction to obtain lysine, and the metabolic pathway involves the regulation of multiple genes. Corynebacterium glutamicum requires the consumption of 4 moles of NADPH for the synthesis of 1 mole of lysine, however, the lack of intracellular NADPH levels limits further increases in lysine production. Thus, increasing intracellular NADPH levels is important for increased lysine production. However, it is still difficult to sufficiently efficiently increase lysine productivity by only this approach.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a construction method of recombinant microorganisms and application thereof.
In a first aspect, the present invention provides a method of constructing a recombinant microorganism comprising:
the expression level of a transcription regulatory factor Cg12680 in the microorganism is reduced, and the expression level of a transhydrogenase gene pntAB is improved.
Further, the transcription regulating factor Cg12680 comprises an amino acid sequence shown as SEQ ID NO. 1; the pntA protein coded by the transhydrogenase gene pntAB comprises an amino acid sequence shown as SEQ ID NO.2, and the coded pntB protein comprises an amino acid sequence shown as SEQ ID NO. 3.
Further, the coding gene of the transcription control factor Cg12680 comprises a nucleotide sequence shown as SEQ ID NO. 4; the pntA coding gene in the transhydrogenase gene pntAB comprises a nucleotide sequence shown as SEQ ID NO.5, and the pntB coding gene comprises a nucleotide sequence shown as SEQ ID NO. 6.
Further, the reducing the expression level of the transcription regulatory factor Cg12680 in the microorganism comprises any one or more of the following:
i) RNA interference;
ii) mutation of the gene;
iii) Gene knockout technology;
v) introducing an antisense oligonucleotide;
the improvement of the expression level of the transhydrogenase gene pntAB comprises any one or more of the following modes:
i) Increasing the copy number;
ii) mutation of the gene;
iii) Replacing the strong promoter;
iv) shortening the distance between the promoter and the coding gene.
Further, the reduction of the expression level of the transcription regulatory factor Cg12680 in the microorganism is realized by gene knockout through homologous recombination technology, and the primer pair comprises:
Cg12680-1f：5’-cgggatccttgaccgtgactacacccc-3’；
Cg12680-1r：5’-ggtgcctctttaatgggc-3’；
Cg12680-2f：5’-ccggcccattaaagaggcaccggacgtcactttccacgag-3’；
Cg12680-2r：5’-cggctagccctggggagatgaccatcaat-3’。
further, the improvement of the expression level of the transhydrogenase gene pntAB is by using a promoter as shown in SEQ ID NO. 20.
Further, the microorganism comprises one or more of corynebacterium, escherichia coli or bacillus subtilis; preferably a coryneform bacterium.
In a second aspect, the invention provides recombinant microorganisms constructed by the construction method.
In a third aspect, the invention provides a method of constructing the recombinant microorganism, and the use of the recombinant microorganism to increase the ability of the microorganism to produce amino acids or derivatives thereof.
Further, the amino acid is an amino acid of the aspartate family; lysine is preferred.
The invention has the following beneficial effects:
according to the research of the invention, the inactivation of the transcription regulatory factor Cg12680 of the coryneform bacterium can improve the lysine production capacity to a certain extent, and after the coding gene of the transhydrogenase pntAB is further over-expressed, the lysine production capacity of the coryneform bacterium is obviously further improved, which has important significance in the field of lysine production, and the method is also suitable for other compounds taking aspartic acid as a precursor.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The names and sequences of the primers involved in the examples are shown in Table 1.
TABLE 1 primer sequences
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
The embodiment of the invention relates to corynebacterium lisurigenes CGMCC No.13407, which is disclosed in Chinese patent CN106635944A, and CGMCC No.11942, which is disclosed in Chinese patent CN105734004B. Corynebacterium glutamicum ATCC 13032 is a strain of Corynebacterium glutamicum model, well known in the art, available from public sources, and genomic sequences have been published, and related information can be queried from NCBI website.
Example 1
1. Inactivation of Cg12680 in model bacteria ATCC 13032
(1) Performing PCR amplification by using a Corynebacterium glutamicum ATCC 13032 genome as a template and a DeltaCg 12680-1 f/DeltaCg12680-1 r primer pair to obtain an upstream homologous arm fragment 13032-DeltaCg 12680-up; the Corynebacterium glutamicum ATCC 13032 genome is used as a template, and a DeltaCg 12680-2 f/DeltaCg12680-2 r primer pair is used for PCR amplification to obtain a downstream homologous arm fragment 13032-DeltaCg 12680-dn.
(2) PCR amplification is carried out by taking a mixture of two fragments of 13032-DeltaCg 12680-up and 13032-DeltaCg 12680-dn as templates and a primer pair of DeltaCg 12680-1 f/DeltaCg12680-2 r to obtain fragments 13032-up-dn, and the 13032-up-dn is subjected to double digestion by BamHI and NheI, and a vector pK18mobsacB (GenBank: FJ437239.1; purchased from a teacher of Shanghai life sciences of China academy of sciences) is subjected to double digestion by the same enzyme. The two digested products were ligated with T4 DNA Ligase, and Trans 1T 1 competent cells were transformed to obtain recombinant plasmid pK18 mobsacB-. DELTA.Cg 12680.
(3) Competent cells of ATCC 13032 were prepared according to Corynebacterium glutamicum Handbook (C.glutamicum Handbook, charpter 23). The recombinant plasmid pK18 mobsacB-. DELTA.Cg 12680 was transformed into the competent cells by electroporation and transformants were selected on BHI selection medium containing 15mg/L kanamycin. The obtained transformant was cultured overnight in a common BHI liquid medium at a temperature of 33℃and shaking-cultured at 220rpm with a shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) Thin, thinThe release solution was spread on a normal BHI solid medium containing 10% sucrose, and was allowed to stand at 33℃for 48 hours. The grown strain does not carry the inserted vector sequence in its genome. The strain with inactivated Cg12680 was obtained by PCR amplification of the sequence of interest and nucleotide sequencing analysis and was designated 13032-DeltaCg 12680.
2. Overexpression of pntAB in 13032
(1) Performing PCR amplification by using a Corynebacterium glutamicum ATCC 13032 genome as a template and a pntAB-1f/pntAB-1r primer pair to obtain an upstream homologous arm fragment 13032-pntAB-up; the downstream homology arm fragment 13032-pntAB-dn is obtained by PCR amplification using Corynebacterium glutamicum ATCC 13032 genome as a template and a pntAB-2f/pntAB-2r primer pair. The mutant promoter Ppyc 1 and the codon optimized pntAB nucleotide sequence were synthesized by Jin Weizhi Biotechnology Inc. (China) (nucleotide sequence of mutant promoter Ppyc 1: ttaagaggtaagatgtctgcaggtggaagcgtttaaatgcgttaaacttggccaaatgtggcaacctttgcaaggtgaaaaactggggcggggttagatcctggggggtttatttcattcactttggcttgaagtcgtgcaggtcaggggagtgttgcccgaaaacattgagaggaaaacaaaaaccgatgtttgattgggggaatcgtgtggtataatggtaggacgcagtgactgctatcacccttggcggtctcttgttgaaaggaataattactcta).
(2) The four fragment mixtures of 13032-pntAB-up, 13032-pntAB-dn, ppyc 1 and pntAB are used as templates, a pntAB-1f/pntAB-2r primer pair is used for PCR amplification to obtain fragments 13032-up-pntAB-dn, the 13032-up-pntAB-dn is subjected to double digestion with BamHI and NheI, and a vector pK18mobsacB (GenBank: FJ437239.1; obtained from a national academy of sciences of China, a benefit of a national academy of life Yang) is purchased from public channels, and the same enzymes are used for double digestion. The two digested products were ligated with T4 DNA Ligase, and Trans 1T 1 competent cells were transformed to obtain recombinant plasmid pK18mobsacB-pntAB.
Referring to the method of example 1, the constructed recombinant plasmid pK18mobsacB-pntAB was transferred into ATCC 13032 to obtain a recombinant strain overexpressing pntAB, which was designated as 13032-pntAB.
3. Inactivation of Cg12680 in 13032 while overexpressing pntAB
Performing PCR amplification by using a Corynebacterium glutamicum ATCC 13032 genome as a template and a DeltaCg 12680-1 f/DeltaCg12680-1 r primer pair to obtain an upstream homologous arm fragment 13032-DeltaCg 12680-up; the downstream homology arm fragment 13032-Cg12680:: pntAB-dn was obtained by PCR amplification using Corynebacterium glutamicum ATCC 13032 genome as a template and a pntAB-2 f/. DELTA.Cg12680-2 r primer pair. The mutant promoter Ppyc 1 and codon optimized pntAB nucleotide sequence were synthesized by Jin Weizhi biotechnology limited (china).
The mixture of four fragments including 13032-DeltaCg 12680-up, 13032-Cg12680:: pntAB-dn, ppyc 1 and pntAB was used as a template, and the PCR amplification was performed with the primer pair of DeltaCg 12680-1 f/DeltaCg12680-2 r to obtain fragment 13032-up-Cg12680:: pntAB-dn, and 13032-up-Cg 12680::: pntAB-dn was digested with BamHI and NheI, and vector pK18mobsacB (GenBank: FJ437239.1; available from the national academy of sciences of China, shanghai sciences of life Yang) was digested with the same enzymes. The two enzyme digestion products are connected by T4 DNA Ligase, and Trans 1T 1 competent cells are transformed to obtain recombinant plasmid pK18mobsacB-Cg 12680:pntAB.
Referring to the procedure of example 1, the constructed recombinant plasmid pK18mobsacB-Cg12680:: pntAB was transferred into ATCC 13032 to obtain a recombinant strain in which Cg12680 was inactivated and pntAB was overexpressed, and the strain was designated as 13032-Cg 12680::: pntAB.
4. Shake flask test of recombinant strain fermentation Performance
The culture medium used for lysine fermentation experiments was as follows:
seed activation medium: BHI 37g/L,18g/L agar powder.
Seed culture medium: 20g/L of sucrose, 5g/L of yeast powder, 10g/L of peptone, 5g/L of urea and 0.4g/L of magnesium sulfate heptahydrate, and adjusting the pH value to 7.0.
Fermentation medium: 60g/L of glucose, 25g/L of ammonium sulfate, 2.0g/L of monopotassium phosphate, 1.0g/L of magnesium sulfate heptahydrate, 10g/L of soybean meal hydrolysate and 30g/L of calcium carbonate, and adjusting the pH value to 7.0.
The lysine fermentation method comprises the following steps:
(1) Seed activation: taking the strain to be verified from the freezing tube, streaking and activating on a seed activation culture medium, and culturing at 33 ℃ for 24 hours;
(2) Seed culture: the plate activated seeds 1 are picked and looped into a 500mL triangular flask filled with 30mL seed culture medium, and shake culture is carried out for 6h at 33 ℃ and 220 r/min;
(3) Fermentation culture: 2mL of the seed solution is inoculated into a 500mL triangular flask filled with 20mL of fermentation medium, and the culture is carried out for 14-15h at 33 ℃ under 220r/min in a shaking way, and three strains are arranged in parallel.
(4)OD 562 And (3) measuring: diluting 100 μl of fermentation broth by a proper multiple, detecting OD at wavelength 562 with spectrophotometer, performing three parallels for each strain, calculating average value, and detecting OD 562 As shown in table 2.
(5) Lysine concentration measurement: 2mL of the fermentation broth was centrifuged (12000 rpm,2 min), the supernatant was collected, the L-lysine content in the fermentation broth of the recombinant bacteria and the control bacteria was measured by HPLC, three bacteria were used in parallel, and the average value was calculated, and the measured lysine concentration was shown in Table 2.
TABLE 2 lysine production and growth assays for recombinant strains
| Strain | L-lysine (g/L) | Sugar acid conversion% | OD 562 |
| ATCC 13032 | 0.52 | 0.87％ | 40.5 |
| 13032-△Cg12680 | 0.60 | 1.00％ | 39.4 |
| 13032-pntAB | 0.59 | 0.98％ | 39.8 |
| 13032-Cg12680::pntAB | 0.70 | 1.17％ | 39.7 |
The lysine content in the fermentation broth is detected, and the lysine concentration in the culture broth of corynebacterium glutamicum carrying the inactivated transcription regulatory factor Cg12680 and the pntAB is higher, but the recombinant strain of the inactivated transcription regulatory factor Cg12680 and the pntAB is higher than the recombinant strain of the inactivated transcription regulatory factor Cg12680 and the pntAB, and the recombinant strain is higher in lysine concentration and higher than the lifting effect of the inactivated transcription regulatory factor Cg12680 and the pntAB.
Example 2
The present example further demonstrates the effect of transformation on lysine-producing bacteria. The verification is carried out by using 2 lysine-producing bacteria CGMCC No.13407 and CGMCC No.11942 preserved in a laboratory. The CGMCC No.13407 and CGMCC No.11942 are obtained by the gene modification of the model bacterium ATCC 13032, and the lysine synthesis related genes are enhanced and modified, and the specific flow is as follows:
1. inactivation of Cg12680 in CGMCC No.13407
Referring to the method of example 1, the constructed recombinant plasmid pK18 mobsacB-DeltaCg 12680 was transferred into CGMCC No.13407 to obtain a recombinant strain of inactivated Cg12680, which was named 13407-DeltaCg 12680.
2. Overexpression of pntAB in CGMCC No.13407
Referring to the method of example 1, the constructed recombinant plasmid pK18mobsacB-pntAB was transferred into CGMCC No.13407 to obtain a recombinant strain overexpressing pntAB, which was named 13407-pntAB.
3. Inactivation of Cg12680 in CGMCC No.13407 while overexpressing pntAB
Referring to the method of example 1, the constructed recombinant plasmid pK18mobsacB-Cg12680:: pntAB was transferred into CGMCC No.13407 to obtain a recombinant strain with inactivated Cg12680 and overexpressed pntAB, which was named 13407-Cg 12680::: pntAB.
4. Inactivation of Cg12680 in CGMCC No.11942
Referring to the method of example 1, the constructed recombinant plasmid pK18 mobsacB-DeltaCg 12680 was transferred into CGMCC No.11942 to obtain a recombinant strain of inactivated Cg12680, which was named 11942-DeltaCg 12680.
5. Overexpression of pntAB in CGMCC No.11942
Referring to the method of example 1, the constructed recombinant plasmid pK18mobsacB-pntAB was transferred into CGMCC No.11942 to obtain a recombinant strain overexpressing pntAB, which was named 11942-pntAB.
6. Inactivation of Cg12680 in CGMCC No.11942 while overexpressing pntAB
Referring to the method of example 1, the constructed recombinant plasmid pK18mobsacB-Cg12680:: pntAB was transferred into CGMCC No.11942 to obtain a recombinant strain with inactivated Cg12680 and overexpressed pntAB, which was named 11942-Cg 12680::: pntAB.
7. Shake flask test of recombinant strain fermentation Performance
Fermenting and verifying CGMCC No.13407, CGMCC No.11942 and recombinant bacteria 13407-DeltaCg 12680, 13407-Cg12680, pntAB, 11942-DeltaCg 12680, 11942-Cg12680, pntAB, 13407-pntAB and 11942-pntAB modified by the same according to the method of the embodiment 3. The lysine fermentation test results are shown in Table 3.
TABLE 3 lysine production and growth assays for recombinant strains
| Strain | L-lysine (g/L) | Sugar acid conversion% | OD 562 |
| CGMCC No.13407 | 15.2 | 25.3％ | 49.0 |
| 13407-△Cg12680 | 16.6 | 27.7％ | 47.7 |
| 13407-pntAB | 15.8 | 26.3％ | 46.9 |
| 13407-Cg12680::pntAB | 17.8 | 29.7％ | 47.9 |
| CGMCC No.11942 | 18.0 | 30.0％ | 39.4 |
| 11942-△Cg12680 | 19.5 | 32.5％ | 38.7 |
| 11942-pntAB | 18.5 | 30.8％ | 39.1 |
| 11942-Cg12680::pntAB | 20.5 | 34.2％ | 38.9 |
The fermentation results show that the same batch of 2 lysine-producing bacteria, the modified bacteria carrying the inactivated transcription regulatory factor Cg12680, the modified bacteria carrying the over-expressed pntAB, the modified bacteria carrying the inactivated transcription regulatory factor Cg12680 and the over-expressed transhydrogenase pntAB are obviously improved in lysine yield and sugar acid conversion rate, and the modified bacteria carrying the inactivated transcription regulatory factor Cg12680 and the over-expressed transhydrogenase pntAB have better effect and are higher than the lifting effect of the independent inactivated transcription regulatory factor Cg12680 and the independent over-expressed transhydrogenase pntAB.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method of constructing a recombinant microorganism, comprising:
the expression level of a transcription regulatory factor Cg12680 in the microorganism is reduced, and the expression level of a transhydrogenase gene pntAB is improved.
2. The method of claim 1, wherein the transcription control factor Cg12680 comprises the amino acid sequence shown in SEQ ID No. 1; and/or the pntA protein coded by the transhydrogenase gene pntAB comprises an amino acid sequence shown as SEQ ID NO.2, and the coded pntB protein comprises an amino acid sequence shown as SEQ ID NO. 3.
3. The construction method according to claim 2, wherein the gene encoding the transcription control factor Cg12680 comprises a nucleotide sequence shown as SEQ ID No. 4; and/or the pntA coding gene in the transhydrogenase gene pntAB comprises a nucleotide sequence shown as SEQ ID NO.5, and the pntB coding gene comprises a nucleotide sequence shown as SEQ ID NO. 6.
4. A method of construction according to any one of claims 1 to 3, wherein said reducing the expression level of the transcription regulatory factor Cg12680 in the microorganism comprises any one or more of the following:
i) RNA interference;
ii) mutation of the gene;
iii) Gene knockout technology;
v) introducing an antisense oligonucleotide;
and/or, said increasing the expression level of the transhydrogenase gene pntAB comprises any one or more of the following:
i) Increasing the copy number;
ii) mutation of the gene;
iii) Replacing the strong promoter;
iv) shortening the distance between the promoter and the coding gene.
5. The method of any one of claims 1 to 4, wherein the reduction of the expression level of the transcription control factor Cg12680 in the microorganism is achieved by gene knockout by homologous recombination techniques using a primer pair comprising:
Cg12680-1f：5’-cgggatccttgaccgtgactacacccc-3’；
Cg12680-1r：5’-ggtgcctctttaatgggc-3’；
Cg12680-2f：5’-ccggcccattaaagaggcaccggacgtcactttccacgag-3’；
Cg12680-2r：5’-cggctagccctggggagatgaccatcaat-3’。
6. the method of any one of claims 1 to 5, wherein the increasing the expression level of the transhydrogenase gene pntAB is by using a promoter as shown in SEQ ID NO. 20.
7. The method of construction according to any one of claims 1 to 6, wherein the microorganism comprises one or more of corynebacterium, escherichia coli, or bacillus subtilis; preferably a coryneform bacterium.
8. Recombinant microorganism constructed by the construction method according to any one of claims 1 to 7.
9. Use of the construction method according to any one of claims 1 to 7, or the recombinant microorganism according to claim 8, for increasing the ability of the microorganism to produce an amino acid or a derivative thereof.
10. The use according to claim 9, wherein the amino acid is an amino acid of the aspartate family; lysine is preferred.
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