CN116904455A - Promoter, recombinant microorganism and application thereof - Google Patents
Promoter, recombinant microorganism and application thereof Download PDFInfo
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- CN116904455A CN116904455A CN202111265985.7A CN202111265985A CN116904455A CN 116904455 A CN116904455 A CN 116904455A CN 202111265985 A CN202111265985 A CN 202111265985A CN 116904455 A CN116904455 A CN 116904455A
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- Prior art keywords
- promoter
- tkt
- gene
- strain
- synthesis
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- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/24—Proline; Hydroxyproline; Histidine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
- C12Y202/01—Transketolases and transaldolases (2.2.1)
- C12Y202/01001—Transketolase (2.2.1.1)
Abstract
The invention relates to the technical field of biology, in particular to a promoter, recombinant microorganism and application thereof. The invention provides a promoter having a sequence as shown in SEQ ID NO1, a nucleotide sequence shown in the specification. The promoter has higher transcription promoting activity than endogenous strong promoter P of corynebacterium glutamicum sod Obviously higher, can drive the gene to be expressed in the microorganism with high efficiency. The natural promoter of the tkt gene is replaced by the promoter, so that the expression of the tkt gene is obviously enhanced, the yield and conversion rate of lysine and proline of the recombinant bacterium constructed by the promoter are obviously improved, and the efficient production of synthesizing the amino acid with the need of reducing power can be promoted.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a promoter, recombinant microorganism and application thereof.
Background
Promoters are DNA sequences that RNA polymerase recognizes, binds to, and initiates transcription, containing sequences required for specific binding and transcription initiation by RNA polymerase. In the biosynthesis process, the promoter is an important element and tool for regulating gene expression, and therefore, development of a high-efficiency promoter is of great importance.
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 organic acids. The history of the use of Corynebacterium glutamicum for the production of amino acids can be traced back to the 60 th century, and as found by Japanese company, corynebacterium glutamicum can produce glutamic acid in natural environment, and the mutagenized Corynebacterium glutamicum can also produce various amino acids such as lysine and valine. Along with the rapid development of metabolic engineering technology and genome sequencing technology, the metabolic path research of corynebacterium glutamicum is clearer, researchers sequentially identify and obtain high-yield mechanisms of various metabolites in genetics, and high-efficiency production of various metabolites is realized.
Efficient production of amino acids can be achieved by a number of routes, for example: relieving feedback inhibition of the product, strengthening key enzymes of terminal synthesis pathways, strengthening cofactor supply, optimizing carbon flow distribution of central metabolism, blocking competition branches and the like. For enhanced expression of genes, conventional strategies are mainly to introduce multiple copies of the gene, or to replace the natural promoter of the gene of interest with a known strong promoter. Currently, the endogenous strong promoters of Corynebacterium glutamicum are commonly used, mainly the promoter encoding the superoxide dismutase gene (sod) and the promoter encoding the elongation factor (tuf). However, these general promoters have a limited number and a fixed expression strength, and thus are far from meeting the requirements of genetic engineering techniques for promoters. In addition, the expression strength of a promoter is also significantly affected by the base sequence and length of the upstream and downstream of the core region, in addition to the base sequence of the core region, on the expression of a gene, which is related to the complicated regulatory mechanism of the gene. Therefore, it is necessary to develop a novel promoter for expression control of a target gene of a target metabolic pathway.
Disclosure of Invention
It is an object of the present invention to provide a promoter having a high transcription initiation activity. Another object of the present invention is to provide a recombinant microorganism containing the promoter and its use.
Specifically, the invention provides the following technical scheme:
the invention provides a promoter which has a nucleotide sequence shown as SEQ ID NO. 1.
The promoter is a mutant promoter obtained by mutation on the basis of a promoter of transketolase (tkt) of corynebacterium glutamicum, and the nuclear promoter has obviously higher activity than a natural promoter of unmutated tkt and can drive the efficient expression of a target gene in microorganisms.
The promoter of the present invention refers to an untranslated nucleic acid sequence located upstream of a gene coding region, which contains at least an RNA polymerase binding site and has an activity of initiating transcription of a downstream gene into mRNA. Also included in the sequence may be 5' untranslated regions, which are the binding regions for certain transcription factors or regulatory factors.
In addition to the above promoters, the present invention provides expression cassettes containing the promoters.
The expression cassette described above may be a recombinant DNA obtained by operably linking a gene of interest downstream of the promoter, or a recombinant DNA obtained by operably linking a gene of interest upstream or downstream of the promoter, other transcription regulatory elements, or translation regulatory elements.
The invention also provides a vector containing the promoter or the expression cassette.
The vectors described above may be expression vectors or cloning vectors, including but not limited to plasmid vectors, phage vectors, transposons, and the like.
The invention also provides a host cell containing the promoter, the expression cassette or the vector.
The host cells described above include, but are not limited to, microbial cells.
The microbial cells are preferably coryneform or Brevibacterium bacteria. Among them, the coryneform bacterium is preferably a bacterium selected from the group consisting of Corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium beijing (Corynebacterium pekinense), corynebacterium validum (Corynebacterium efficiens), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes) and Corynebacterium ammoniagenes (Corynebacterium aminogenes). The Brevibacterium bacteria are preferably bacteria selected from Brevibacterium flavum (Breviabacterium flavum) or Brevibacterium lactofermentum (Brevibacterium lactofermentum).
Based on the function of the promoter provided by the invention, the invention provides any one of the following applications of the promoter, the expression cassette, the vector or the host cell:
(1) Use in driving gene expression;
(2) Use in enhancing gene expression;
(3) The application in improving the yield and conversion rate of the microbial metabolites;
(4) The application in breeding the production strain of the microorganism metabolite;
(5) Use in enhancing the reductive synthesis of microorganisms;
(6) The application in fermentation production of microbial metabolites.
The use described in (1) above is in particular to drive gene expression in microorganisms.
The use as described in (2) above is specifically to enhance gene expression in microorganisms.
In the above (3), (4) and (6), the metabolite may be any metabolite that can be synthesized by a microorganism, including but not limited to amino acids, organic acids, nucleosides, and the like.
In the above (5), the reducing force is preferably NADPH.
The microorganism mentioned above is preferably a coryneform bacterium. More preferably Corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium beijing (Corynebacterium pekinense), corynebacterium validum (Corynebacterium efficiens), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium ammoniagenes thermophilum (Corynebacterium thermoaminogenes) or Corynebacterium ammoniagenes (Corynebacterium aminogenes).
The present invention provides a recombinant microorganism having the promoter in an intergenic region upstream of a transketolase gene (tkt).
The transketolase gene (tkt) is an important gene involved in the production of reducing power in microorganisms. The invention discovers that compared with the natural promoter of the tkt gene, the promoter provided by the invention has higher activity. The natural promoter region of the tkt gene is replaced by the promoter, so that the expression of the tkt gene can be obviously enhanced, and the synthesis of metabolic products which consume reducing power and are needed for synthesis of lysine, proline, threonine, isoleucine and the like can be promoted.
The above-mentioned region between the upstream genes of the transketolase gene is specifically a nucleotide sequence between the start codon of the transketolase gene on the chromosome of the microorganism and the coding region of the upstream gene.
Preferably, in said recombinant microorganism, the transketolase gene is transcribed driven by said promoter provided by the present invention.
The expression level of transketolase of the recombinant microorganism is significantly increased as compared with that before the introduction of the promoter, thereby promoting the synthesis and accumulation of metabolites requiring the consumption of reducing power for synthesis or metabolites whose synthesis precursors are metabolites of pentose phosphate pathway, including but not limited to lysine, proline, threonine, isoleucine or derivatives thereof.
The invention also provides a construction method of the recombinant microorganism, which comprises the following steps: the natural promoter of the transketolase gene of the original strain is replaced by the promoter provided by the invention.
Preferably, the starting strain is a strain capable of accumulating metabolites that require consumption of reducing power for synthesis or for synthesis of precursors to metabolites of the pentose phosphate pathway.
The above-described metabolites requiring the consumption of reducing power for synthesis include, but are not limited to, lysine, proline, threonine, isoleucine, etc.
The synthetic precursors described above are metabolites of pentose phosphate pathway metabolites including, but not limited to, aromatic amino acids (tryptophan, phenylalanine, tyrosine), histidine, and the like.
As an implementation mode of the invention, the starting strain is lysine-producing bacteria, and the preservation number of the starting strain is CGMCC No.13407. The strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at 11 and 30 days of 2016, and has the address of China academy of sciences of China including national academy of sciences of China No.3, which is the Korean area of Beijing, and the classification designation: corynebacterium glutamicum, corynebacterium glutamicum, which strain is disclosed in patent CN 106635944A. For the strain, the promoter provided by the invention is adopted to drive the expression of the transketolase coding gene, and the lysine yield and the conversion rate of the modified strain are obviously improved.
As another embodiment of the invention, the starting strain is proline producing strain, and the preservation number of the starting strain is CGMCC No.13757. The strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) in 3 months and 15 days of 2017, and has the address of China academy of sciences of China including national academy of sciences of China No.3, including North Chen West Lu 1, chat, beijing, and the classification designation: corynebacterium glutamicum, corynebacterium glutamicum, which strain is disclosed in patent CN 107227283B. For the strain, the promoter provided by the invention is adopted to drive the expression of the transketolase coding gene, and the proline yield and the conversion rate of the modified strain are obviously improved.
The invention provides any one of the following applications of the recombinant microorganism:
(1) The application in the fermentation production of microbial metabolites;
(2) The application in breeding the production strain of the microorganism metabolite.
The above-mentioned microbial metabolites are metabolites whose synthesis requires consumption of reducing power, or metabolites whose synthesis precursors are metabolites of pentose phosphate pathway.
The above-described metabolites requiring the consumption of reducing power for synthesis include, but are not limited to, lysine, proline, threonine, isoleucine, etc.
The synthetic precursors described above are metabolites of pentose phosphate pathway metabolites including, but not limited to, aromatic amino acids (tryptophan, phenylalanine, tyrosine), histidine, and the like.
The derivatives mentioned above include, but are not limited to, trifluoroacetyl lysine, acetyl-L-threonine, N-acetyl-L-isoleucine, hydroxyproline, and the like.
The present invention also provides a method for fermentatively producing an amino acid or a derivative thereof, the method comprising: culturing the recombinant microorganism, and isolating the amino acid or derivative thereof from the culture.
Preferably, the amino acid is an amino acid whose synthesis requires consumption of reducing power, or an amino acid whose synthesis precursor is a pentose phosphate pathway metabolite.
Amino acids requiring reducing power for the synthesis described above include, but are not limited to, lysine, proline, threonine, isoleucine, and the like.
Amino acids for which the synthetic precursors described above are metabolites of the pentose phosphate pathway include, but are not limited to, aromatic amino acids (tryptophan, phenylalanine, tyrosine), histidine, and the like.
Specifically, the method for producing an amino acid or a derivative thereof by fermentation comprises: inoculating the recombinant microorganism into a seed culture medium for seed culture to obtain seed liquid, inoculating the seed liquid into a fermentation culture medium for culture to obtain fermentation liquor, and separating and extracting the fermentation liquor to obtain amino acid or derivatives thereof.
The present invention also provides a method for expressing a target gene, comprising: operably linking a target gene or a DNA fragment upstream and downstream thereof with the promoter to obtain a recombinant DNA, introducing the recombinant DNA into a host cell, and expressing the target gene.
Specifically, the gene of interest is operably linked downstream of the promoter; alternatively, the DNA fragments upstream and downstream of the gene of interest on the chromosome are operably linked upstream and downstream of the promoter, respectively.
The target gene of the invention can be any protein coding gene (such as amino acid synthesis related genes and the like) or RNA molecule (such as interfering RNA molecules, regulatory RNA molecules, sgRNA and the like).
The invention has the beneficial effects that: the promoter provided by the invention has higher transcription initiation activity, and the promoter activity is higher than that of an endogenous strong promoter P of corynebacterium glutamicum sod Obviously higher, can drive the gene to be expressed in the microorganism with high efficiency. The natural promoter of the tkt gene is replaced by the promoter, so that the expression of the tkt gene is obviously enhanced, the yield and conversion rate of lysine and proline of the recombinant bacterium constructed are obviously improved, and the efficient production of synthesizing amino acids (lysine, proline, threonine, isoleucine and the like) with the need of reducing power can be promoted.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The following examples are not to be construed as limiting the specific techniques or conditions set forth in the art, or as per the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
The strain with the preservation number of CGMCC No.13407 used in the following examples was preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 11 and 30 days in 2016, and has the address of China academy of sciences of China, national institute of sciences of China, no.3, north Chen West Lu 1, korea, beijing, and the classification designation: corynebacterium glutamicum, corynebacterium glutamicum, which strain is disclosed in patent application CN 106635944A.
The strain with the preservation number of CGMCC No.13757 used in the following examples has been preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 3 months 15 days in 2017, and has an address of China institute of microbiological culture Collection, national academy of sciences, national institute of sciences, 1 st Caragana, 3 rd, beijing, chaoyang, and classification designation: corynebacterium glutamicum, corynebacterium glutamicum, which strain is disclosed in patent CN 107227283B.
The primer sequence information used in the following examples is shown in Table 1.
TABLE 1 primer sequence information
Example 1 promoter and Activity detection thereof
This example provides a promoter having a nucleotide sequence shown in SEQ ID NO. 1. The promoter is a mutant transketolase gene (tkt) promoter (P tkt* ). The activity of the promoter was examined as follows:
1. construction of Green fluorescent protein expression plasmid
(1) Mutant promoter P tkt* Is synthesized by Jin Weizhi Biotechnology Co., ltd (China). PCR amplification is carried out by taking the Corynebacterium glutamicum ATCC 13032 genome as a template and Psod-f/Psod-r and Ptkt-f/Ptkt-r primer pairs to respectively obtain a sod gene promoter P sod (SEQ ID NO. 3) and the native promoter P of the tkt gene tkt (SEQ ID NO. 2). PCR amplification was performed using the pEF-GFP plasmid (Shanghai Korea Biotechnology Co., ltd., china) as a template and a GFP-f/GFP-r primer pair to obtain the open reading frame (ORF, SEQ ID NO. 5) of the GFP gene.
(2) The GFP gene was digested with SacI and EcoRI, and vector pEKEx2 (GenBank: AY585307.1; available from the Shanghai Seisakusho of China academy of sciences of life Yang, or purchased from a commercial source) 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 the recombinant plasmid pEKEx2-GFP without promoter before GFP gene.
(3) P obtained in the step (1) tkt* 、P sod And P tkt The fragment was digested with BamHI and KpnI, and vector pEKEx2-GFP was digested with the same enzymes. The two enzyme cutting products are connected by T4 DNA Ligase to transform Trans 1T 1 competent cells to obtain recombinant plasmid pEKEx2-P respectively tkt* -GFP、pEKEx2-P sod- GFP and pEKEx2-P tkt -GFP。
2. Construction of Green fluorescent protein expression Strain
(1) Competent cells of CGMCC No.13407 and ATCC 13032 were prepared according to the Corynebacterium glutamicum Handbook (C.glutamicum Handbook, charpter 23). Recombinant plasmid pEKEx2-GFP, pEKEx2-P tkt* -GFP、pEKEx2-P sod GFP and pEKEx2-P tkt GFP was electroporated to transform competent cells and transformants were selected on BHI selection medium containing 15mg/L kanamycin.
(2) Amplifying target sequence by PCR and performing nucleotide sequencing analysis to obtain recombinant strains, namely CGMCC No.13407/GFP (transformed pEKEx 2-GFP) and CGMCC No.13407/P tkt* GFP (transformation of pEKEx 2-P) tkt* -GFP)、CGMCC No.13407/P sod GFP (transformation of pEKEx 2-P) sod -GFP)、CGMCC No.13407/P tkt GFP (transformation of pEKEx 2-P) tkt -GFP), ATCC 13032/GFP (transformed pEKEx 2-GFP), ATCC 13032/P tkt* GFP (transformation of pEKEx 2-P) tkt* -GFP)、ATCC 13032/P sod GFP (transformation of pEKEx 2-P) sod -GFP) and ATCC 13032/P tkt GFP (transformation of pEKEx 2-P) tkt -GFP)。
3. Characterization of promoter Activity by Green fluorescent protein
Taking the strain to be measured (recombinant strain constructed in the above 2) from the freezing tube, streaking and activating on a seed activation medium, and culturing at 33 ℃ for 12 hours; selecting seed activated strain 1, inoculating into a 50mL triangular flask containing 10mL BHI liquid culture medium, and culturing at 33deg.C under 220r/min shaking for 15 hr; 1mL of seed solution is inoculated into a 500mL triangular flask filled with 30mL of BHI liquid culture medium, the culture is carried out at 33 ℃ and 220r/min until the logarithmic phase, bacterial cells are collected and washed three times by PBS, and the strain OD 562 Diluting to 1, collecting 200ul thallus suspension to 96-well plate, and using multifunctionalThe enzyme-labeled instrument detects the fluorescence intensity (excitation wavelength 484nm, emission wavelength 507 nm), fluorescence intensity value (RFU/OD 562 ) The measured fluorescence intensity values for the fluorescence intensity/cell density of the samples are shown in Table 2.
TABLE 2 GFP fluorescence intensity assay
The results showed that in Corynebacterium glutamicum CGMCC No.13407 and Corynebacterium glutamicum ATCC 13032, the enzyme was expressed as P tkt* When used as a promoter, the recombinant strain exhibits a fluorescence intensity of P tkt More than 4 times of the fluorescence intensity of (C) and P tkt* Stronger promoter P than known Corynebacterium glutamicum sod Has significantly higher priming activity, has significant difference (P < 0.05), and therefore, P tkt* Is a strong promoter capable of driving the expression of a target gene in Corynebacterium glutamicum.
Example 2 promoter for enhanced lysine synthesis
In the embodiment, a corynebacterium glutamicum (with the preservation number of CGMCC No. 13407) with high lysine yield is used as an initial strain, and a promoter with the sequence shown as SEQ ID No.1 is introduced into the strain by utilizing a genetic engineering technology and is used for up-regulating expression of a target gene tkt (SEQ ID No. 4).
1. Construction of pK18mobsacB-P tkt* And pK18mobsacB-P sod Engineering plasmid
Performing PCR amplification by using a Corynebacterium glutamicum ATCC 13032 genome as a template and a tkt-1f/tkt-1r primer pair to obtain an upstream fragment tkt-up; the Corynebacterium glutamicum ATCC 13032 genome is used as a template, and a tkt-2f/tkt-2r primer pair is used for PCR amplification to obtain a downstream fragment tkt-dn. With tkt-up, P tkt* And the mixture of the three fragments of tkt-dn is used as a template, and a primer pair of tkt-1f/tkt-2r is used for PCR amplification to obtain a promoter P with an upstream and a downstream homology arm tkt* Fragments. The fragment was digested with BamHI and SphI, and vector pK18mobsacB (GenBank: FJ437239.1; obtained from a teacher of the national academy of sciences Shanghai Seisakusho Yang, or purchased from a commercial source) 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-P tkt* 。
Construction of recombinant plasmid pK18mobsacB-P by the same procedure sod 。
2. Construction of pK18mobsacB-P tkt* And pK18mobsacB-P sod Engineering strain
Competent cells of CGMCC No.13407 were prepared according to Corynebacterium glutamicum Handbook (C.glutamicum Handbook, charpter 23). Recombinant plasmid pK18mobsacB-P tkt* And pK18mobsacB-P sod The competent cells were transformed 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 ) The diluted solution was spread on a normal BHI solid medium containing 10% sucrose, and was subjected to stationary culture at 33℃for 48 hours. The grown strain does not carry the inserted vector sequence in its genome. By PCR amplification of the target sequence and nucleotide sequencing analysis, the introduction of the promoter P before the tkt gene is obtained tkt* And P sod Strains (respectively with P) tkt* And P sod The natural promoter replacing the tkt gene) and designated 13407-P tkt* ::P tkt -tkt and 13407-P sod ::P tkt -tkt。
3. Testing lysine fermentation capacity of engineering bacteria by shaking flask fermentation
For the above constructed strain 13407-P tkt* ::P tkt -tkt and 13407-P sod ::P tkt -tkt for lysine fermentation production ability test using the strain CGMCC No.13407 as a control, a culture medium such asThe following steps:
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) Growth measurement: 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 3.
(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 3.
TABLE 3 lysine production and growth assays for recombinant strains
| Strain | L-lysine (g/L) | Sugar acid conversion% | OD 562 |
| CGMCC No.13407 | 15.3 | 25.4 | 46.7 |
| 13407-P tkt* ::P tkt -tkt | 18.4 | 30.6 | 46.8 |
| 13407-P sod ::P tkt -tkt | 17.2 | 28.3 | 46.6 |
The results showed that recombinant strain 13407-P tkt* ::P tkt Compared with the control group strain CGMCC No.13407, the productivity and the conversion rate of lysine are greatly improved, and the productivity of L-lysine is improved by 3.1g/L, and the conversion rate is improved by 5.2 percent, wherein the productivity and the conversion rate of lysine are obviously different (P is less than 0.05); 13407-P tkt* ::P tkt The L-lysine yield and the conversion rate of the-tkt are obviously higher than those of 13407-P sod ::P tkt Tkt, with significant differences (P < 0.05), the final OD of the recombinant strain was comparable to the control strain. It can be seen that the strong promoter P tkt* The tkt gene is reinforced, so that the regeneration capacity of NADPH is enhanced, sufficient NADPH is provided for lysine synthesis, and the yield of lysine is further improved.
Example 3 promoter for enhanced proline synthesis
In this embodiment, a high yield of proline is obtainedCorynebacterium glutamicum (collection number CGMCC No. 13757) was used as the starting strain, and the recombinant plasmid pK18mobsacB-P was obtained by the method of example 2 tkt* And pK18mobsacB-P sod Competent cells of the strain CGMCC No.13757 transferred respectively. Amplifying target sequence by PCR and performing nucleotide sequencing analysis to obtain the promoter P introduced before tkt gene tkt* And P sod Strains (respectively with P) tkt* And P sod The natural promoter replacing the tkt gene) and designated 13757-P tkt* ::P tkt -tkt and 13757-P sod ::P tkt -tkt。
Engineering bacteria 13757-P tested by shaking flask fermentation tkt* ::P tkt -tkt and 13757-P sod ::P tkt The proline fermenting capacity of tkt is compared with the strain CGMCC No.13757.
The culture medium used for the proline fermentation experiments was as follows:
seed activation medium: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH 7.2.
Seed culture medium: corn steep liquor 2.5%, glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.1%, urea 0.1%, caCO 3 0.5%, and water in balance, pH 7.2.
Fermentation medium: corn steep liquor 0.6%, glucose 12.0%, ammonium sulfate 3.7%, magnesium sulfate 0.05%, monopotassium phosphate 0.1%, caCO 3 4%, VH 70 μg/L, VB 1 & HCl 80 μg/L, and water in balance, and pH 7.2.
The proline 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 20mL seed culture medium, and shake culture is carried out for 16-22h at 33 ℃ and 220 r/min;
(3) Fermentation culture: 2mL of the seed solution was inoculated into a 500mL triangular flask containing 20mL of fermentation medium, and the culture was performed at 33℃and 220r/min for 72 hours with shaking, and three strains were made in parallel.
(4) Growth measurement: 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 4.
(5) Proline concentration determination: 2mL of the fermentation broth was centrifuged (12000 rpm,2 min), the supernatant was collected, the proline content in the fermentation broth of the recombinant bacteria and the control bacteria was measured by HPLC, three bacteria were used in parallel, the average value was calculated, and the detected proline concentration was shown in Table 4.
TABLE 4 proline yield and growth assays for recombinant strains
| Strain | Proline (g/L) | Sugar acid conversion% | OD 562 |
| CGMCC No.13757 | 38.0 | 30.0 | 45.5 |
| 13757-P tkt* ::P tkt -tkt | 41.1 | 34.2 | 46.4 |
| 13757-P sod ::P tkt -tkt | 39.2 | 32.5 | 46.5 |
The results showed that recombinant strain 13757-P tkt* ::P tkt Compared with the control group strain CGMCC No.13757, the proline yield and the conversion rate of the tkt are greatly improved, and the significant difference (P is less than 0.05) exists, wherein the proline yield is improved by 3.1g/L, and the conversion rate is improved by 4.2%;13757-P tkt* ::P tkt The proline yield and conversion of-tkt are higher than 13757-P sod ::P tkt Tkt, with significant differences (P < 0.05), the final OD of the recombinant strain was comparable to the control strain.
The above results indicate that compared with the known strong promoters, the novel promoter P provided by the invention tkt* Can obviously enhance the expression of target genes in recombinant microorganisms and promote the efficient synthesis of amino acids.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
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gagtaccgcc acaccaaggg cgttgagatc accactggcc ctcttggcca gggtcttgca 420
tctgcagttg gtatggccat ggctgctcgt cgtgagcgtg gcctattcga cccaaccgct 480
gctgagggcg aatccccatt cgaccaccac atctacgtca ttgcttctga tggtgacctg 540
caggaaggtg tcacctctga ggcatcctcc atcgctggca cccagcagct gggcaacctc 600
atcgtgttct gggatgacaa ccgcatctcc atcgaagaca acactgagat cgctttcaac 660
gaggacgttg ttgctcgtta caaggcttac ggctggcaga ccattgaggt tgaggctggc 720
gaggacgttg cagcaatcga agctgcagtg gctgaggcta agaaggacac caagcgacct 780
accttcatcc gcgttcgcac catcatcggc ttcccagctc caactatgat gaacaccggt 840
gctgtgcacg gtgctgctct tggcgcagct gaggttgcag caaccaagac tgagcttgga 900
ttcgatcctg aggctcactt cgcgatcgac gatgaggtta tcgctcacac ccgctccctc 960
gcagagcgcg ctgcacagaa gaaggctgca tggcaggtca agttcgatga gtgggcagct 1020
gccaaccctg agaacaaggc tctgttcgat cgcctgaact cccgtgagct tccagcgggc 1080
tacgctgacg agctcccaac atgggatgca gatgagaagg gcgtcgcaac tcgtaaggct 1140
tccgaggctg cacttcaggc actgggcaag acccttcctg agctgtgggg cggttccgct 1200
gacctcgcag gttccaacaa caccgtgatc aagggctccc cttccttcgg ccctgagtcc 1260
atctccaccg agacctggtc tgctgagcct tacggccgta acctgcactt cggtatccgt 1320
gagcacgcta tgggatccat cctcaacggc atttccctcc acggtggcac ccgcccatac 1380
ggcggaacct tcctcatctt ctccgactac atgcgtcctg cagttcgtct tgcagctctc 1440
atggagaccg acgcttacta cgtctggacc cacgactcca tcggtctggg cgaagatggc 1500
ccaacccacc agcctgttga aaccttggct gcactgcgcg ccatcccagg tctgtccgtc 1560
ctgcgtcctg cagatgcgaa cgagaccgcc caggcttggg ctgcagcact tgagtacaag 1620
gaaggcccta agggtcttgc actgacccgc cagaacgttc ctgttctgga aggcaccaag 1680
gagaaggctg ctgaaggcgt tcgccgcggt ggctacgtcc tggttgaggg ttccaaggaa 1740
accccagatg tgatcctcat gggctccggc tccgaggttc agcttgcagt taacgctgcg 1800
aaggctctgg aagctgaggg cgttgcagct cgcgttgttt ccgttccttg catggattgg 1860
ttccaggagc aggacgcaga gtacatcgag tccgttctgc ctgcagctgt gaccgctcgt 1920
gtgtctgttg aagctggcat cgcaatgcct tggtaccgct tcttgggcac ccagggccgt 1980
gctgtctccc ttgagcactt cggtgcttct gcggattacc agaccctgtt tgagaagttc 2040
ggcatcacca ccgatgcagt cgtggcagcg gccaaggact ccattaacgg ttaa 2094
<210> 5
<211> 720
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 6
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
aggatccacc acagttggtt ataaaat 27
<210> 7
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
aggtaccggt ggtcaatcct tcctggg 27
<210> 8
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
aggatcctag ctgccaatta ttccggg 27
<210> 9
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
aggtaccggg taaaaaatcc tttcgta 27
<210> 10
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
agagctcatg gtgagcaagg gcgagga 27
<210> 11
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
agaattctta cttgtacagc tcgtcca 27
<210> 12
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
aggatccgac acacggcaaa ttagtcg 27
<210> 13
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
attttataac caactgtggt tcaaatgtgt caaaggtgtg 40
<210> 14
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
cccaggaagg attgaccacc ttgacgctgt cacctgaact 40
<210> 15
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
agcatgcatg tggtggtcga atgggga 27
<210> 16
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
cccggaataa ttggcagcta tcaaatgtgt caaaggtgtg 40
<210> 17
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
tacgaaagga ttttttaccc ttgacgctgt cacctgaact 40
Claims (10)
1. Promoter, characterized in that it has the nucleotide sequence shown as SEQ ID No. 1.
2. An expression cassette comprising the promoter of claim 1.
3. A vector comprising the promoter of claim 1 or the expression cassette of claim 2.
4. A host cell comprising the promoter of claim 1 or the expression cassette of claim 2 or the vector of claim 3.
5. Use of the promoter of claim 1 or the expression cassette of claim 2 or the vector of claim 3 or the host cell of claim 4, for any of the following:
(1) Use in driving gene expression;
(2) Use in enhancing gene expression;
(3) The application in improving the yield and conversion rate of the microbial metabolites;
(4) The application in breeding the production strain of the microorganism metabolite;
(5) Use in enhancing the reductive synthesis of microorganisms;
(6) The application in fermentation production of microbial metabolites.
6. A recombinant microorganism comprising the promoter according to claim 1 in an intergenic region upstream of a transketolase gene;
preferably, the transketolase gene of the recombinant microorganism is expressed driven by the promoter of claim 1.
7. The method for constructing a recombinant microorganism according to claim 6, wherein the promoter according to claim 1 is replaced with a natural promoter of the transketolase gene of the starting strain;
preferably, the starting strain is a strain capable of accumulating metabolites that require consumption of reducing power for synthesis or for synthesis of precursors to metabolites of the pentose phosphate pathway.
8. Use of the recombinant microorganism of claim 6 for any of the following:
(1) The application in the fermentation production of microbial metabolites;
(2) The application in breeding the production strain of the microorganism metabolite;
the microbial metabolite is a metabolite which needs to consume reducing power for synthesis, or a metabolite of which synthesis precursor is a pentose phosphate pathway metabolite.
9. A method for producing an amino acid or a derivative thereof by fermentation, characterized by culturing the recombinant microorganism according to claim 6, and isolating the amino acid or the derivative thereof from the culture;
preferably, the amino acid is an amino acid whose synthesis requires consumption of reducing power, or an amino acid whose synthesis precursor is a pentose phosphate pathway metabolite.
10. A method of expressing a gene of interest, comprising: operably linking a gene of interest or a DNA fragment upstream and downstream thereof to the promoter of claim 1 to obtain a recombinant DNA, introducing the recombinant DNA into a host cell, and expressing the gene of interest.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111265985.7A CN116904455A (en) | 2021-10-28 | 2021-10-28 | Promoter, recombinant microorganism and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111265985.7A CN116904455A (en) | 2021-10-28 | 2021-10-28 | Promoter, recombinant microorganism and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116904455A true CN116904455A (en) | 2023-10-20 |
Family
ID=88367398
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111265985.7A Pending CN116904455A (en) | 2021-10-28 | 2021-10-28 | Promoter, recombinant microorganism and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116904455A (en) |
-
2021
- 2021-10-28 CN CN202111265985.7A patent/CN116904455A/en active Pending
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