CN116904453A - Nucleic acid molecules, recombinant microorganisms and uses thereof - Google Patents
Nucleic acid molecules, recombinant microorganisms and uses thereof Download PDFInfo
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 13
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of biotechnology, in particular to a nucleic acid molecule, a recombinant microorganism and application thereof. The invention provides a nucleic acid molecule which has a nucleotide sequence shown as SEQ ID NO. 1. The nucleic acid molecule has higher promoter activity than the endogenous strong promoter P of corynebacterium glutamicum tuf Obviously higher, can drive the gene to be expressed in the microorganism with high efficiency. The nucleic acid molecule is utilized to replace the natural promoter of the argS gene, so that the expression of the argS gene is obviously enhanced, the yield and the conversion rate of lysine of the recombinant bacterium constructed by the nucleic acid molecule are obviously improved, and the efficient production of lysine can be promoted.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a nucleic acid molecule, a recombinant microorganism and application thereof.
Background
In the fermentation production of microbial metabolites, efficient production of the metabolites can be achieved by engineering production strains, including: 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. Among them, for the enhanced expression of genes, conventional strategies include introducing multiple copies of the gene, or replacing the natural promoter of the gene of interest with a known strong promoter, and the like.
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. Currently, common endogenous strong promoters for the cereal bars include promoters encoding the superoxide dismutase gene (sod), and promoters encoding the elongation factor (tuf). However, the number of general promoters is limited, and the expression strength is fixed, so that the requirement of the genetic engineering technology on the promoters can not be met far. In addition, the expression strength of the promoter is related to the nucleotide sequence of the core region, the nucleotide sequence and length of the upstream and downstream thereof have a significant effect on the expression of the gene, which is related to the complex regulatory mechanism of the gene. Therefore, the development of new promoters is continuously required, and the regulation of expression of specific genes applied to specific metabolic pathways is necessary.
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. Wherein L-lysine is a basic essential amino acid with the molecular formula of C 6 H 14 N 2 O 2 The appearance is white or almost white crystalline powder, darkens at 210 ℃, decomposes at 224.5 ℃, is easily soluble in water, slightly soluble in alcohol, and insoluble in ether. L-lysine is widely used in animal feed, medicine and food industry, and about 90% of L-lysine currently produced is used in feed industry, and 10% is used in food and medicine industry. When the L-lysine is used as an animal feed additive, the L-lysine can promote the animal body to absorb other amino acids, so that the feed quality is improved. 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. The microbial fermentation method has the advantages of low cost of raw materials, mild reaction conditions, easy realization of large-scale production and the like, and development of efficient production strains has important significance for improving the production efficiency of the microbial fermentation method.
Disclosure of Invention
It is an object of the present invention to provide a nucleic acid molecule having a high promoter activity. Another object of the present invention is to provide a recombinant microorganism containing the nucleic acid molecule and its use.
Specifically, the invention provides the following technical scheme:
the invention provides a nucleic acid molecule which has a nucleotide sequence shown as SEQ ID NO. 1.
The nucleic acid molecule is obtained by mutating an arginine tRNA synthetase gene (argS) promoter of corynebacterium glutamicum to obtain a mutant promoter, has obviously higher promoter activity than an unmutated argS natural promoter, 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.
Based on the above nucleic acid molecules, the present invention provides expression cassettes containing the nucleic acid molecules.
The expression cassette described above may be a recombinant DNA obtained by operably linking a gene of interest downstream of the nucleic acid molecule, or a recombinant DNA obtained by operably linking a gene of interest, upstream or downstream of the promoter, or other transcriptional regulatory elements, translational regulatory elements.
The invention also provides vectors containing the nucleic acid molecules or the expression cassettes.
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 nucleic acid molecule, 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 nucleic acid molecule provided by the invention, the invention provides any one of the following applications of the nucleic acid molecule, 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) 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 (5), 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.
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 invention provides a recombinant microorganism comprising the nucleic acid molecule in an intergenic region upstream of an arginyl-tRNA synthetase gene of the recombinant microorganism.
aminoacyl-tRNA synthetases play a central role in the translation machinery of genetic information, and are involved indirectly in a variety of cellular regulatory processes, such as bacterial cell wall synthesis, control of RNA formation stability, initiation of DNA replication, and control of protein degradation. Arginine tRNA synthetases are a class of aminoacyl-tRNA synthetases that catalyze an ATP-PPi exchange reaction. The present invention has found that the above nucleic acid molecule provided by the present invention has a higher promoter activity than the natural promoter of argS. The natural promoter region of argS gene is replaced by the nucleic acid molecule, so that the synthesis of metabolic products such as lysine can be obviously enhanced.
The above-described intergenic region of the arginyl-tRNA synthetase gene is specifically the nucleotide sequence between the initiation codon of the arginyl-tRNA synthetase gene on the chromosome of the microorganism and the coding region of the gene upstream thereof.
Preferably, in the recombinant microorganism, the arginyl-tRNA synthetase gene is expressed driven by the nucleic acid molecule.
The recombinant microorganism described above has significantly increased arginyl-tRNA synthetase expression levels compared to those prior to introduction of the nucleic acid molecule, thereby facilitating synthesis and accumulation of metabolites, including but not limited to, amino acids of the aspartate family or derivatives thereof, having a synthesis efficiency that correlates with arginyl-tRNA synthetase expression levels.
The invention also provides a construction method of the recombinant microorganism, which comprises the following steps: the natural promoter of the arginyl-tRNA synthetase gene of the starting strain is replaced by the nucleic acid molecule.
Specifically, the method comprises the following steps: in the starting strain, the natural promoter of the arginyl tRNA synthetase gene is replaced by the nucleic acid molecule by using genetic engineering technology.
The starting strain of the recombinant microorganism described above is preferably a microorganism capable of synthesizing and accumulating the target metabolite.
Preferably, the starting strain is a strain capable of accumulating amino acids of the aspartate family or derivatives thereof. More preferred are strains capable of accumulating lysine or derivatives thereof.
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 application CN 106635944A. For the strain, the nucleic acid molecule provided by the invention drives the expression of the arginyl-tRNA synthetase coding gene, and the lysine 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) Use in the fermentative production of amino acids of the aspartate family or derivatives thereof;
(2) The application in breeding the production strain of amino acid of aspartic acid family or its derivative.
The above-mentioned amino acids of the aspartic acid family include aspartic acid, lysine, homoserine, threonine, methionine or isoleucine.
Derivatives of the above-mentioned amino acids of the aspartic acid family include trifluoroacetyl lysine, acetyl-L-threonine, S-adenosyl Gan Ji methionine, N-acetyl-L-isoleucine, glycyl-L-aspartic acid and the like.
The present invention also provides a method for producing lysine or its derivatives by fermentation, the method comprising: culturing the recombinant microorganism, and separating lysine or its derivative from the culture.
Specifically, the method for producing lysine or its derivatives 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 lysine or derivatives thereof.
The present invention also provides a method for expressing a target gene, comprising: operably linking a gene of interest or a DNA fragment upstream and downstream thereof to said nucleic acid molecule to obtain a recombinant DNA, introducing said recombinant DNA into a host cell, and expressing said gene of interest.
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, respectively, of the nucleic acid molecule.
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 nucleic acid molecule provided by the invention has higher promoter activity than the endogenous strong promoter P of corynebacterium glutamicum tuf Obviously higher, can drive the gene to be expressed in the microorganism with high efficiency. The nucleic acid molecule is used for replacing the argS gene natural promoter, so that the expression of the argS gene is obviously enhanced, the yield and the conversion rate of lysine of the recombinant bacterium constructed by the nucleic acid molecule are obviously improved, and the efficient production of lysine 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 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 was the promoter (P) of the mutated arginyl tRNA synthetase gene (argS) argS* )。
The activity of the promoter was examined as follows:
1. construction of beta-galactosidase expression plasmid
(1) Mutant promoter P argS* Is synthesized by Jin Weizhi Biotechnology Co., ltd (China). PCR amplification was performed using the Corynebacterium glutamicum ATCC13032 genome as a template and Ptuf-f/Ptuf-r and PargS-f/PargS-r primer pairs, respectively, to obtain tuf gene promoters P tuf (SEQ ID NO. 3) and the native promoter P of the argS gene argS (SEQ ID NO. 2). The E.coli MG1655 genome was used as a template, and a lacZ-f/lacZ-r primer set was used for PCR amplification to obtain an open reading frame (ORF, SEQ ID NO. 4) of the lacZ gene.
(2) The lacZ gene was digested with BamHI and KpnI, and vector pEKEx2 (GenBank: AY585307.1; available from the university of Shanghai Seisakusho of China academy of sciences Yang, or purchased from commercial sources) was digested with the same enzymes. The two cleavage products are connected by T4DNA Ligase, and Trans 1T 1 competent cells are transformed to obtain recombinant plasmid pEKEx2-lacZ without promoter before lacZ gene.
(3) P obtained in the step (1) argS* 、P argS And P tuf The fragment was digested with PstI and SalI, and vector pEKEx2-lacZ was digested with the same enzymes. The two enzyme cutting products are connected by T4DNA Ligase to transform Trans 1T 1 competent cells to obtain recombinant plasmid pEKEx2-P respectively argS* -lacZ、pEKEx2-P argS lacZ and pEKEx2-P tuf -lacZ。
2. Construction of beta-galactosidase-expressing Strain
(1) Competent cells of ATCC13032 were prepared according to Corynebacterium glutamicum Handbook (C.glutamicum Handbook, charpter 23). Recombinant plasmid pEKEx2-lacZ, pEKEx2-P argS* -lacZ、pEKEx2-P argS lacZ and pEKEx2-P tuf lacZ was transformed into competent cells by electroporation and transformants were selected on BHI selection medium containing 15mg/L kanamycin.
(2) The target sequence was amplified by PCR and subjected to nucleotide sequencing analysis to obtain recombinant strains, i.e., ATCC 13032/lacZ (transformed with pEKEx 2-lacZ), ATCC 13032/P, respectively argS* lacZ (transformation of pEKEx2-P argS* -lacZ)、ATCC 13032/P argS lacZ (transformation of pEKEx2-P argS -lacZ)、ATCC 13032/P tuf lacZ (transformation of pEKEx2-P tuf -lacZ)。
3. Characterization of promoter Activity by beta-galactosidase
The fluorogenic substrate 4-methylumbelliferyl-beta-D-galactopyranoside (MUG) was used to determine the galactosidase activity. The strain ATCC 13032/lacZ, ATCC 13032/P to be determined is taken from the frozen tube argS* -lacZ、ATCC 13032/P argS -lacZ、ATCC 13032/P tuf lacZ, streaking activation on seed activation medium, culturing at 33℃for 12h; 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 the seed solution was inoculated into a 500mL Erlenmeyer flask containing 30mL of BHI liquid medium, cultured at 33℃and 220r/min to logarithmic phase, 40. Mu.L of the bacterial solution was transferred into a 96-well plate containing 80. Mu.L of a buffer (40 mM sodium dihydrogen phosphate, 60mM disodium hydrogen phosphate, 10mM potassium chloride, 50mM magnesium sulfate-mercaptoethanol, pH 7.0), and the OD was measured 595 To assess cell density, 20. Mu.l of MUG dissolved in dimethyl sulfoxide was added to each well, incubated at room temperature for 15min, 1M sodium carbonate was added to stop the reaction, and the fluorescence intensity generated by beta-galactosidase-dependent MUG hydrolysis was quantitatively determined in a microfluorometer (excitation wavelength 360nm, emission wavelength 460 nm), according to literature (Vidal-Aroca F, giannattasio M, brunelli E, et al, one-step high-throughput assay for quantitative detection of beta-galactosidase activity in intact gram-negative) bacteria,yeast,and mammalian cells.[J]The method reported in Biotechniques,2006,40 (4): 433-4,436,438 passim.) determines the beta-galactosidase activity. The beta-galactosidase activity was calculated as unit MUG and the measured beta-galactosidase activity is shown in table 2.
TABLE 2 determination of beta-galactosidase Activity
| Strain | Beta-galactosidase Activity |
| ATCC 13032/lacZ | 0 |
| ATCC 13032/P argS *-lacZ | 1302 |
| ATCC 13032/P argS -lacZ | 430 |
| ATCC 13032/P tuf -lacZ | 1088 |
The results showed that in Corynebacterium glutamicum ATCC13032, the enzyme was used as P argS* The beta-galactosidase activity when used as a promoter is P argS More than 3 times and P argS* Stronger promoter P than known Corynebacterium glutamicum tuf Has significantly higher enzyme activity (with significant differences, P < 0.05), and therefore, P argS* Is a strong promoter capable of driving the expression of a target gene in Corynebacterium glutamicum.
Example 2 promoter for enhancing expression of arginyl-tRNA synthetase Gene
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 nucleotide sequence shown as SEQ ID No.1 is introduced into the strain by utilizing a genetic engineering technology and is used for up-regulating and expressing a target gene arginyl-tRNA synthetase gene.
1. Construction of arginyl-tRNA synthetase genetic engineering plasmid
Performing PCR amplification by using a Corynebacterium glutamicum ATCC13032 genome as a template and an argS-1f/argS-1r primer pair to obtain an upstream fragment argS-up; the downstream fragment argS-dn was obtained by PCR amplification using the Corynebacterium glutamicum ATCC13032 genome as a template and the argS-2f/argS-2r primer pair. With argS-up, P argS* And the mixture of the argS-dn three fragments is used as a template, and PCR amplification is carried out by using the argS-1f/argS-2r primer pair to obtain the promoter P with the upstream and downstream homology arms argS* Fragments. The fragment was digested with BamHI and HindI, and vector pK18mobsacB (GenBank: FJ437239.1; available from the university of Shanghai Seisakusho-Miao, china academy of life sciences Yang, or purchased from commercial sources) was digested with the same enzymes. The two enzyme digestion products are connected by T4DNA Ligase, and Trans 1T 1 competent cells are transformed to obtain recombinant plasmid pK18mobsacB-P argS* 。
Recombinant plasmid pK18mobsacB-P was constructed by the same procedure as described above tuf 。
2. Construction of arginyl-tRNA synthetase genetic engineering Strain
Competent cells of CGMCC No.13407 were prepared according to Corynebacterium glutamicum Handbook (C.glutamicum Handbook, charpter 23). Recombinant plasmid pK18mobsacB-P argS* And pK18mobsacB-P tuf 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 sequence of interest and nucleotide sequencing analysis, the introduction of the promoter P before the argS gene was obtained argS* And P tuf Strains (respectively with P) argS* And P tuf Replacement of the native promoter of the argS gene) and was designated 13407-P argS* ::P argS -argS and 13407-P tuf ::P argS -argS。
3. Testing lysine fermentation capacity of engineering bacteria by shaking flask fermentation
For strain 13407-P argS* ::P argS -argS and 13407-P tuf ::P argS argS was tested for lysine fermentation capacity using strain CGMCC No.13407 as a control and the following media were used for the lysine fermentation experiments:
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 detectingOD 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 | 15.4 | 25.7 | 46.6 |
| CGMCC No.13407/P argS *-argS | 18.2 | 30.2 | 46.7 |
| CGMCC No.13407/P tuf -argS | 17.6 | 29.2 | 46.7 |
The results showed that recombinant strain 13407-P argS* ::P argS -argS and 13407-P tuf ::P argS The lysine yield and conversion rate of argS are greatly improved compared with those of the control strain CGMCC No.13407, and the mutant strain has significant difference (P is less than 0.05), and the final OD of the recombinant strain is equivalent to that of the control strain. Wherein 13407-P argS* ::P argS The lysine yield and conversion rate of argS are highest, 2.8g/L and 4.5% higher than CGMCC No.13407, and 13407-P argS* ::P argS Lysine production and conversion of argS were also higher than those of 13407-P tuf ::P argS argS, with significant differences (P < 0.05). The verification result of the shake flask fermentation experiment shows that the genetic engineering strain adopting the mutant promoter with the sequence shown as SEQ ID NO.1 to modify argS has higher sugar acid conversion rate and fermentation strength of lysine compared with the natural argS promoter.
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
<110> Feng Bo
<120> nucleic acid molecules, recombinant microorganisms and uses thereof
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tcgaggcggc aagaactgct actacccttt ttattgtcga acggggcatt acggctccaa 180
ggacgtttgt tttctgggtc agttacccca aaaagcatat acagagacca atgatttttc 240
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<210> 14
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
caagcttgaa gtagtattcg cgggtcac 28
<210> 15
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
acttacccta cgcgcctact cccgggggca cgtgtgtccg 40
<210> 16
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
gaagtccagg aggacataca atgacaccag ctgatctcgc 40
Claims (10)
1. A nucleic acid molecule, characterized in that it has a nucleotide sequence as shown in SEQ ID No. 1.
2. An expression cassette comprising the nucleic acid molecule of claim 1.
3. Vector, characterized in that it contains a nucleic acid molecule according to claim 1 or an expression cassette according to claim 2.
4. A host cell comprising the nucleic acid molecule of claim 1 or the expression cassette of claim 2 or the vector of claim 3.
5. Use of the nucleic acid molecule 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) The application in fermentation production of microbial metabolites.
6. A recombinant microorganism comprising the nucleic acid molecule of claim 1 in an intergenic region upstream of its arginyl-tRNA synthetase gene;
preferably, the arginyl-tRNA synthetase gene of the recombinant microorganism is driven by the nucleic acid molecule of claim 1.
7. The method for constructing a recombinant microorganism according to claim 6, wherein the nucleic acid molecule according to claim 1 is replaced with a natural promoter of the arginyl-tRNA synthetase gene of the starting strain;
preferably, the starting strain is a strain capable of accumulating amino acids of the aspartate family or derivatives thereof.
8. Use of the recombinant microorganism of claim 6 for any of the following:
(1) Use in the fermentative production of amino acids of the aspartate family or derivatives thereof;
(2) The application in breeding the production strain of amino acid of aspartic acid family or its derivative.
9. A method for producing lysine or a derivative thereof by fermentation, comprising culturing the recombinant microorganism according to claim 6 and separating lysine or a derivative thereof from the culture.
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 nucleic acid molecule of claim 1 to obtain recombinant DNA, introducing said recombinant DNA into a host cell, and expressing said gene of interest.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111264638.2A CN116904453A (en) | 2021-10-28 | 2021-10-28 | Nucleic acid molecules, recombinant microorganisms and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111264638.2A CN116904453A (en) | 2021-10-28 | 2021-10-28 | Nucleic acid molecules, recombinant microorganisms and uses thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116904453A true CN116904453A (en) | 2023-10-20 |
Family
ID=88353593
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111264638.2A Pending CN116904453A (en) | 2021-10-28 | 2021-10-28 | Nucleic acid molecules, recombinant microorganisms and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116904453A (en) |
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2021
- 2021-10-28 CN CN202111264638.2A patent/CN116904453A/en active Pending
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