CN114317538A - Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and application thereof - Google Patents
Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and application thereof Download PDFInfo
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Abstract
A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and application thereof, the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 consists of a Corynebacterium glutamicum promoter and a heat-resistant functional gene, the Corynebacterium glutamicum promoter is a strong promoter Ptuf, and the heat-resistant functional gene is derived from a TTHA0571 gene in Thermus thermophilus CICC 10647; the TTHA0571 gene sequence is shown in SEQ NO.1, and the strong promoter Ptuf gene sequence is shown in SEQ NO. 2. The heat-resistant component Ptuf-TTHA0571 of the corynebacterium glutamicum can greatly improve the heat stability of the corynebacterium glutamicum, enhance the heat resistance of strains, greatly reduce the energy consumption of cooling water in the fermentation process and reduce the production cost when being applied to industrial production of the strains, and has better industrial application prospect.
Description
Technical Field
The invention relates to a corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and application thereof, and belongs to the technical field of biological engineering.
Background
Corynebacterium glutamicum (Corynebacterium glutamicum) is currently used for producing glutamic acid, lysine, branched chain amino acid, vitamin D, organic acid and the like, and is widely applied to the fields of food, medicine, agriculture and the like. Corynebacterium glutamicum is an important industrial microorganism, and is often used as a microbial cell factory to produce amino acids, organic acids, vitamins, fuel ethanol and the like by using carbon sources such as glucose, sucrose, xylose and the like, and has become an important biological platform.
The most suitable culture temperature of the corynebacterium glutamicum is generally 26-37 ℃, the normal fermentation production is generally controlled at about 32 ℃, a series of cooling measures such as circulating water cooling, freezing water cooling and the like need to be taken to control the fermentation temperature of the corynebacterium glutamicum to be 32 ℃, particularly in high-temperature seasons in summer, the environmental temperature is generally high, a large amount of refrigerant media need to be consumed in the fermentation process of the corynebacterium glutamicum, the cooling effect cannot be achieved frequently, the fermentation is abnormal, a large amount of cost is increased for a cooling system, the yield can be directly influenced by the abnormal fermentation, and the production and operation cost is invisibly increased.
Disclosure of Invention
The invention aims to provide a corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and application thereof.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571, the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 consists of a Corynebacterium glutamicum promoter and a heat-resistant functional gene, the Corynebacterium glutamicum promoter is a strong promoter Ptuf, and the heat-resistant functional gene is derived from a TTHA0571 gene in Thermus thermophilus CICC 10647; the TTHA0571 gene sequence is shown in SEQ NO.1, and the strong promoter Ptuf gene sequence is shown in SEQ NO. 2.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: the application of the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 disclosed in claim 1, and the application of the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 in improving the heat resistance of corynebacterium glutamicum.
The preferable technical scheme is as follows: the Corynebacterium glutamicum thermotolerant element Ptuf-TTHA0571 is introduced into a plasmid and then expressed in Corynebacterium glutamicum.
The preferable technical scheme is as follows: the Corynebacterium glutamicum thermotolerant element Ptuf-TTHA0571 is integrated on the genome of Corynebacterium glutamicum.
The preferable technical scheme is as follows: the high-temperature acclimation is carried out on the corynebacterium glutamicum with the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571, so that the heat resistance of the corynebacterium glutamicum is improved.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. according to the invention, a large number of corynebacterium glutamicum heat-resistant components are constructed, so that a corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 with a good heat-resistant effect is screened, the culture temperature of strains is increased by applying the component in corynebacterium glutamicum, so that the energy consumption and the production cost can be reduced, the practical application value of production is obvious, and a good scientific reference value is provided for stress resistance research of corynebacterium glutamicum and construction design of stress-resistant components.
2. The heat-resistant component Ptuf-TTHA0571 of the corynebacterium glutamicum can greatly improve the heat stability of the corynebacterium glutamicum, enhance the heat resistance of strains, greatly reduce the energy consumption of cooling water in the fermentation process and reduce the production cost when being applied to industrial production of the strains, and has better industrial application prospect.
Drawings
FIG. 1 shows the first control of growth of cells after high temperature (42 ℃ C.) culture.
FIG. 2 shows the growth of cells after high-temperature (42 ℃ C.) culture as a control.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-2. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1: corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and application thereof
The method for constructing the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 comprises the following steps:
step 1: activating Thermus thermophilus CICC10647, inoculating to liquid culture medium, and culturing in a shaking table at 70-80 deg.C for 20-28 hr to obtain seed culture solution; inoculating the seed culture solution into a liquid culture medium, and extracting a genome from a bacterial solution in a logarithmic phase;
liquid medium composition: 4.0g of yeast powder; 8.0g of polypeptone; NaCl2.0 g; 1L of distilled water; the pH was adjusted to 7.0.
The bacterial genome extraction method comprises the following steps:
using a bacterial Genomic DNA extraction Kit (Wizard Genomic DNA Purification Kit A1120), the procedure was performed as follows:
(1) taking out the pretreated corynebacterium glutamicum cells from-80 ℃, adding 600 mu L of cell lysate into each tube, uniformly dispersing the cells in the lysate by turning over the cells in a gentle reverse manner or blowing and beating the cells by using a pipette, and putting an EP tube in a constant-temperature water bath at 80 ℃ for incubation for 5min to accelerate the cell lysis.
(2) The lysis system was cooled to room temperature, 2. mu.L of RNase solution (using RNase solution provided in kit or prepared by itself at 50 mg/mL) was added to each tube, and incubated in a water bath at 37 ℃ for 30-90 min.
(3) Adding 200 mu L of protein separation liquid into each tube, quickly turning over to fully mix the solution, placing 1.5mL of EP tube in an ice bath for 2-3min after obvious white flocculent precipitate appears, centrifuging at 4 ℃ for 10-20min at the rotation speed of 10000-13000r/min, and transferring 400-600 mu L of supernatant into a new 1.5mL of EP tube.
(4) Adding equal volume of isopropanol or double volume of absolute ethyl alcohol into a 1.5mL EP tube, and turning the tube in a gentle and gentle way to ensure that the solution is fully mixed, and then the genome DNA can be separated out in a macroscopic form, and whether the extraction of the genome DNA is successful or not can be visually judged by observation.
(5) A1.5 mL EP tube was centrifuged at 4 ℃ for 1min at 12000r/min and the supernatant was discarded.
(6) To a 1.5mL EP tube was added 700. mu.L of 70% ethanol solution (-20 ℃), gently inverted and the genomic DNA was brought into contact with the ethanol solution sufficiently, centrifuged at 4 ℃ for 1min at 12000r/min, the supernatant was discarded, and the procedure of the step was repeated 2 to 3 times.
(7) Carefully sucking the residual ethanol solution as clean as possible by a pipette, and allowing a 1.5mL EP tube to stand open for 1-2h to fully volatilize ethanol.
(8) To a 1.5mL EP tube was added 40-100. mu.L of a buffer solution (pH 7.0-8.0) or ddH2And O, standing for 1h at room temperature or standing for 12h at 4 ℃ to fully dissolve the genome DNA.
(9) mu.L of the genomic DNA solution was transferred to a new 1.5mL EP tube for quality control analysis using a pipette, and the remaining solution was stored at-80 ℃.
Step 2: taking the genome extracted in the step 1 as a template, and carrying out PCR amplification by using the upper primer TTHA0571-1 and the lower primer TTHA 0571-2;
upper primer TTHA 0571-1:
5’-CGAAGTCCAGGAGGAAAGCTT ATGCTGGAGCGCCACGAC-3’；
the lower primer TTHA 0571-2:
5’-TATCATCACAGAGTGCAACCAGATC CTACGCCTCCTTTAGGGGAAG-3’。
and step 3: activating Corynebacterium glutamicum ATCC13032, inoculating to a liquid LB culture medium, placing in a shaking table at 30-34 ℃ and culturing for 20-28h to obtain a seed culture solution; inoculating the seed culture solution into a liquid LB culture medium, and extracting a genome from a bacterial solution in a logarithmic phase; the genome extraction method is the same as above.
And 4, step 4: performing PCR amplification by using the genome extracted in the step 3 as a template and using the primer Ptuf-1 and the primer Ptuf-2;
the upper primer Ptuf-1:
5’-TGTTGACCTCAAATATCGGAAGTACAAGCGCGTGCCCTCTTTGCTGCAG-3’；
the lower primer Ptuf-2: 5'-GTCGTGGCGCTCCAGCAT AAGCTTTCCTCCTGGACTTCG-3' are provided.
And 5: the promoter Ptuf is connected with a DNA fragment of a heat-resistant functional gene TTHA 0571; the primer pairs used were: an upper primer up-1 and a lower primer down-2;
the upper primer up-1: 5'-ACCACGTCGATGTCTTTTACCTG-3', respectively;
the following primers down-2: 5'-GAGAAGTTAGCAAAGCCACGAGTAG-3' are provided.
Step 6: and C, performing combined transformation on the Corynebacterium glutamicum.
In step 2, a PCR amplification system of the heat-resistant functional gene TTHA 0571: a50. mu.L enzyme digestion reaction system was prepared by first adding 20. mu.L of ddH to a 0.2mL PCR tube2O; add 10. mu.L of 5 XPrimeSTAR Buffer solution; adding 4 mu L dNTP solution; adding 2 mu L of each solution of an upper primer TTHA0571-1 and a lower primer TTHA 0571-2; adding template with total DNA not more than 200ng(ii) a Add 0.5. mu.L PrimeSTAR HS DNA Polymerase solution; by ddH2O, complementing the reaction system to 50 mu L, and repeatedly blowing and sucking by a pipettor to mix uniformly; the PCR amplification parameters of the heat-resistant functional gene TTHA0571 are as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles comprising denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s and extension at 72 ℃ for 30 s; extension at 72 ℃ for 10 min.
In step 4, the PCR amplification system of the promoter Ptuf: a50. mu.L enzyme digestion reaction system was prepared by first adding 20. mu.L of ddH to a 0.2mL PCR tube2O; add 10. mu.L of 5 XPrimeSTAR Buffer solution; adding 4 mu L dNTP solution; adding 2 mu L of each of the upper primer Ptuf-1 and the lower primer Ptuf-2 solution; adding template with total DNA not more than 200 ng; add 0.5. mu.L of PrimeSTAR HS DNA Polymerase solution; by ddH2O, complementing the reaction system to 50 mu L, and repeatedly blowing and sucking by a pipettor to mix uniformly; the PCR amplification parameters of the promoter Ptuf are as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles comprising denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s and extension at 72 ℃ for 30 s; extension at 72 ℃ for 10 min.
In step 5, 50. mu.L of the enzyme digestion reaction system was prepared by first adding 20. mu.L of ddH to a 0.2mL PCR tube2O; add 10. mu.L of 5 XPrimeSTAR Buffer solution; adding 4 mu L dNTP solution; adding 2 mu L of each of the upper primer up-1 and the lower primer down-2 solution; adding 1. mu.L of up template, 2.5. mu.L of Ptuf template (amplification product of step 4), 0.9. mu.L of TTHA0571 template (amplification product of step 2), and 1.7. mu.L of down template; add 0.5. mu.L PrimeSTAR HS DNA Polymerase solution; by ddH2O, complementing the reaction system to 50 mu L, and repeatedly blowing and sucking by a pipettor to mix uniformly; the parameters of the PCR instrument are set as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles comprising denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s and extension at 72 ℃ for 2 min; extension at 72 ℃ for 10 min.
up template sequence:
ACCACGTCGATGTCTTTTACCTGCTCGATCACACAACCAGCCCTCAGATCACGGTGCTGCCGCACTCAATGGATTATTTCGATCAAACGCGCAGCGATGCTGTTATGGCTGCCATCATTGAGCAAAACCCTGCGTTCGCAGAAATTAAAGGCTCACCCATTACAACCGCAGATGTAGCCCTCCACAATCTCGGTGACACCAACGCCAACCGACGCTGGCAGTCTAACGTGCTGCTTGCCCGGCTACTCGGGGGTATTAGTGTGCGCGGAGAGGTACCTGAGCACCAGAGCCACAACCATCTCGCCAAGCAGTTTGCCGAGGCAACCTTGGTCACCAGGGACTTCGATGTGAATTATGATCCAACAAGCGCTCACCCTTTTACTGCTGGCTTCAACTCGATCAACTATGACACCACCTTGCTCAGCCTGTACTTCGCAATGTTGACCTCAAATATCGGAAGTAC。
down template sequence:
GATCTGGTTGCACTCTGTGATGATATTCGCGGTGTACTTGATCGAGGTTTAGAGATCTCATCTCCGAATCATCATGAGATGGTGGATGCTATGCGCAAGCAGCTGCACTATATTCAGGCATTTTACCGTGCCTGGGGACCCATTCAACGCCGCTTCAATGACGCTGACCCAGCGGTGACCCATCCGCATCTCACAGTGATCTACCCACCGCTCACCCCTGCATCCGCAGAGAAATTCAACAAGATCACCTCAGTCGCTGCTGTGAGCAAGCGCCCAACCACCCTGCCGTATTTCCGTGCAGATGGTTCACCTACTCGTGGCTTTGCTAACTTCTC。
the step 6 specifically comprises the following steps: prepare 20. mu.L in vitro ligation system by first adding 5. mu.L of ddH to a 0.2mL PCR tube2O; add 4. mu.L of 5 × CE button solution; adding 50-200 ng of pk18mobrpsL plasmid; adding 20-200 ng of the connected target DNA fragment obtained in the step (5); 2. mu.L of ExnaseTMII was added; by ddH2O, complementing the reaction system to 20 mu L; water bath at 37 deg.C for 30 min; coli DH5 alpha competent cells were added and ice-cooled for 20 min; placing in 42 deg.C water bath, heat-shocking for 60s, and ice-bathing for 2 min; adding 900 μ L of preheated SOC resuscitation solution into sterile EP tube, culturing at 37 deg.C for 1h at 220 r/min; taking 100 mu L of bacterial liquid, coating the bacterial liquid on a kanamycin-resistant plate, and culturing for 12h at 37 ℃; randomly selecting a single colony in a plate, carrying out colony PCR identification, screening a correct target strain, namely Escherichia coli DH5 alpha-pk 18mobrpsL-Ptuf-TTHA0571, and culturing the plate at 37 ℃ overnight; inoculating the correctly identified strain into LB liquid culture medium with kanamycin, culturing for 10h, centrifuging the thallus and extracting plasmid; precooling a 2mm electric rotating cup on ice for about 20min, adding the corynebacterium glutamicum ATCC13032 to be frozen into the electric rotating cup to melt competent cells and add plasmids into the electric rotating cup, and carrying out ice bath for 20 min; after electrotransfer, resuspending the thallus in the electrotransfer cup with 900 μ L of preheated BHIS resuscitating fluid, transferring into an EP tube, thermally shocking for 6min at 46 ℃, resuscitating for 2h at 32 ℃ and 220r/min, coating a BHI flat plate containing kanamycin resistance, and culturing for 24h at 32 ℃; randomly selecting single colony in the plate by using toothpick, scratching the single colony in a solid culture medium plate, dissolving the colony on the toothpick in a prepared colony PCR reaction system, carrying out colony PCR amplification on the reaction system, screening a correct target strain through a PCR identification result, and naming the strain as a glutamic acid rodThe strain ATCC13032-pk18mobrpsL-Ptuf-TTHA0571, the plate was incubated overnight at 32 ℃; inoculating the correctly identified strain into a BHI liquid culture medium with kanamycin, and culturing for 10-12 h; sucking 3-5 mu L of bacterial liquid into 900 mu L of BHI resuscitation liquid, sucking 100 mu L of resuscitation liquid to spread on a streptomycin resistant plate, and culturing for 24h at 32 ℃; randomly selecting a single colony in a plate by using a toothpick, scratching the single colony in a solid culture medium plate, dissolving the colony on the toothpick in a prepared colony PCR reaction system, carrying out colony PCR amplification on the reaction system, screening a correct target strain according to a PCR identification result, and storing the correctly identified strain, wherein the correctly identified strain is named as corynebacterium glutamicum ATCC 13032-Ptuf-TTHA 0571.
The plasmid extraction method comprises the following steps:
the operation was carried out according to the instructions provided by the Plasmid Mini Kit I, and the main procedure was as follows:
(1) centrifuging 1.5-5mL of bacterial liquid in a 1.5mL EP tube to collect thalli cells, wherein the centrifugation condition is 12000r/min and 3 min;
(2) removing supernatant, adding Solution I (containing RNase A) provided by 250 μ L kit, and re-suspending thallus cells by repeated suction or vortex oscillation;
(3) adding 250 μ L Solution II, mixing by gentle inversion for 3-5 times, and standing at room temperature for 2 min;
(4) adding 250 μ L Solution III, mixing by gentle inversion until white precipitate is generated, ice-cooling for 2min, and centrifuging at 12000r/min for 15 min;
(5) transferring 600 μ L of the supernatant to an adsorption column, centrifuging for 1min at 12000r/min, and discarding the filtrate;
(6) adding 500 μ L Buffer HB, centrifuging for 1min at 12000r/min, and discarding the filtrate;
(7) adding 700 μ L Buffer WB (diluted with ethanol), centrifuging at 12000r/min for 1min, discarding the filtrate, and repeating the step once;
(8) centrifuging the adsorption column at 12000r/min for 2min, and replacing the collection tube with a clean 1.5mL EP tube;
(9) 50-100. mu.L of Buffer EB or ddw was added to the adsorption column and centrifuged at 12000r/min for 1 min.
Verification of the heat resistance of Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA 0571:
corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 is a strain in which Corynebacterium glutamicum thermotolerant element Ptuf-TTHA0571 is integrated into the genome of Corynebacterium glutamicum ATCC 13032.
Streaking Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 and Corynebacterium glutamicum ATCC13032 from glycerol tube to plate, culturing at 32 deg.C for 24h, picking single colony from plate, inoculating to 3mL liquid culture medium, culturing at 32 deg.C for 16h, and detecting OD600Press OD600The inoculum size was 200. mu.L/10, inoculated into a 500mL fermentation flask containing 20mL of liquid medium, incubated at 42 ℃ for 24h on a shaker at 200rpm, and the OD measured600The value is obtained. As shown in FIG. 1, it can be seen from the fruit of FIG. 1 that the bacterial cells OD of ATCC 13032-Ptuf-TTHA0571 were obtained after culturing Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 and Corynebacterium glutamicum ATCC13032 at high temperature (42 ℃) for 24 hours600Compared with ATCC13032, the growth rate of ATCC 13032-Ptuf-TTHA0571 thalli is improved by 19.32 percent, and the growth rate of the thalli of ATCC 13032-Ptuf-TTHA0571 thalli at high temperature (42 ℃) is obviously better than that of ATCC 13032.
C.glutamicum ATCC13032-pk18mobrpsL-Ptuf-TTHA0571 heat resistance verification:
corynebacterium glutamicum ATCC13032-pk18mobrpsL-Ptuf-TTHA0571 is a strain in which Corynebacterium glutamicum thermotolerant element Ptuf-TTHA0571 is introduced into plasmid pk18mobrpsL vector and then expressed in Corynebacterium glutamicum ATCC 13032.
Streaking Corynebacterium glutamicum ATCC13032-pk18mobrpsL-Ptuf-TTHA0571 and Corynebacterium glutamicum ATCC13032 from glycerol tube to plate, culturing at 32 deg.C for 24h, picking single colony from plate, inoculating to 3mL liquid medium, culturing at 32 deg.C for 16h, detecting OD600Press OD600The inoculum size was 200. mu.L/10, inoculated into a 500mL fermentation flask containing 20mL of liquid medium, incubated at 42 ℃ for 24h on a shaker at 200rpm, and the OD measured600The value is obtained. As shown in FIG. 2, it can be seen from the results that Corynebacterium glutamicum ATCC13032-pk18mobrpsL-Ptuf-TTHA0571 and Corynebacterium glutamicum ATCC13032 were cultured at high temperature (42 ℃) for 24 hours, and then ATCC13032-pk18mobrpOD of sL-Ptuf-TTHA0571 cells600The growth rate of the thalli of ATCC13032-pk18mobrpsL-Ptuf-TTHA0571 is obviously superior to that of ATCC13032 at high temperature (42 ℃), which is 18.72% higher than that of ATCC 13032.
Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 high-temperature domestication
The Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 is streaked from a glycerol tube to be activated by a flat plate, is cultured for 24 hours at 32 ℃, is subjected to multiple times of high-temperature (42 ℃) culture acclimation, the cultured fermentation broth is subjected to gradient dilution, 50 mu L of the fermentation broth subjected to gradient dilution is sucked and coated on the flat plate, the flat plate is cultured for 16 hours at 32 ℃, and single colonies with good thallus morphology and growth are selected from the flat plate for preservation. The strain after high-temperature acclimatization is named as Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 variant. The Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571, Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 and Corynebacterium glutamicum ATCC13032 were subjected to culture control test under the same conditions of 42 ℃ and tested for OD after 24h of culture600Value, OD600The results of the values show that ATCC 13032-Ptuf-TTHA0571 variant > ATCC 13032-Ptuf-TTHA0571 > ATCC13032, indicating that the thermotolerance of Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 variant was further enhanced after acclimation at high temperature.
The corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and the application thereof in corynebacterium glutamicum can obviously improve the heat resistance of corynebacterium glutamicum under a high-temperature culture condition. On one hand, the culture problem of corynebacterium glutamicum can be improved, the growth and the propagation of thalli can be accelerated, and the energy consumption required by cooling in the fermentation culture process can be reduced; on the other hand, under the sudden condition of meeting the high temperature in summer or the failure of a cooling system in the fermentation culture process of the corynebacterium glutamicum, the strain with the heat-resistant component can greatly improve the stability of the strain, so that the influence on the growth of the strain under the abnormal high temperature condition is reduced to the minimum, and the stable production of the fermentation of the corynebacterium glutamicum is facilitated. The corynebacterium glutamicum heat-resistant component can be widely applied to production of products such as amino acid, organic acid, vitamin, fuel ethanol and the like with corynebacterium glutamicum as a biotechnology platform.
1, SEQ No. 1: TTHA0571 gene sequence:
ATGCTGGAGC GCCACGACCG CCTGGAAACC CTGAGAAAGC TGAAGGAACT GCAGGAGCGC ATCGCCGAGC TCGCCTACCT CCTCACCGGG GAGGAGCCCG CCGCCTGGAC CCCCAGGGTG GACCTTCTGG AAACCGAGGA GCACTACGTC CTCCTCGTGG ACCTTCCCGG GGTGCGCCCC GAGGACCTGG AGCTTCTGGA GGAGGGGCAG CGGGTGACCC TGGCCGGGGT GCGCCACCCC CTGCCCGGCA CCTACCTCTT GGAGGAGAGG CCCATGGGCA CCTTCCGCCG CACCCTGGAC CTCCCCGGGC CCATTGAGGA GGGGACGGCC CAGGCCACCC TGCGGAACGG GGTCTTGGAG GTCCGCTTCC GCAAGAGGCC GGCCACGGCC CTTCCCCTAA AGGAGGCGTA G。
SEQ NO. 2: promoter Ptuf Gene sequence
GTTAACAGAT CGTTTAGATC CGAAGGAAAA CGTCGAAAAG CAATTTGCTT TTCGACGCCC CACCCCGCGC GTTTTAGCGT GTCAGTAGGC GCGTAGGGTA AGTGGGGTAG CGGCTTGTTA GATATCTTGA AATCGGCTTT CAACAGCATT GATTTCGATG TATTTAGCTG GCCGTTACCC TGCGAATGTC CACAGGGTAG CTGGTAGTTT GAAAATCAAC GCCGTTGCCC TTAGGATTCA GTAACTGGCA CATTTTGTAA TGCGCTAGAT CTGTGTGCTC AGTCTTCCAG GCTGCTTATC ACAGTGAAAG CAAAACCAAT TCGTGGCTGC GAAAGTCGTA GCCACCACGA AGTCCAGGAG GAAAGCTT。
Example 2: corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and application thereof
The primers and materials used were the same as in example 1.
A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571, the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 consists of a Corynebacterium glutamicum promoter and a heat-resistant functional gene, the Corynebacterium glutamicum promoter is a strong promoter Ptuf, and the heat-resistant functional gene is derived from a TTHA0571 gene in Thermus thermophilus CICC 10647; the TTHA0571 gene sequence is shown in SEQ NO.1, and the strong promoter Ptuf gene sequence is shown in SEQ NO. 2.
The application of the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 in improving the heat resistance of corynebacterium glutamicum is provided.
The Corynebacterium glutamicum thermotolerant element Ptuf-TTHA0571 is introduced into a plasmid and then expressed in Corynebacterium glutamicum.
The Corynebacterium glutamicum thermotolerant element Ptuf-TTHA0571 is integrated on the genome of Corynebacterium glutamicum.
The high-temperature acclimation is carried out on the corynebacterium glutamicum with the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571, so that the heat resistance of the corynebacterium glutamicum is improved.
Activating and inoculating thermus thermophilus CICC10647 in a liquid LB culture medium, and culturing in a shaker at 75 ℃ for 24h to obtain a seed culture solution; inoculating the seed culture solution into a liquid culture medium, and extracting the genome from the bacterial solution in the logarithmic phase.
The method for constructing the Corynebacterium glutamicum heat-resistant element Ptuf-TTHA0571 comprises the following steps:
PCR amplification of the heat-resistant functional gene TTHA 0571: a50. mu.L enzyme digestion reaction system was prepared by first adding 20. mu.L of ddH to a 0.2mL PCR tube2O; add 10. mu.L of 5 XPrimeSTAR Buffer solution; adding 4. mu.L dNTP solution; adding 2 mu L of each solution of an upper primer TTHA0571-1 and a lower primer TTHA 0571-2; adding template with total DNA not more than 200 ng; add 0.5. mu.L PrimeSTAR HS DNA Polymerase solution; by ddH2O, complementing the reaction system to 50 mu L, and repeatedly blowing and sucking by a pipettor to mix uniformly; the parameters of the PCR instrument are set as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles comprising denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s and extension at 72 ℃ for 30 s; extension at 72 ℃ for 10 min.
Activating and inoculating corynebacterium glutamicum ATCC13032 to a liquid LB culture medium, and culturing for 24 hours in a shaking table at 32 ℃ to obtain a seed culture solution; inoculating the seed culture solution into a liquid culture medium, and extracting the genome from the bacterial solution in the logarithmic phase.
PCR amplification of promoter Ptuf: a50. mu.L enzyme digestion reaction system was prepared by first adding 20. mu.L of ddH to a 0.2mL PCR tube2O; add 10. mu.L of 5 XPrimeSTAR Buffer solution; adding 4. mu.L dNTP solution; adding 2 mu L of each of the upper primer Ptuf-1 and the lower primer Ptuf-2 solution; adding template with total DNA not more than 200 ng; add 0.5. mu.L PrimeSTAR HS DNA Polymerase solution; by ddH2O, complementing the reaction system to 50 mu L, and repeatedly blowing and sucking by a pipettor to mix uniformly; the parameters of the PCR instrument are set as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles comprising denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s and extension at 72 ℃ for 30 s; 72 ℃ extensionStretching for 10 min.
The promoter Ptuf is connected with a DNA fragment of a heat-resistant functional gene TTHA 0571: a50. mu.L enzyme digestion reaction system was prepared by first adding 20. mu.L of ddH to a 0.2mL PCR tube2O; add 10. mu.L of 5 XPrimeSTAR Buffer solution; adding 4. mu.L dNTP solution; adding 2 mu L of each of the upper primer up-1 and the lower primer down-2 solution; adding 1 mu L of up template, 2.5 mu L of Ptuf template, 0.9 mu L of TTHA0571 template and 1.7 mu L of down template; add 0.5. mu.L PrimeSTAR HS DNA Polymerase solution; by ddH2O, complementing the reaction system to 50 mu L, and repeatedly blowing and sucking by a pipettor to mix uniformly; the parameters of the PCR instrument are set as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles comprising denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s and extension at 72 ℃ for 2 min; extension at 72 ℃ for 10 min.
C, corynebacterium glutamicum binding transformation: prepare 20. mu.L in vitro ligation system by first adding 5. mu.L ddH to a 0.2mL PCR tube2O; add 4. mu.L of 5 × CE button solution; adding 50-200 ng of pk18mobrpsL plasmid; adding 20-200 ng of the connected target DNA fragment; adding ExnaseTMII, 2 mu L; by ddH2O, complementing the reaction system to 20 mu L; water bath at 37 deg.C for 30 min; coli DH5 alpha competent cells were added and ice-cooled for 20 min; placing in 42 deg.C water bath, heat-shocking for 60s, and ice-bathing for 2 min; adding 900 μ L of preheated SOC resuscitation solution into sterile EP tube, culturing at 37 deg.C for 1h at 220 r/min; taking 100 mu L of bacterial liquid, coating the bacterial liquid on a kanamycin-resistant plate, and culturing for 12h at 37 ℃; randomly selecting a single colony in a plate by using a toothpick, scratching the single colony in a solid culture medium plate, dissolving the colony on the toothpick in a prepared colony PCR reaction system, carrying out colony PCR amplification on the reaction system, screening a correct target strain through a PCR identification result, and naming the strain as escherichia coli DH5 alpha-pk 18mobrpsL-Ptuf-TTHA0571, and placing the plate at 37 ℃ for overnight culture; inoculating the correctly identified strain into LB liquid culture medium with kanamycin, culturing for 10h, centrifuging the thallus and extracting plasmid; a2 mm electric rotor was precooled on ice for about 20min, and the Corynebacterium glutamicum ATCC13032 to be frozen was thawed and the plasmid was added thereto, both in the electric rotor, in ice bath for 20 min. After electrotransfer, the thalli in the electrotransfer cup are resuspended by 900 mu L of preheated BHIS resuscitating fluid, transferred into an EP tube, thermally shocked for 6min at 46 ℃, and then resuscitated for 2min at 32 ℃ and 220r/minh, coating a BHI plate containing kanamycin resistance, and culturing at 32 ℃ for 24 h; randomly selecting a single colony in a plate by using a toothpick, scratching the single colony in a solid culture medium plate, dissolving the colony on the toothpick in a prepared colony PCR reaction system, carrying out colony PCR amplification on the reaction system, screening a correct target strain through a PCR identification result, named as Corynebacterium glutamicum ATCC13032-pk18mobrpsL-Ptuf-TTHA0571, and placing the plate at 32 ℃ for overnight culture; inoculating the correctly identified strain into a BHI liquid culture medium with kanamycin, and culturing for 10-12 h; sucking 3-5 mu L of bacterial liquid into 900 mu L of BHI resuscitation liquid, sucking 100 mu L of resuscitation liquid to spread on a streptomycin resistant plate, and culturing for 24h at 32 ℃; randomly selecting a single colony in a plate by using a toothpick, scratching the single colony in a solid culture medium plate, dissolving the colony on the toothpick in a prepared colony PCR reaction system, carrying out colony PCR amplification on the reaction system, screening a correct target strain according to a PCR identification result, and storing the correctly identified strain, wherein the correctly identified strain is named as corynebacterium glutamicum ATCC 13032-Ptuf-TTHA 0571.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
SEQUENCE LISTING
<110> AoChua Biotechnology Ltd of Jiangsu
<120> Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and application thereof
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 411
<212> DNA
<213> TTHA0571 Gene sequence
<400> 1
atgctggagc gccacgaccg cctggaaacc ctgagaaagc tgaaggaact gcaggagcgc 60
atcgccgagc tcgcctacct cctcaccggg gaggagcccg ccgcctggac ccccagggtg 120
gaccttctgg aaaccgagga gcactacgtc ctcctcgtgg accttcccgg ggtgcgcccc 180
gaggacctgg agcttctgga ggaggggcag cgggtgaccc tggccggggt gcgccacccc 240
ctgcccggca cctacctctt ggaggagagg cccatgggca ccttccgccg caccctggac 300
ctccccgggc ccattgagga ggggacggcc caggccaccc tgcggaacgg ggtcttggag 360
gtccgcttcc gcaagaggcc ggccacggcc cttcccctaa aggaggcgta g 411
<210> 2
<211> 368
<212> DNA
<213> promoter Ptuf Gene sequence
<400> 2
gttaacagat cgtttagatc cgaaggaaaa cgtcgaaaag caatttgctt ttcgacgccc 60
caccccgcgc gttttagcgt gtcagtaggc gcgtagggta agtggggtag cggcttgtta 120
gatatcttga aatcggcttt caacagcatt gatttcgatg tatttagctg gccgttaccc 180
tgcgaatgtc cacagggtag ctggtagttt gaaaatcaac gccgttgccc ttaggattca 240
gtaactggca cattttgtaa tgcgctagat ctgtgtgctc agtcttccag gctgcttatc 300
acagtgaaag caaaaccaat tcgtggctgc gaaagtcgta gccaccacga agtccaggag 360
gaaagctt 368
<210> 3
<211> 39
<212> DNA
<213> primer TTHA0571-1
<400> 3
cgaagtccag gaggaaagct tatgctggag cgccacgac 39
<210> 4
<211> 46
<212> DNA
<213> primer TTHA0571-2
<400> 4
tatcatcaca gagtgcaacc agatcctacg cctcctttag gggaag 46
<210> 5
<211> 49
<212> DNA
<213> primer Ptuf-1
<400> 5
tgttgacctc aaatatcgga agtacaagcg cgtgccctct ttgctgcag 49
<210> 6
<211> 39
<212> DNA
<213> primer Ptuf-2
<400> 6
gtcgtggcgc tccagcataa gctttcctcc tggacttcg 39
<210> 7
<211> 23
<212> DNA
<213> primer up-1
<400> 7
accacgtcga tgtcttttac ctg 23
<210> 8
<211> 25
<212> DNA
<213> lower primer down-2
<400> 8
gagaagttag caaagccacg agtag 25
<210> 9
<211> 463
<212> DNA
<213> up template sequence
<400> 9
accacgtcga tgtcttttac ctgctcgatc acacaaccag ccctcagatc acggtgctgc 60
cgcactcaat ggattatttc gatcaaacgc gcagcgatgc tgttatggct gccatcattg 120
agcaaaaccc tgcgttcgca gaaattaaag gctcacccat tacaaccgca gatgtagccc 180
tccacaatct cggtgacacc aacgccaacc gacgctggca gtctaacgtg ctgcttgccc 240
ggctactcgg gggtattagt gtgcgcggag aggtacctga gcaccagagc cacaaccatc 300
tcgccaagca gtttgccgag gcaaccttgg tcaccaggga cttcgatgtg aattatgatc 360
caacaagcgc tcaccctttt actgctggct tcaactcgat caactatgac accaccttgc 420
tcagcctgta cttcgcaatg ttgacctcaa atatcggaag tac 463
<210> 10
<211> 335
<212> DNA
<213> down template sequence
<400> 10
gatctggttg cactctgtga tgatattcgc ggtgtacttg atcgaggttt agagatctca 60
tctccgaatc atcatgagat ggtggatgct atgcgcaagc agctgcacta tattcaggca 120
ttttaccgtg cctggggacc cattcaacgc cgcttcaatg acgctgaccc agcggtgacc 180
catccgcatc tcacagtgat ctacccaccg ctcacccctg catccgcaga gaaattcaac 240
aagatcacct cagtcgctgc tgtgagcaag cgcccaacca ccctgccgta tttccgtgca 300
gatggttcac ctactcgtgg ctttgctaac ttctc 335
Claims (5)
1. A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 is characterized in that: the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 consists of a corynebacterium glutamicum promoter and a heat-resistant functional gene, the corynebacterium glutamicum promoter is a strong promoter Ptuf, and the heat-resistant functional gene is derived from a TTHA0571 gene in Thermus thermophilus CICC 10647; the TTHA0571 gene sequence is shown in SEQ NO.1, and the strong promoter Ptuf gene sequence is shown in SEQ NO. 2.
2. The application of the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 of claim 1, wherein the temperature of the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 is as follows: the application of the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 in improving the heat resistance of corynebacterium glutamicum is provided.
3. The use of the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 of claim 2, wherein: the Corynebacterium glutamicum thermotolerant element Ptuf-TTHA0571 is introduced into a plasmid and then expressed in Corynebacterium glutamicum.
4. The use of the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 of claim 2, wherein: the Corynebacterium glutamicum thermotolerant element Ptuf-TTHA0571 is integrated on the genome of Corynebacterium glutamicum.
5. The use of the corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 of claim 2, wherein: the high-temperature acclimation is carried out on the corynebacterium glutamicum with the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571, so that the heat resistance of the corynebacterium glutamicum is improved.
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