CN114317538A - A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and its application - Google Patents

Abstract

A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and an application thereof, the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 is composed of a Corynebacterium glutamicum promoter and a heat-resistant functional gene, and the The promoter of Corynebacterium acidophilus is a strong promoter Ptuf, and the thermostable function gene is the TTHA0571 gene derived from Thermus thermophilus CICC10647; the TTHA0571 gene sequence is shown in SEQ NO.1, and the strong promoter The Ptuf gene sequence is shown in SEQ NO.2. The heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum in the present invention can greatly improve the thermal stability of Corynebacterium glutamicum, enhance the heat resistance of the strain, and can be applied to industrialized production of strains, which can greatly reduce the amount of time in the fermentation process. The energy consumption of cooling water is reduced, and the production cost is reduced, and it has a good industrial application prospect.

Description

A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and its application

technical field

The invention relates to a heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum and its application, belonging to the technical field of biological engineering.

Background technique

Corynebacterium glutamicum is currently used for the production of glutamic acid, lysine, branched-chain amino acids, vitamin D, organic acids, etc., and has been widely used in food, medicine, agriculture and other fields. Corynebacterium glutamicum is not only an important industrial microorganism, it is often used as a microbial cell factory, using carbon sources such as glucose, sucrose, xylose to produce amino acids, organic acids, vitamins and fuel ethanol, etc., has become an important biological platform.

The most suitable culture temperature of Corynebacterium glutamicum is generally 26℃-37℃, and the normal fermentation production is generally controlled at about 32℃. To control the fermentation temperature of Corynebacterium glutamicum to 32℃, a series of cooling measures need to be taken. For example, circulating water cooling, freezing water cooling, etc., especially in the high temperature season in summer, the ambient temperature is generally high, and the fermentation process of Corynebacterium glutamicum needs to consume a large amount of cold medium, which often fails to achieve the cooling effect, resulting in abnormal fermentation, not only cooling The system increases a lot of costs, and abnormal fermentation will directly affect the yield, which will increase production and operation costs.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum and its application.

In order to achieve the above-mentioned purpose and other related purposes, the technical scheme provided by the present invention is: a heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum, the heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum is composed of glutamic acid. Corynebacterium promoter and heat-resistant functional gene are composed, the Corynebacterium glutamicum promoter is a strong promoter Ptuf, and the heat-resistant functional gene is the TTHA0571 gene derived from Thermus thermophilus CICC10647; the TTHA0571 gene The sequence is shown in SEQ NO.1, and the sequence of the strong promoter Ptuf gene is shown in SEQ NO.2.

In order to realize the above-mentioned purpose and other related purposes, the technical scheme provided by the invention is: a kind of application of the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 described in claim 1, the Corynebacterium glutamicum heat-resistant element Application of device Ptuf-TTHA0571 in improving heat resistance of Corynebacterium glutamicum.

The preferred technical scheme is as follows: the heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum is introduced into a plasmid and then expressed in Corynebacterium glutamicum.

The preferred technical solution is: integrating the heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum into the genome of Corynebacterium glutamicum.

The preferred technical scheme is as follows: high-temperature domestication of Corynebacterium glutamicum with heat-resistant components Ptuf-TTHA0571 of Corynebacterium glutamicum to improve the heat resistance of Corynebacterium glutamicum.

Due to the application of the above-mentioned technical solutions, the advantages of the present invention compared with the prior art are:

1. The present invention selects a Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 with better heat-resistance effect by constructing a large number of heat-resistant components of Corynebacterium glutamicum. The application of raising the culture temperature of strains can reduce energy consumption and production costs, and has obvious practical application value in production. reference value.

2. The heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum in the present invention can greatly improve the thermal stability of Corynebacterium glutamicum, enhance the heat resistance of strains, and can greatly reduce fermentation when applied to industrialized production strains. The energy consumption of cooling water in the process is reduced, the production cost is reduced, and the invention has a good industrial application prospect.

Description of drawings

Figure 1. Control condition 1 of bacterial growth after high temperature (42°C) culture.

Figure 2. Control condition 2 of bacterial growth after high temperature (42°C) culture.

Detailed ways

The embodiments of the present invention are described below by specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

See Figure 1-2. It should be noted that the structures, proportions, sizes, etc. shown in the drawings in this specification are only used to cooperate with the contents disclosed in the specification, so as to be understood and read by those who are familiar with the technology, and are not used to limit the implementation of the present invention. Restricted conditions, it does not have technical substantive significance, any structural modification, proportional relationship change or size adjustment, without affecting the effect that the present invention can produce and the purpose that can be achieved, should still fall within the present invention. The disclosed technical content must be within the scope of coverage. At the same time, the terms such as "up", "down", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description and clarity, and are not used to limit this specification. The implementable scope of the invention, and the change or adjustment of the relative relationship thereof, shall also be regarded as the implementable scope of the present invention without substantially changing the technical content.

Example 1: A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and its application

The construction method of the heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum includes the following steps:

Step 1: The Thermus thermophilus CICC10647 is activated and inoculated into a liquid medium, and then placed in a shaker at 70-80°C for 20-28 hours to obtain a seed culture solution; the seed culture solution is inoculated into the liquid culture medium , take the bacterial liquid in the logarithmic growth phase to extract the genome;

Liquid medium components: yeast powder 4.0g; polypeptone 8.0g; NaCl 2.0g; distilled water 1L; adjust pH to 7.0.

Bacterial genome extraction method:

Using the Bacterial Genomic DNA Purification Kit (Wizard Genomic DNA Purification KitA1120), follow these steps:

(1) Take out the pretreated Corynebacterium glutamicum cells from -80°C, add 600 μL of cell lysate to each tube, and gently invert or use a pipette to evenly disperse the cells in the lysate. In the solution, the EP tube was placed in a constant temperature water bath at 80 °C and incubated for 5 min to accelerate the lysis of the cells.

(2) Cool the lysis system to room temperature, add 2 μL of RNase solution to each tube (use the RNase solution with a concentration of 50 mg/mL provided by the kit or prepared by yourself), and incubate in a water bath at 37°C for 30-90 min.

(3) Add 200 μL of protein separation solution to each tube, and quickly invert the solution to fully mix the solution. After obvious white flocculent precipitation appears, place the 1.5 mL EP tube in an ice bath for 2-3 minutes, at 4°C Centrifuge for 10-20 min at 10,000-13,000 r/min, and transfer 400-600 μL of the supernatant to a new 1.5 mL EP tube.

(4) Add an equal volume of isopropanol or twice the volume of absolute ethanol to the 1.5mL EP tube, and gently invert the solution to fully mix the solution. At this time, the genomic DNA will be precipitated in a form visible to the naked eye. Intuitively judge whether the extraction of genomic DNA is successful.

(5) Centrifuge the 1.5 mL EP tube at 4°C for 1 min, with a rotating speed of 12000 r/min, and discard the supernatant.

(6) Add 700 μL of 70% ethanol solution (-20°C) to a 1.5mL EP tube, invert gently to make the genomic DNA fully contact the ethanol solution, and centrifuge at 4°C for 1 min at 12000 r /min, discard the supernatant, and repeat this step 2-3 times.

(7) Carefully suck up the residual ethanol solution as much as possible with a pipette, and leave the 1.5mL EP tube open for 1-2 hours to fully evaporate the ethanol.

(8) Add 40-100 μL buffer solution (pH 7.0-8.0) or ddH ₂ O to a 1.5 mL EP tube, and let stand for 1 h at room temperature or 12 h at 4° C. to fully dissolve the genomic DNA.

(9) Transfer 10 μL of genomic DNA solution to a new 1.5 mL EP tube with a pipette for quality control analysis, and store the remaining solution at -80°C.

Step 2: Using the genome extracted in step 1 as a template, the above primer TTHA0571-1 and the lower primer TTHA0571-2 are used for PCR amplification;

On primer TTHA0571-1:

5'-CGAAGTCCAGGAGGAAAGCTT ATGCTGGAGCGCCACGAC-3';

Under primer TTHA0571-2:

5'-TATCATCACAGAGTGCAACCAGATCCTACGCCTCCTTTAGGGGAAG-3'.

Step 3: Inoculate the activated Corynebacterium glutamicum ATCC 13032 into a liquid LB medium, place it in a shaker at 30-34°C for 20-28 hours, and obtain a seed culture solution; inoculate the seed culture solution into the liquid LB medium , take the bacterial liquid in the logarithmic growth phase to extract the genome; the genome extraction method is the same as above.

Step 4: using the genome extracted in step 3 as a template, the above primer Ptuf-1 and the lower primer Ptuf-2 are used for PCR amplification;

On primer Ptuf-1:

5'-TGTTGACCTCAAATATCGGAAGTACAAGCGCGTGCCCTCTTTGCTGCAG-3';

Lower primer Ptuf-2: 5'-GTCGTGCGCTCCAGCAT AAGCTTTCCTCCTGGACTTCG-3'.

Step 5: The promoter Ptuf is connected to the DNA fragment of the heat-resistant functional gene TTHA0571; the primer pairs used are: upper primer up-1, lower primer down-2;

Upper primer up-1: 5'-ACCACGTCGATGTCTTTTACCTG-3';

Lower primer down-2: 5'-GAGAAGTTAGCAAAGCCACGAGTAG-3'.

Step 6: C. glutamicum conjugation transformation.

In step 2, the PCR amplification system of the heat-resistant functional gene TTHA0571: to prepare a 50 μL enzyme digestion reaction system, first add 20 μL ddH ₂ O to a 0.2 mL PCR tube; add 10 μL of 5× PrimeSTAR Buffer solution; add 4 μL of dNTP solution; add 2 μL of upper primer TTHA0571-1 and lower primer TTHA0571-2 solution; add template with total DNA not exceeding 200 ng; add 0.5 μL PrimeSTAR HS DNA Polymerase solution; add 50 μL of ddH ₂ O to the reaction system and pipette Repeated pipetting and mixing; PCR amplification parameters of the heat-resistant functional gene TTHA0571 were: 95°C pre-denaturation for 5 min; 30 cycles including 98°C denaturation for 10s, 55°C annealing for 5s and 72°C extension for 30s; 72°C extension for 10 min.

In step 4, the PCR amplification system of the promoter Ptuf: to prepare a 50 μL enzyme digestion reaction system, first add 20 μL of ddH ₂ O to a 0.2 mL PCR tube; add 10 μL of 5× PrimeSTAR Buffer solution; add 4 μL of dNTP solution; Add 2 μL each of the upper primer Ptuf-1 and the lower primer Ptuf-2 solution; add a template with a total DNA not exceeding 200 ng; add 0.5 μL PrimeSTAR HS DNA Polymerase solution; add 50 μL of ddH ₂ O to the reaction system and blow it repeatedly with a pipette Aspirate and mix well; the PCR amplification parameters of the promoter Ptuf are: pre-denaturation at 95°C for 5 min; 30 cycles including denaturation at 98°C for 10s, annealing at 55°C for 5s and extension at 72°C for 30s; extension at 72°C for 10 min.

In step 5, prepare a 50 μL digestion reaction system, first add 20 μL ddH ₂ O to a 0.2 mL PCR tube; add 10 μL 5× PrimeSTAR Buffer solution; add 4 μL dNTP solution; add upper primer up-1 and lower primer 2 μL of each down-2 solution; add 1 μL of up template, 2.5 μL of Ptuf template (amplification product of step 4), 0.9 μL of TTHA0571 template (amplified product of step 2), and 1.7 μL of down template; add 0.5 μL PrimeSTAR HS DNA Polymerase solution; add 50 μL of ddH ₂ O to the reaction system and mix by pipetting repeatedly; the parameters of the PCR instrument are set as: 95°C pre-denaturation for 5 min; 30 cycles include 98°C denaturation for 10s and 55°C annealing for 5s and 72°C for 2 min; 72°C for 10 min.

up template sequence:

ACCACGTCGATGTCTTTTACCTGCTCGATCACACAACCAGCCCTCAGATCACGGTGCTGCCGCACTCAATGGATTATTTCGATCAAACGCGCAGCGATGCTGTTATGGCTGCCATCATTGAGCAAAACCCTGCGTTCGCAGAAATTAAAGGCTCACCCATTACAACCGCAGATGTAGCCCTCCACAATCTCGGTGACACCAACGCCAACCGACGCTGGCAGTCTAACGTGCTGCTTGCCCGGCTACTCGGGGGTATTAGTGTGCGCGGAGAGGTACCTGAGCACCAGAGCCACAACCATCTCGCCAAGCAGTTTGCCGAGGCAACCTTGGTCACCAGGGACTTCGATGTGAATTATGATCCAACAAGCGCTCACCCTTTTACTGCTGGCTTCAACTCGATCAACTATGACACCACCTTGCTCAGCCTGTACTTCGCAATGTTGACCTCAAATATCGGAAGTAC。

down template sequence:

GATCTGGTTGCACTCTGTGATGATATTCGCGGTGTACTTGATCGAGGTTTAGAGATCTCATCTCCGAATCATCATGAGATGGTGGATGCTATGCGCAAGCAGCTGCACTATATTCAGGCATTTTACCGTGCCTGGGGACCCATTCAACGCCGCTTCAATGACGCTGACCCAGCGGTGACCCATCCGCATCTCACAGTGATCTACCCACCGCTCACCCCTGCATCCGCAGAGAAATTCAACAAGATCACCTCAGTCGCTGCTGTGAGCAAGCGCCCAACCACCCTGCCGTATTTCCGTGCAGATGGTTCACCTACTCGTGGCTTTGCTAACTTCTC。

Step 6 specifically includes: preparing a 20 μL in vitro ligation system, first adding 5 μL of ddH ₂ O to a 0.2 mL PCR tube; adding 4 μL of 5×CE butter solution; adding 50-200 ng of pk18mobrpsL plasmid; adding the ligated purpose obtained in step 5 DNA fragment 20～200ng; add ExnaseTMII 2μL; make up the reaction system with ddH ₂ O to 20μL; 37℃ water bath for 30min; add E.coli DH5α competent cells, ice bath for 20min; put in 42℃ water bath, heat shock 60s, ice Bath for 2 min; add 900 μL of pre-warmed SOC resuscitation solution to a sterile EP tube, incubate for 1 h at 37 °C, 220 r/min; take 100 μL of bacterial solution, spread it on kanamycin-resistant plates, and incubate at 37 °C for 12 h; randomly Select a single colony in the plate, carry out colony PCR identification, screen the correct target strain, name it Escherichia coli DH5α-pk18mobrpsL-Ptuf-TTHA0571, and place the plate at 37 °C for overnight cultivation; In the LB liquid medium of glutamic acid, cultured for 10h, centrifuged the cells to extract plasmids; pre-cooled the 2mm electroporation cup on ice for about 20min, thawed the competent cells of Corynebacterium glutamicum ATCC13032 to be frozen, added the plasmids, and added electroporation. After electroporation, resuspend the bacteria in the electroporation cup with 900 μL of preheated BHIS recovery solution, transfer it into an EP tube, heat shock at 46 °C for 6 min, recover at 32 °C and 220 r/min for 2 h, apply Cloth the BHI plate containing kanamycin resistance and incubate at 32°C for 24h; randomly select a single colony on the plate with a toothpick, first put it on the solid medium plate, and then dissolve the colony on the toothpick in the prepared colony PCR In the reaction system, the reaction system was subjected to colony PCR amplification, and the correct target strain was screened by the PCR identification results, named Corynebacterium glutamicum ATCC 13032-pk18mobrpsL-Ptuf-TTHA0571, and the plate was placed at 32 °C for overnight cultivation; the identification was correct. The bacterial strains were inoculated into the BHI liquid medium with kanamycin, and cultivated for 10-12 h; suck 3-5 μL of the bacterial solution into 900 μL of BHI recovery solution, and suck 100 μL of the above-mentioned recovery solution to streptomycin-resistant plate coating, 32 Cultivated at ℃ for 24h; randomly select single colonies on the plate with toothpicks, first put them on the solid medium plate, then dissolve the colonies on the toothpick in the prepared colony PCR reaction system, and perform colony PCR amplification on the reaction system. The correct target strains were screened by PCR identification results, and the correctly identified strains were saved and named as Corynebacterium glutamicum ATCC13032-Ptuf-TTHA0571.

Plasmid extraction method:

According to the instructions provided by the plasmid extraction kit (Plasmid Mini Kit I), the main process is as follows:

(1) Take 1.5-5 mL of bacterial liquid and centrifuge it in a 1.5 mL EP tube to collect bacterial cells, and the centrifugation conditions are 12000 r/min for 3 min;

(2) Remove the supernatant, add 250 μL of the Solution I solution (containing RNase A) provided by the kit, and resuspend the bacterial cells by repeated pipetting or vortexing;

(3) Add 250 μL of Solution II solution, gently invert and mix 3-5 times, and let stand for 2 min at room temperature;

(4) Add 250 μL of Solution III solution, invert and mix gently until a white precipitate is formed, ice bath for 2 min, and centrifuge at 12000 r/min for 15 min;

(5) Transfer 600 μL of the supernatant to the adsorption column, centrifuge for 1 min at 12000 r/min, and discard the filtrate;

(6) Add 500 μL of Buffer HB, centrifuge at 12000 r/min for 1 min, and discard the filtrate;

(7) Add 700 μL of Buffer WB (diluted with ethanol), centrifuge at 12000 r/min for 1 min, discard the filtrate, and repeat this step once;

(8) Centrifuge the adsorption column at 12000r/min for 2min, and replace the collection tube with a clean 1.5mL EP tube;

(9) Add 50-100 μL of Buffer EB or ddw to the adsorption column, and centrifuge at 12000 r/min for 1 min.

Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 heat resistance verification:

Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 is a strain of Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 integrated into the genome of Corynebacterium glutamicum ATCC 13032.

Streak Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 and Corynebacterium glutamicum ATCC 13032 from a glycerol tube to a plate, culture at 32°C for 24h, pick a single colony from the plate and inoculate it in 3mL liquid medium, and culture at 32°C 16h, detect _OD600 , according to _OD600 =10, inoculate 200μL volume of inoculum in 500mL fermentation shake flask with 20mL liquid medium, place in 200rpm shaker for 24h at 42°C, measure its _OD600 value. As shown in Figure 1, it can be seen from Figure 1 that after C. glutamicum ATCC 13032-Ptuf-TTHA0571 and C. glutamicum ATCC 13032 were cultured at high temperature (42°C) for 24 hours, Compared with ATCC 13032, the OD ₆₀₀ of ATCC 13032-Ptuf-TTHA0571 was significantly better than that of ATCC 13032 at high temperature (42℃).

Corynebacterium glutamicum ATCC 13032-pk18mobrpsL-Ptuf-TTHA0571 heat resistance verification:

Corynebacterium glutamicum ATCC 13032-pk18mobrpsL-Ptuf-TTHA0571 is a strain of Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 introduced into the plasmid pk18mobrpsL vector and then expressed in Corynebacterium glutamicum ATCC 13032.

Streak Corynebacterium glutamicum ATCC 13032-pk18mobrpsL-Ptuf-TTHA0571 and Corynebacterium glutamicum ATCC13032 from a glycerol tube to a plate, incubate at 32°C for 24h, pick a single colony from the plate and inoculate it in 3mL liquid medium at 32°C Incubate for 16h, detect _OD600 , according to _OD600 =10, inoculate 200μL volume of inoculum in 500mL fermentation shake flask with 20mL liquid medium, place in 200rpm shaker for 24h at 42°C, and measure its _OD600 value. As shown in Fig. 2, it can be seen from the results that after C. glutamicum ATCC 13032-pk18mobrpsL-Ptuf-TTHA0571 and C. glutamicum ATCC 13032 were cultured at The OD ₆₀₀ of TTHA0571 was 18.72% higher than that of ATCC13032, and the growth of ATCC 13032-pk18mobrpsL-Ptuf-TTHA0571 at high temperature (42℃) was significantly better than that of ATCC 13032.

High temperature acclimation of Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571

Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 was streaked from a glycerol tube to activated on a plate, cultured at 32°C for 24 hours, and after multiple high temperature (42°C) cultivation and acclimation, the cultured fermentation broth was serially diluted, and 50 μL was drawn. The serially diluted fermentation broth was coated on a plate, cultured at 32°C for 16 hours, and a single colony with a cell morphology and good growth was selected from the plate for preservation. The strain after high temperature acclimation was named as Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 variant. The Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571 variant, Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571, and Corynebacterium glutamicum ATCC 13032 were cultured under the same conditions at 42°C for control experiments, and the OD ₆₀₀ was detected after culturing for 24 hours. The results of OD ₆₀₀ value showed that ATCC 13032-Ptuf-TTHA0571 variant>ATCC 13032-Ptuf-TTHA0571>ATCC13032, indicating that after high temperature acclimation, the heat resistance of C. glutamicum ATCC 13032-Ptuf-TTHA0571 variant was further enhanced .

The Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and its application in Corynebacterium glutamicum according to the invention can significantly improve the heat resistance of Corynebacterium glutamicum under high temperature culture conditions. On the one hand, it can improve the cultivation problem of Corynebacterium glutamicum, accelerate the growth and reproduction of bacteria, and reduce the energy consumption required for cooling in the fermentation and cultivation process; In the event of a system failure, the strain with the heat-resistant components can greatly improve the stability of the strain, minimize the impact on the growth of the bacteria under abnormally high temperature conditions, and is conducive to the stability of the fermentation of Corynebacterium glutamicum. Production. The Corynebacterium glutamicum heat-resistant component can be widely used in the production of amino acids, organic acids, vitamins, fuel ethanol and other products using Corynebacterium glutamicum as a biotechnology platform. The technology of the invention is suitable for industrialized production and promotion, and has significant advantages. advanced nature and better application prospects.

SEQ NO.1: TTHA0571 gene sequence:

ATGCTGGAGC GCCACGACCG CCTGGAAACC CTGAGAAAGC TGAAGGAACT GCAGGAGCGCATCGCCGAGC TCGCCTACCT CCTCACCGGG GAGGAGCCCG CCGCCTGGAC CCCCAGGGTG GACCTTCTGGAAACCGAGGA GCACTACGTC CTCCTCGTGG ACCTTCCCGG GGTGCGCCCC GAGGACCTGG AGCTTCTGGAGGAGGGGCAG CGGGTGACCC TGGCCGGGGT GCGCCACCCC CTGCCCGGCA CCTACCTCTT GGAGGAGAGGCCCATGGGCA CCTTCCGCCG CACCCTGGAC CTCCCCGGGC CCATTGAGGA GGGGACGGCC CAGGCCACCCTGCGGAACGG GGTCTTGGAG GTCCGCTTCC GCAAGAGGCC GGCCACGGCC CTTCCCCTAA AGGAGGCGTAG。

SEQ NO.2: promoter Ptuf gene sequence

GTTAACAGAT CGTTTAGATC CGAAGGAAAA CGTCGAAAAG CAATTTGCTT TTCGACGCCCCACCCCGCGC GTTTTAGCGT GTCAGTAGGC GCGTAGGGTA AGTGGGGTAG CGGCTTGTTA GATATCTTGAAATCGGCTTT CAACAGCATT GATTTCGATG TATTTAGCTG GCCGTTACCC TGCGAATGTC CACAGGGTAGCTGGTAGTTT GAAAATCAAC GCCGTTGCCC TTAGGATTCA GTAACTGGCA CATTTTGTAA TGCGCTAGATCTGTGTGCTC AGTCTTCCAG GCTGCTTATC ACAGTGAAAG CAAAACCAAT TCGTGGCTGC GAAAGTCGTAGCCACCACGA AGTCCAGGAG GAAAGCTT。

Example 2: A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and its application

The primers and materials used were the same as those in Example 1.

A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571, the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 is composed of a Corynebacterium glutamicum promoter and a heat-resistant functional gene, and the glutamic acid stick The Bacillus promoter is a strong promoter Ptuf, and the thermostable function gene is the TTHA0571 gene derived from Thermus thermophilus CICC10647; the TTHA0571 gene sequence is shown in SEQ NO.1, and the strong promoter Ptuf gene sequence As shown in SEQ NO.2.

The application of the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 in improving the heat resistance of Corynebacterium glutamicum.

The heat-resistant component Ptuf-TTHA0571 of C. glutamicum was introduced into the plasmid and then expressed in C. glutamicum.

The thermostable component Ptuf-TTHA0571 of Corynebacterium glutamicum was integrated into the genome of Corynebacterium glutamicum.

The high temperature acclimation of Corynebacterium glutamicum with the heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum improves the heat resistance of Corynebacterium glutamicum.

Thermus thermophilus CICC 10647 was activated and inoculated into liquid LB medium and cultured in a shaker at 75°C for 24 hours to obtain a seed culture solution; the seed culture solution was inoculated into the liquid culture medium, and the bacteria in the logarithmic growth phase were taken. Liquid extracted genome.

The construction method of the heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum includes:

PCR amplification of heat-resistant functional gene TTHA0571: To prepare a 50 μL digestion reaction system, first add 20 μL ddH ₂ O to a 0.2 mL PCR tube; add 10 μL 5×PrimeSTAR Buffer solution; add 4 μL dNTP solution; add primers TTHA0571-1, Add 2 μL of primer TTHA0571-2 solution each; add template with total DNA not exceeding 200 ng; add 0.5 μL PrimeSTAR HS DNA Polymerase solution; add 50 μL of ddH ₂ O to the reaction system and mix by pipette repeatedly; the parameters of the PCR instrument are set to : Pre-denaturation at 95°C for 5 min; 30 cycles including denaturation at 98°C for 10s, annealing at 55°C for 5s and extension at 72°C for 30s; extension at 72°C for 10 min.

Corynebacterium glutamicum ATCC 13032 was activated and inoculated in liquid LB medium and placed in a shaker at 32°C for 24 hours to obtain a seed culture solution; the seed culture solution was inoculated into the liquid medium, and the bacteria in the logarithmic growth phase were taken. Liquid extracted genome.

PCR amplification of promoter Ptuf: To prepare a 50 μL digestion reaction system, first add 20 μL ddH ₂ O to a 0.2 mL PCR tube; add 10 μL 5× PrimeSTAR Buffer solution; add 4 μL dNTP solution; add upper primer Ptuf-1 and lower primer Ptuf -2 solution of 2 μL each; add template with total DNA not exceeding 200 ng; add 0.5 μL PrimeSTAR HS DNAPolymerase solution; add 50 μL of ddH ₂ O to the reaction system and mix by pipette repeatedly; the parameters of the PCR instrument are set to: 95°C Pre-denaturation for 5 min; 30 cycles including denaturation at 98 °C for 10 s, annealing at 55 °C for 5 s and extension at 72 °C for 30 s; extension at 72 °C for 10 min.

Ligation of the promoter Ptuf and the DNA fragment of the thermostable gene TTHA0571: To prepare a 50 μL digestion reaction system, first add 20 μL ddH ₂ O to a 0.2 mL PCR tube; add 10 μL 5×PrimeSTAR Buffer solution; add 4 μL dNTP solution; add primers 2 μL each of up-1 and down-2 primers; add 1 μL up template, 2.5 μL Ptuf template, 0.9 μL TTHA0571 template, 1.7 μL down template; add 0.5 μL PrimeSTAR HS DNA Polymerase solution; add ddH ₂ O The reaction system was supplemented with 50 μL and mixed with a pipette repeatedly; the parameters of the PCR instrument were set as: 95°C pre-denaturation for 5 min; 30 cycles including 98°C denaturation for 10s, 55°C annealing for 5s and 72°C extension for 2min; 72°C extension for 10min .

Combined transformation of Corynebacterium glutamicum: To prepare 20 μL of in vitro ligation system, first add 5 μL of ddH ₂ O to a 0.2 mL PCR tube; add 4 μL of 5×CE butter solution; add 50-200 ng of pk18mobrpsL plasmid; add the above ligated target DNA fragment 20～200ng; add Exnase ^TM II 2μL; make up the reaction system with ddH ₂ O to 20μL; 37°C water bath for 30min; add E.coli DH5α competent cells, ice bath for 20min; place in 42°C water bath, heat shock for 60s, ice Bath for 2 min; add 900 μL of pre-warmed SOC resuscitation solution to a sterile EP tube, incubate for 1 h at 37 °C, 220 r/min; take 100 μL of bacterial solution, spread it on kanamycin-resistant plates, and incubate at 37 °C for 12 h; The toothpick randomly selects a single colony in the plate, firstly draws it on the solid medium plate, and then dissolves the colony on the toothpick in the prepared colony PCR reaction system. The correct target strain was named Escherichia coli DH5α-pk18mobrpsL-Ptuf-TTHA0571, and the plate was cultured at 37°C overnight; the correctly identified strain was inoculated into LB liquid medium with kanamycin, cultured for 10 hours, and centrifuged. Extract the plasmid; pre-cool the 2mm electric rotor cup on ice for about 20 minutes, thaw the competent cells of Corynebacterium glutamicum ATCC13032 to be frozen and add the plasmid to the rotor cup, and ice bath for 20 minutes. After electroporation, the cells in the electroporation cup were resuspended with 900 μL of preheated BHIS recovery solution, transferred to EP tubes, heat-shocked at 46°C for 6 min, recovered at 32°C and 220 r/min for 2 h, and coated with kanamycin. Resistant BHI plates were incubated at 32°C for 24 hours; single colonies on the plate were randomly selected with toothpicks, firstly placed on solid medium plates, and then the colonies on the toothpicks were dissolved in the prepared colony PCR reaction system, and the reaction Colony PCR amplification was carried out in the system, and the correct target strain was screened according to the PCR identification results, which was named as Corynebacterium glutamicum ATCC13032-pk18mobrpsL-Ptuf-TTHA0571, and the plate was placed at 32 °C for overnight cultivation; the correctly identified strain was inoculated with kana In the BHI liquid medium of BHI, culture for 10-12 h; suck 3-5 μL of bacterial solution into 900 μL of BHI resuscitation solution, draw 100 μL of the above-mentioned resuscitation solution to streptomycin-resistant plate coating, and incubate at 32 °C for 24 h; randomize with toothpicks Pick a single colony on the plate, first put it on the solid medium plate, then dissolve the colony on the toothpick in the prepared colony PCR reaction system, carry out the colony PCR amplification of the reaction system, and screen the correct purpose according to the PCR identification result. The strains identified correctly were saved and named as Corynebacterium glutamicum ATCC 13032-Ptuf-TTHA0571.

The above descriptions are only used to explain the preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Therefore, any modification or change of the present invention should be made within the same spirit of the invention. , all should still be included in the scope of the intended protection of the present invention.

SEQUENCE LISTING

<110> Jiangsu Aochuang Biotechnology Co., Ltd.

<120> A Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 and its application

<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> Lower 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> Lower 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> down 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 is made up of a Corynebacterium glutamicum promoter and a heat-resistant functional gene, The C. glutamicum promoter is a strong promoter Ptuf, and the heat-resistant functional gene is the TTHA0571 gene derived from Thermus thermophilus CICC10647; the TTHA0571 gene sequence is shown in SEQ NO. The strong promoter Ptuf gene sequence is shown in SEQ NO.2. 2. the application of the described Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 of claim 1, it is characterized in that: described Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 is improving the Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 heat resistance applications. 3. the application of Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 according to claim 2, is characterized in that: Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 is imported into plasmid and then in Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 internal expression. 4. the application of the heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum according to claim 2, is characterized in that: the heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum is integrated on the genome of Corynebacterium glutamicum . 5. the application of Corynebacterium glutamicum heat-resistant component Ptuf-TTHA0571 according to claim 2, is characterized in that: carry out the Corynebacterium glutamicum with the heat-resistant component Ptuf-TTHA0571 of Corynebacterium glutamicum High temperature acclimation improves the heat resistance of Corynebacterium glutamicum.

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