Heat-resistant element from Thermus thermophilus and application of heat-resistant element

Abstract

The invention belongs to the technical field of biology, and particularly relates to a heat-resistant element newly derived from Thermus thermophilus and application of the heat-resistant element. The heat-resistant gene is derived from Thermus thermophilus, is connected with a promoter and is transformed into Corynebacterium glutamicum. Experimental results show that after the corynebacterium glutamicum is cultured at the high temperature of 42 DEG C for 24 hours, the OD600 of the corynebacterium glutamicum carrying the heat-resistant elements and plasmids is obviously higher than that of a control strain ATCC 13032, which indicates that the corynebacterium glutamicum has obviously better growth capability under the high-temperature condition than that of a starting strain, and the effectiveness of the heat-resistant elements in the aspect of improving the heat resistance of the strains is verified. The method is of great significance to improvement of the thermal stability of the corynebacterium glutamicum and application prospects of the corynebacterium glutamicum in industrial production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a new heat-resistant element derived from Thermus thermophilus and application thereof. Background Art

[0002] In the field of industrial fermentation, culturing Corynebacterium glutamicum at higher temperatures can bring a variety of potential benefits to the fermentation process. This includes reducing cooling costs, reducing the consumption of water and electricity resources, optimizing fermentation conditions, and reducing pollution risks. In some cases, increasing the fermentation temperature may also accelerate the growth of microorganisms and increase the overall biomass, making it particularly important to enhance the strain's high temperature tolerance in industrial applications.

[0003] Metabolic engineering aims to transform the existing metabolic pathways of cells, mainly through gene knockout, introduction of exogenous genes and overexpression of key genes, to increase the content of specific metabolites, or give cells the ability to synthesize new metabolites, thereby optimizing product generation and cell performance, so that they can adapt to various environmental pressures. Thermus thermophilus CICC 10647 is a thermophilic bacterium with great heat resistance, which is widely used in high-temperature industrial processes and biotechnology. It contains a variety of heat-resistant and stress-resistant elements, such as heat shock proteins and specific membrane proteins, which help cells cope with the stress caused by high temperatures and maintain the function and integrity of cells. Therefore, metabolic engineering technology can be used to extract these effective heat-resistant genes, transfer them into Corynebacterium glutamicum, and use the resistance of exogenous genes to environmental stress to fully express them in Corynebacterium glutamicum cells to adapt to high temperature environmental stress. Summary of the invention

[0004] The present invention obtains heat-resistant genes from Thermus thermophilus CICC 10647 through research, including TTHA0151, TTHA0675, TTHA0700, TTHA1480, TTHA1488 and TTHA1621. The nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.6 respectively.

[0005] The TTHA0151 gene is named MoaD, which is a sulfur carrier protein that helps bacterial cells balance sulfur metabolism in high temperature environments;

[0006] The TTHA0675 gene is named ThiS, which is a sulfur carrier protein. Its full name is thiamine biosynthetic protein. Thiamine is a cofactor of many enzymes and participates in important biochemical reactions such as energy metabolism and protein synthesis.

[0007] The TTHA0700 gene is named ATPase, which provides energy to cells by hydrolyzing ATP;

[0008] The TTHA1480 and TTHA1621 genes both belong to the HSP20 family and are heat shock proteins;

[0009] The TTHA1488 gene is named CbpM, which is a chaperone regulatory protein and is DnaK-DnaJ assembly factor A.

[0010] Furthermore, the present invention provides a heat-resistant element, characterized in that it consists of a promoter and a heat-resistant gene, and the heat-resistant gene is selected from TTHA0151, TTHA0675, TTHA0700, TTHA1480, TTHA1488 or TTHA1621 derived from Thermus thermophilus, or a gene of a degenerate sequence thereof.

[0011] In particular, the promoter is a constitutive or inducible promoter, in particular the promoter Ptuf.

[0012] The present invention also provides a vector containing the heat-resistant element, such as an expression vector, and is particularly suitable for transforming Corynebacterium glutamicum.

[0013] The present invention further provides use of the heat-resistant element or the vector in improving the heat resistance of Corynebacterium glutamicum.

[0014] The present invention particularly provides a Corynebacterium glutamicum with improved heat resistance, which is achieved by introducing the heat-resistant element into Corynebacterium glutamicum to improve the heat resistance of Corynebacterium glutamicum.

[0015] Specifically, the method of introduction is to transform Corynebacterium glutamicum by combining the vector.

[0016] More specifically, the specific operation of introduction is to add the competent cells of Corynebacterium glutamicum and the vector into the electroporation cup, resuspend them in BHIS after ice bath, protect them at 46°C for 5-8min, resuscitate them at 30-34°C and 200-240r / min, then spread them on BHI plates, and culture them at 30-34°C to obtain single colonies for identification.

[0017] Optionally, the obtained single colony strain is further verified for heat resistance, specifically by culturing at a high temperature of 40-44°C to observe the growth.

[0018] The advantages of the present invention compared with the prior art are:

[0019] 1. The present invention obtains a variety of heat-resistant elements from Corynebacterium glutamicum by screening, and then successfully develops Corynebacterium glutamicum with excellent heat resistance. This innovative method not only provides a new perspective for the study of stress resistance of Corynebacterium glutamicum, but also lays a solid foundation for the design and development of related stress resistance elements.

[0020] 2. The Corynebacterium glutamicum of the present invention significantly improves thermal stability, so that it can still maintain high growth activity under high temperature environment. This characteristic is crucial for many industrial applications, which can improve production efficiency and reduce yield losses caused by temperature fluctuations.

[0021] 3. The heat-resistant element and modified strain provided by the present invention have wide application potential and can play an important role in many fields such as food and medicine. Its superior heat resistance makes it possible to be used in high-temperature fermentation and other extreme conditions, providing new technical support for the development of related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The growth of Corynebacterium glutamicum containing heat-resistant elements and plasmids compared with Corynebacterium glutamicum ATCC 13032 under high temperature environment. DETAILED DESCRIPTION

[0023] Example 1: Mining and amplification of heat-resistant genes of Thermus thermophilus

[0024] 1. Extraction of the genome of Thermusthermophilus CICC 10647

[0025] The composition of the culture medium is 4.0g of yeast powder, 8.0g of polypeptone, and 2.0g of NaCl per 1L of distilled water, and the pH is adjusted to neutral. Seed culture fluid extraction method: After Thermusthermophilus CICC 10647 is activated, it is inoculated into the liquid culture medium and cultured on a shaker at 70-80°C for 20-28h to obtain the seed culture fluid. The seed culture fluid is inoculated into a new liquid culture medium, and the bacterial fluid is extracted during the logarithmic growth phase to obtain the genome. The genome extraction method is operated according to the kit (Wizard Genomic DNA Purification KitA1120).

[0026] 2. Screening of heat-resistant genes from Thermusthermophilus CICC 10647

[0027] By searching the literature, we collected 10 genes related to heat resistance in Thermusthermophilus CICC 10647, including TTHA0059 (MoaE-MoaD fusion protein), TTHA0151 (MoaD), TTHA0508 (HspR), TTHA0571 (Hsp20), TTHA0675 (ThiS), TTHA0700 (ATPase), TTHA0894 (Hsp33), TTHA1480 (Hsp20), TTHA1488 (CbpM) and TTHA1621 (Hsp20). After preliminary research and verification, we finally selected six genes, TTHA0151, TTHA0675, TTHA0700, TTHA1480, TTHA1488 and TTHA1621, for subsequent experiments.

[0028] 3. Amplification of heat-resistant genes from Thermusthermophilus CICC 10647

[0029] The Thermusthermophilus CICC 10647 genome extracted in the first part was used as a template. Forward primers and reverse primers were designed for the genes screened in the second part, and PCR amplification was performed.

[0030] The PCR amplification system is to add 20 μL of deionized water (ddH2O), 10 μL of 5× PrimeSTAR buffer, 4 μL of dNTP, 2 μL of upper and lower primers, and template DNA (total amount less than 200 ng) into the PCR tube, and add 0.5 μL of PrimeSTAR HSDNA polymerase. The total volume of the enzyme digestion reaction system is 50 μL, which is supplemented with deionized water. The specific parameters of amplification are: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 55℃ annealing for 5 s and 72℃ extension for 30 s, 30 cycles; and finally extension at 72℃ for 10 min.

[0031] Table 1 Primer list

[0032]

[0033] Example 2: Construction and expression of the heat-resistant element of Corynebacterium glutamicum

[0034] 1. Construction of the heat-resistant element of Corynebacterium glutamicum

[0035] First, Corynebacterium glutamicum ATCC 13032 was activated and then inoculated in LB medium. Corynebacterium glutamicum ATCC 13032 was cultured in a shaking incubator at 30 to 34°C for 20 to 28 hours to obtain a seed culture solution. The seed culture solution was inoculated into a new LB medium, and the bacterial solution in the logarithmic growth phase was extracted therefrom to obtain a genome. The genome extraction method was performed according to the kit (Wizard Genomic DNA Purification Kit A1120).

[0036] PCR amplification of the aforementioned extracted genome of Corynebacterium glutamicum ATCC 13032 was used as a template. Both constitutive and inducible promoters can be expressed in this embodiment, but the strong promoter Ptuf has the best effect, so it is used as an example for subsequent experiments.

[0037] Primers were designed for Ptuf (nucleotide sequence as shown in SEQ ID NO.7) to amplify the promoter Ptuf fragment, and primers were further designed to connect the promoter Ptuf with the heat-resistant gene DNA fragment to obtain the heat-resistant element. The up template sequence and the down template sequence are shown in SEQ NO.8-SEQ ID NO.9.

[0038] 2. Conjugative transformation of Corynebacterium glutamicum

[0039] In a PCR tube, the components used include 5 μL of deionized water (ddH2O), 4 μL of 5×CE buffer, 50-200 ng of pk18mobrpsL plasmid, 20-200 ng of the target DNA fragment, i.e., the heat-resistant element constructed in the first part, and Exnase TM II 2 μL. The total volume of the in vitro ligation system is 20 μL, which is made up to this volume with deionized water.

[0040] The system was reacted in a 37°C water bath for 30 minutes, then E. coli DH5α competent cells were added, ice bathed for 20 minutes, then treated in a 42°C water bath for 60 seconds, and finally ice bathed for 2 minutes. Subsequently, 900 μL of SOC recovery solution was added to the EP tube and cultured at 37°C and 220 r / min for 1 hour. 100 μL of bacterial solution was taken to coat the plate and cultured at 37°C for 12 hours. Single colony PCR identification was then performed, and the plate was cultured overnight at 37°C.

[0041] The identified strains were inoculated into LB liquid medium, cultured for 10 hours, and then centrifuged to extract the plasmid. For the competent cells and plasmids of Corynebacterium glutamicum ATCC 13032, they were added to the electroporation cup, ice-bathed for 20 minutes, resuspended in BHIS, and transferred to EP tubes, treated in a 46°C water bath for 6 minutes, then revived at 32°C and 220r / min for 2 hours, and finally coated on BHI plates and cultured at 32°C for 24 hours. Pick a single colony, streak it on the plate first, then dissolve it in the PCR reaction system for amplification, and culture the plate at 32°C. The strain was inoculated in BHI liquid medium and cultured for 10 to 12 hours; 3 to 5 μL of bacterial solution was added to the BHI recovery solution, and 100 μL of the recovery solution was coated on the plate and cultured at 32°C for 24 hours. Plasmid extraction was performed according to the instructions of the PlasmidMini Kit I kit.

[0042] Example 3: Verification of heat resistance of Corynebacterium glutamicum containing heat-resistant elements

[0043] The Corynebacterium glutamicum and Corynebacterium glutamicum ATCC 13032 obtained from the heat-resistant element in Example 2 were streaked on a plate and cultured at 32°C for 24 h. A single colony was picked and inoculated into 3 mL of culture medium, cultured at 32°C for 16 h, and the OD was measured. 600 The inoculated bacterial solution (OD 600 =10) was transferred to a fermentation shake flask (20 mL / 500 mL), cultured at 200 rpm and 42 °C for 24 h, and the OD was measured again. 600 The specific results are shown in Figure 1 Among them, OD ATCC13032 =2.95,OD pk18mobrpsL Ptuf TTHA0151 =8.11>OD pk18mobrpsL Ptuf TTHA0700 =8.08>OD pk18mobrpsL Ptuf TTHA1488 =7.79>OD pk18mobrpsL Ptuf TTHA1480 =7.42>OD pk18mobrpsL Ptuf TTHA1621 =6.98>OD pk18mobrpsL Ptuf TTHA0675 =5.57>OD ATCC 13032 Compared with the starting bacteria ATCC 13032, the OD values ​​increased by 1.75 times, 1.74 times, 1.64 times, 1.52 times, 1.37 times and 0.89 times, respectively.

[0044] The results showed that after culturing at 42°C for 24 h, the OD of the heat-resistant element obtained in Example 2 was 600 It is significantly improved compared with Corynebacterium glutamicum ATCC 13032, and its growth at 42℃ is significantly better than that of Corynebacterium glutamicum ATCC13032, with Ptuf TTHA0151 heat-resistant element having the best effect.

Claims

1. A heat-resistant gene, characterized in that: It is derived from Thermus thermophilus ( Thermusthermophilus )'s Ptuf-TTHA0151, Ptuf-TTHA0675, Ptuf-TTHA0700, Ptuf-TTHA1480, Ptuf-TTHA1488 or Ptuf-TTHA1621 gene, or a gene of a degenerate sequence thereof.

2. A heat-resistant component, characterized in that: Its promoter and heat-resistant gene composition, the heat-resistant gene is selected from the group consisting of those derived from Thermus thermophilus ( Thermusthermophilus )'s Ptuf-TTHA0151, Ptuf-TTHA0675, Ptuf-TTHA0700, Ptuf-TTHA1480, Ptuf-TTHA1488 or Ptuf-TTHA1621 gene, or a gene of a degenerate sequence thereof.

3. The heat-resistant element according to claim 2, characterized in that The promoter is a constitutive or inducible promoter, specifically the promoter Ptuf.

4. A vector containing the heat-resistant element according to claim 2 or 3, for example an expression vector, particularly an expression vector suitable for transforming Corynebacterium glutamicum.

5. Use of the heat-resistant element according to claim 2 or 3, or the vector according to claim 4, in improving the heat resistance of Corynebacterium glutamicum.

6. A Corynebacterium glutamicum with improved heat resistance, characterized in that The method comprises introducing the heat-resistant element as claimed in claim 2 or 3 into Corynebacterium glutamicum to improve the heat resistance of Corynebacterium glutamicum.

7. The Corynebacterium glutamicum according to claim 6, characterized in that The method of introduction is to transform Corynebacterium glutamicum by combining the vector as claimed in claim 4.

8. The Corynebacterium glutamicum according to claim 7, characterized in that The specific operation of introduction is to add the competent cells of Corynebacterium glutamicum and the vector into the electroporation cup, resuspend in BHIS after ice bath, keep at 46°C for 5-8min, resuscitate at 30-34°C and 200-240r / min, then spread on BHI plate, culture at 30-34°C to obtain single colonies for identification.

9. The Corynebacterium glutamicum according to claim 7, characterized in that The heat resistance of the obtained single colony strains was further verified by observing their growth at a high temperature of 40-44°C.

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