Corynebacterium glutamicum-derived heat-resistant element and application thereof

Abstract

The invention discloses three novel corynebacterium glutamicum heat-resistant elements and application thereof, and belongs to the technical field of bioengineering. Three heat-resistant genes are obtained from corynebacterium glutamicum research. The heat-resistant gene and a promoter Ptuf are connected and are combined and transformed in corynebacterium glutamicum. The heat-resistant element is introduced into a plasmid and is expressed in corynebacterium glutamicum. After 24 hours of high-temperature culture at 42 DEG C, the OD600 of the corynebacterium glutamicum containing the heat-resistant element and the plasmid is improved, and the growth condition of the corynebacterium glutamicum at 42 DEG C is obviously superior to that of the starting corynebacterium glutamicum. Therefore, the heat-resistant elements can obviously improve the heat resistance of corynebacterium glutamicum under a high-temperature culture condition. The method has important application value in improvement of the thermal stability of corynebacterium glutamicum and industrial production prospect.

Description

Technical Field

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

[0002] Corynebacterium glutamicum is a kind of bacterium widely used in industrial fermentation production.As a kind of food safety grade microorganism, it has the advantages of easy cultivation, fast growth, no endotoxin, strong environmental adaptability, easy separation of fermentation products, and can be used for the production of various amino acids, nucleic acids and vitamins.But in the fermentation production process, Corynebacterium glutamicum is often faced with multiple environmental stresses, such as high temperature and pH changes, and these factors may cause cell metabolism to be suppressed, production efficiency to decrease, and product output, production intensity and quality to reduce.How to make it grow under adverse conditions and can produce by fermentation in large quantities become the problem that needs to be solved today.

[0003] When facing adverse environments, ideal industrial strains can effectively regulate themselves and have characteristics such as high product yield, high vitality and high production intensity. Endowing microorganisms with environmental stress tolerance is very beneficial for industrial microbial fermentation, especially high temperature tolerance. Normal growth of cells in a high temperature environment is a prerequisite for achieving high product yields. For a long time, how to improve the adaptability of strains at higher fermentation temperatures has been a focus of attention.

[0004] Microorganisms respond to high temperatures by expressing various genes encoding molecular chaperones and heat shock proteins (HSPs), which are universal defenses against heat stress. By inducing the synthesis of molecular chaperones or heat shock proteins, stress-damaged proteins can be repaired or degraded, thereby preventing further damage to microbial cells. With the rapid development of genetic engineering and omics analysis, through efficient and accurate screening methods, the potential heat-resistant elements of Corynebacterium glutamicum can be quickly located and tested, which can understand the mechanism by which strains acquire heat resistance and achieve the development of robust strains with ideal heat-resistant traits. Summary of the invention

[0005] The first object of the present invention is to provide a new heat-resistant element of Corynebacterium glutamicum, namely cg2611, cg3097 and cg3100. The nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.3 respectively;

[0006] The heat-resistant functional genes cg2611, cg3097 and cg3100 are derived from Corynebacterium glutamicum ATCC 13032; the cg2611 gene is named hscA, which is a molecular chaperone belonging to the HSP70 family; the cg3097 gene is named hspR, which is a MerR family transcriptional regulatory factor, and mainly helps bacteria cope with heat stress by regulating the expression of heat shock proteins; the cg3100 gene is named dnaK, which belongs to the HSP70 family.

[0007] Therefore, the present invention provides a heat-resistant gene, which is derived from the cg2611, cg3097 or cg3100 gene of Corynebacterium glutamicum, or a gene of a degenerate sequence thereof.

[0008] Furthermore, the present invention provides a heat-resistant element, characterized in that it consists of a promoter and a heat-resistant gene, wherein the heat-resistant gene is selected from the cg2611, cg3097 or cg3100 gene derived from Corynebacterium glutamicum, or a gene of a degenerate sequence thereof, and its specific nucleotide sequence is shown in SEQ ID NO: 1-3.

[0009] In particular, the promoter is a constitutive or inducible promoter, in particular the promoter Ptuf.

[0010] 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.

[0011] The present invention further provides use of the heat-resistant element or the vector in improving the heat resistance of Corynebacterium glutamicum.

[0012] 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.

[0013] Specifically, the method of introduction is to transform Corynebacterium glutamicum by combining the vector.

[0014] 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.

[0015] 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.

[0016] The advantages of the present invention compared with the prior art are:

[0017] 1. By constructing a variety of heat-resistant elements, the present invention successfully cultivated heat-resistant Corynebacterium glutamicum. This innovative method provides a new theoretical basis for the study of stress resistance of Corynebacterium glutamicum and also opens up a new direction for the development of related stress-resistant elements.

[0018] 2. The heat-resistant element of the present invention and the modified Corynebacterium glutamicum have broad application prospects and can be applied to multiple industries such as food, pharmaceuticals, and chemicals. Its superior heat resistance provides new technical support for industry innovation.

[0019] 3. During the fermentation process, the application of the heat-resistant element of the present invention can significantly reduce the use of cooling water and energy consumption, thereby reducing production costs. This feature not only improves the utilization efficiency of resources, but also has important significance for the sustainable development of enterprises.

[0020] 4. The Corynebacterium glutamicum of the present invention exhibits excellent growth ability under high temperature conditions, greatly enhancing its thermal stability. This is particularly important for industrial applications, as it can maintain efficient productivity during high temperature fermentation, thereby improving overall production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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

[0022] Example 1: Mining and amplification of heat-resistant genes of Corynebacterium glutamicum

[0023] 1. Extraction of the genome of Corynebacterium glutamicum ATCC 13032

[0024] After activation, Corynebacterium glutamicum ATCC 13032 was inoculated into LB medium. The culture was cultured in a shaker at 30-34°C for 20-28 hours to obtain a seed culture solution. The seed culture solution was inoculated into a liquid culture medium, and the bacterial solution in the logarithmic growth phase was used to extract the genome. The genome extraction method was performed according to the kit (Wizard Genomic DNA Purification KitA1120).

[0025] 2. Screening of heat-resistant genes from Corynebacterium glutamicum ATCC 13032

[0026] Through literature search and analysis, we collected genes related to heat resistance in Corynebacterium glutamicum ATCC 13032, and finally focused on six genes, including cg0123 (HtpG), cg0691 (groEL'), cg2330 (ribosome-associated heat shock protein), cg2611 (hscA), cg3097 (hspR) and cg3100 (dnaK). After preliminary research and verification, we finally selected three genes, cg2611, cg3097 and cg3100, for subsequent experiments.

[0027] 3. Amplification of heat-resistant genes from Corynebacterium glutamicum ATCC 13032

[0028] The genome of Corynebacterium glutamicum ATCC 13032 extracted in the first part is used as a template. Forward primers and reverse primers are designed for the genes screened in the second part, and PCR amplification is performed.

[0029] The PCR amplification system includes 20 μL of ddH2O, 10 μL of 5× PrimeSTAR Buffer, 4 μL of dNTP, 2 μL of upper and lower primers, template (total DNA < 200 ng), and 0.5 μL of PrimeSTAR HSDNA Polymerase. The enzyme digestion reaction system is 50 μL in total, which is supplemented with ddH2O. PCR amplification parameters: 95°C pre-denaturation for 5 min; 98°C denaturation for 10 s, 55°C annealing for 5 s, and 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min.

[0030] Table 1 Primer list

[0031]

[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] PCR amplification of the aforementioned extracted genome of Corynebacterium glutamicum ATCC 13032 was used as a template. A number of constitutive or inducible promoters were tried, all of which could be expressed, but the strong promoter ptuf had an outstanding effect, so primers were designed for promoter Ptuf (nucleotide sequence as shown in SEQ ID NO.7) to amplify a fragment of promoter Ptuf.

[0036] The primers are 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 used. The nucleotide sequences are shown in SEQ ID NO.5-SEQ ID NO.6 respectively.

[0037] 2. Conjugative transformation of Corynebacterium glutamicum

[0038] In a PCR tube, the components used include ddH2O 5μL, 5×CEbutter 4μL, pk18mobrpsL plasmid 50-200ng, target DNA fragment, i.e., the heat-resistant element constructed in the first part 20-200ng, and Exnase TM II 2 μL. The total volume of the in vitro ligation system is 20 μL, which is made up with ddH2O.

[0039] The system was placed in a water bath at 37°C for 30 minutes, E. coli DH5α competent cells were added, ice bathed for 20 minutes, then in a water bath at 42°C for 60 seconds, and 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, spread on the plate, and cultured at 37°C for 12 hours. Subsequently, single colony PCR identification was performed, and the plate was cultured overnight at 37°C.

[0040] The identified strains were inoculated into LB liquid medium, cultured for 10 hours, centrifuged, and plasmids were extracted. For Corynebacterium glutamicum ATCC13032 competent cells and plasmids, add electroporation cups and ice bath for 20 minutes; resuspend in BHIS, transfer to EP tubes, 46℃ for 6 minutes, recover at 32℃, 220r / min for 2 hours, apply BHI plates, and culture at 32℃ for 24 hours. Pick a single colony, first streak it on a plate, then dissolve it in the PCR reaction system, amplify it, and it is Corynebacterium glutamicum containing the heat-resistant element and plasmid described in purpose 1, and the plate is cultured at 32℃. The strain was inoculated into BHI liquid medium and cultured for 10-12 hours; 3-5μL of bacterial solution was taken to BHI recovery solution, 100μL of recovery solution was applied to the plate, and cultured at 32℃ for 24 hours. Plasmid extraction was performed according to the instructions of the Plasmid Mini Kit I kit.

[0041] Example 3: Verification of heat resistance of Corynebacterium glutamicum containing heat-resistant elements

[0042] The heat-resistant element obtained in Example 2, Corynebacterium glutamicum and Corynebacterium glutamicum ATCC 13032, were streaked on a plate and cultured at 32°C for 24 h. A single colony was picked and placed in 3 mL of culture medium and cultured at 32°C for 16 h. The OD 600 Inoculation (OD 600= 10) in a fermentation shake flask (20 mL / 500 mL) at 200 rpm and 42 °C for 24 h. 600 The specific results are shown in Figure 1 Among them, OD ATCC 13032 =2.95,OD pk18mobrpsL Ptuf cg2611 =7.59, 1.58 times higher than the control bacteria; OD pk18mobrpsL Ptuf cg3097 =7.15, an increase of about 1.43 times; OD pk18mobrpsL Ptuf cg3100 =5.05, which is 0.71 times higher than ACTT 13032.

[0043] 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. The heat-resistant element Ptuf cg2611 has the best effect.

Claims

1. A heat-resistant gene, characterized in that: It is a gene derived from cg2611, cg3097 or cg3100 of Corynebacterium glutamicum, or a gene of a degenerate sequence thereof.

2. A heat-resistant component, characterized in that: The promoter and the heat-resistant gene are composed, and the heat-resistant gene is selected from the cg2611, cg3097 or cg3100 gene derived from Corynebacterium glutamicum, or the gene of the 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 the 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.

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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