A heat-resistant element derived from corynebacterium glutamicum and application thereof
By introducing heat-resistant elements cg2611, cg3097 and cg3100 into Corynebacterium glutamicum, the problem of low production efficiency under high temperature conditions was solved, achieving efficient high-temperature fermentation production and reducing energy consumption and production costs.
Patent Information
- Application Number
- CN202411706675.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-26
- Publication Date
- 2025-11-25
- Estimated Expiration
- 2044-11-26
AI Technical Summary
Existing Corynebacterium glutamicum exhibits decreased production efficiency, reduced product yield, and lower production intensity when faced with environmental stresses such as high temperature and pH changes, and there is a lack of effective methods to improve its heat resistance.
The thermostable elements cg2611, cg3097, and cg3100 from Corynebacterium glutamicum were constructed and introduced. The thermal stability of the strain was improved by expressing the promoter Ptuf and the thermostable genes cg2611, cg3097, or cg3100.
It significantly improved the growth capacity and production efficiency of Corynebacterium glutamicum under high temperature conditions, reduced cooling water consumption and energy consumption, and enhanced the thermal stability and productivity of the strain.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a novel heat-resistant element derived from Corynebacterium glutamicum and its application. Background Technology
[0002] Corynebacterium glutamicum is a bacterium widely used in industrial fermentation production. As a food-grade microorganism, it has advantages such as easy cultivation, rapid growth, no endotoxins, strong environmental adaptability, and easy separation of fermentation products, and can be used for the production of various amino acids, nucleic acids, and vitamins. However, during fermentation production, Corynebacterium glutamicum often faces various environmental stresses, such as high temperature and pH changes. These factors may inhibit cell metabolism, reduce production efficiency, and decrease product yield, production intensity, and quality. How to enable it to grow under adverse conditions and achieve large-scale fermentation production has become a problem that needs to be solved.
[0003] When faced with adverse environments, ideal industrial microbial strains can effectively regulate themselves and possess characteristics such as high product yield, high activity, and high production intensity. Imparting environmental stress tolerance to microorganisms is highly beneficial for industrial microbial fermentation, especially high-temperature tolerance, as normal cell growth in high-temperature environments is a prerequisite for achieving high product yields. For a long time, how to improve the adaptability of strains to higher fermentation temperatures has been a major focus.
[0004] Microorganisms respond to high temperatures by expressing various genes encoding molecular chaperones and heat shock proteins (HSPs), which are universal defense mechanisms 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, efficient and precise screening methods can be used to quickly locate and test potential heat-resistant elements in Corynebacterium glutamicum, enabling the understanding of the mechanisms by which strains acquire heat resistance and the development of robust strains with ideal heat-resistant traits. Summary of the Invention
[0005] The first objective of this invention is to provide a novel thermostable element for Corynebacterium glutamicum, namely cg2611, cg3097, and cg3100. The nucleotide sequences of the elements are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.
[0006] The heat-resistance functional genes cg2611, cg3097, and cg3100 are derived from Corynebacterium glutamicum ATCC 13032; the cg2611 gene, named hscA, is a molecular chaperone belonging to the HSP70 family; the cg3097 gene, named hspR, is a MerR family transcription factor that mainly helps bacteria cope with heat stress by regulating the expression of heat shock proteins; and the cg3100 gene, named dnaK, 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 with a degenerate sequence thereof.
[0008] Furthermore, the present invention provides a heat-resistant element, characterized in that its promoter and heat-resistant gene composition, wherein the heat-resistant gene is selected from cg2611, cg3097 or cg3100 genes derived from Corynebacterium glutamicum, or genes with degenerate sequences thereof, and the specific nucleotide sequence is shown in SEQ ID NO: 1-3.
[0009] Specifically, the promoter is a constitutive or inductive promoter, specifically the promoter Ptuf.
[0010] The present invention also provides a carrier containing the heat-resistant element described above, such as an expression vector, particularly suitable for transforming Corynebacterium glutamicum expression vectors.
[0011] The present invention further provides the application of the heat-resistant element or the carrier described herein in improving the heat resistance of Corynebacterium glutamicum.
[0012] The present invention particularly provides a heat-resistant Corynebacterium glutamicum, which is obtained by introducing the heat-resistant element into Corynebacterium glutamicum to improve the heat resistance of Corynebacterium glutamicum.
[0013] Specifically, the introduction is carried out by combining the vector with transformed Corynebacterium glutamicum.
[0014] More specifically, the specific procedure for introduction is to add competent Corynebacterium glutamicum cells and the vector to an electroporation vessel, resuspend them in BHIS after an ice bath, incubate at 46°C for 5-8 min, revive at 30-34°C and 200-240 r / min, then spread them on BHI plates and incubate at 30-34°C to obtain single colonies for identification.
[0015] Optionally, the heat resistance of the obtained single colony strains can be further verified by observing their growth at a high temperature of 40-44℃.
[0016] The advantages of this invention compared to the prior art are:
[0017] 1. By constructing various heat-resistant elements, this invention successfully cultivated heat-resistant Corynebacterium glutamicum. This innovative method provides a new theoretical basis for the study of the stress resistance of Corynebacterium glutamicum and opens up new directions for the development of related stress-resistant elements.
[0018] 2. The heat-resistant element and the modified Corynebacterium glutamicum of this invention have broad application prospects and can be applied to multiple industries such as food, pharmaceuticals, and chemicals. Their superior heat resistance provides new technical support for industry innovation.
[0019] 3. During the fermentation process, the application of the heat-resistant element of this invention can significantly reduce the amount of cooling water used and energy consumption, thereby reducing production costs. This feature not only improves resource utilization efficiency but also has important significance for the sustainable development of enterprises.
[0020] 4. The Corynebacterium glutamicum of this invention exhibits excellent growth capacity under high-temperature conditions, greatly enhancing its thermal stability. This is particularly important for industrial applications, as it can maintain high productivity during high-temperature fermentation, thereby improving overall production efficiency. Attached Figure Description
[0021] Figure 1 The growth of Corynebacterium glutamicum containing heat-resistant elements and plasmids compared to Corynebacterium glutamicum ATCC 13032 under high-temperature conditions. Detailed Implementation
[0022] Example 1: Discovery and amplification of thermostable genes in Corynebacterium glutamicum
[0023] I. Extraction of the genome of Corynebacterium glutamicum ATCC 13032 strain
[0024] Corynebacterium glutamicum ATCC 13032 was activated and inoculated into LB medium. It was cultured in a shaker at 30–34°C for 20–28 h to obtain a seed culture. The seed culture was then inoculated into liquid medium, and the genome was extracted from the bacterial culture in the logarithmic growth phase. The genome extraction method followed the procedures outlined in the Wizard Genomic DNA Purification Kit A1120.
[0025] II. Screening for heat-resistant genes from Corynebacterium glutamicum ATCC 13032
[0026] Through literature review and analysis, genes related to heat resistance in *Corynebacterium glutamicum* ATCC 13032 were collected. The genes ultimately focused on for further study included cg0123 (HtpG), cg0691 (groEL'), cg2330 (ribosome-associated heat shock protein), cg2611 (hscA), cg3097 (hspR), and cg3100 (dnaK), totaling six genes. Based on preliminary research and validation, cg2611, cg3097, and cg3100 were selected for subsequent experiments.
[0027] III. Amplification of the thermostable gene from Corynebacterium glutamicum ATCC 13032
[0028] The genome of Corynebacterium glutamicum ATCC 13032 extracted in Part I was used as a template. Forward and reverse primers were designed for the genes screened in Part II, and PCR amplification was performed.
[0029] The PCR amplification system consisted of 20 μL ddH2O, 10 μL 5×PrimeSTAR Buffer, 4 μL dNTPs, 2 μL upper and lower primers, a template (total DNA < 200 ng), and 0.5 μL PrimeSTAR HSDNA Polymerase. The total enzyme digestion reaction volume was 50 μL, which was brought to a final volume with ddH2O. PCR amplification parameters were: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, and 72℃ extension for 30 s, for 30 cycles; and a final extension at 72℃ for 10 min.
[0030] Table 1 Primer Table
[0031]
[0032]
[0033] Example 2: Construction and expression of thermostable elements in Corynebacterium glutamicum
[0034] I. Construction of thermostable elements for Corynebacterium glutamicum
[0035] The genome of Corynebacterium glutamicum ATCC 13032 extracted earlier was used as a template for PCR amplification. Several constitutive or inducible promoters were tried, and expression was achieved, but the strong promoter ptuf showed outstanding results. Therefore, primers were designed targeting promoter Ptuf (nucleotide sequence shown in SEQ ID NO.7) to amplify the fragment of promoter Ptuf.
[0036] Further primers were designed to ligate the promoter Ptuf to the thermostable gene DNA fragment, resulting in a thermostable element. Up and down template sequences were used. The nucleotide sequences are shown in SEQ ID NO. 5-SEQ ID NO. 6, respectively.
[0037] II. Conjugation and Transformation of Corynebacterium glutamicum
[0038] The components used in the PCR tube include 5 μL ddH2O, 4 μL 5×CEbutter, 50–200 ng pk18 mobrpsL plasmid, 20–200 ng of the target DNA fragment (i.e., the heat-resistant element constructed in the first part), and Exnase. TM II 2μL. The total volume of the in vitro ligation system was 20μL, which was made up using ddH2O.
[0039] The system was incubated at 37°C for 30 min, followed by the addition of E. coli DH5α competent cells, then incubated on ice for 20 min, then incubated at 42°C for 60 s, and then on ice for 2 min. Subsequently, 900 μL of SOC resuscitation medium was added to EP tubes and incubated at 37°C and 220 rpm for 1 h. 100 μL of the bacterial culture was then plated and incubated at 37°C for 12 h. Single colony PCR identification was then performed, and the plates were incubated overnight at 37°C.
[0040] The identified strains were inoculated into LB liquid medium and cultured for 10 h. After centrifugation, plasmids were extracted. For *Corynebacterium glutamicum* ATCC13032 competent cells and plasmids, the cells were added to an electroporation vessel and incubated on ice for 20 min. The cells were resuspended in BHIS, transferred to EP tubes, and incubated at 46℃ for 6 min. After recovery at 32℃ and 220 rpm for 2 h, the cells were plated on BHI plates and incubated at 32℃ for 24 h. Single colonies were picked, streaked onto plates, and then dissolved in the PCR reaction system for amplification to obtain *Corynebacterium glutamicum* containing the heat-resistant element and plasmid described in Target 1. The plates were incubated at 32℃. The strains were inoculated into BHI liquid medium and cultured for 10–12 h. 3–5 μL of the bacterial culture was transferred to BHI recovery buffer, and 100 μL of the recovery buffer was plated and incubated at 32℃ for 24 h. Plasmid extraction was performed according to the Plasmid Mini Kit I instructions.
[0041] Example 3: Verification of the heat resistance of Corynebacterium glutamicum containing heat-resistant elements
[0042] The *Corynebacterium glutamicum* and *Corynebacterium glutamicum* ATCC 13032 containing the heat-resistant elements obtained in Example 2 were streaked onto plates and incubated at 32°C for 24 h. Single colonies were picked and cultured in 3 mL of culture medium, incubated at 32°C for 16 h, and OD was measured. 600 Inoculation (OD) 600=10) In a fermentation shake flask (20 mL / 500 mL), incubate at 200 rpm and 42 °C for 24 h, then measure the OD. 600 Value. See the detailed results below. Figure 1 Among them, OD ATCC 13032 =2.95, OD pk18mobrpsL Ptuf cg2611 =7.59, which is 1.58 times higher than the control bacteria; OD pk18mobrpsL Ptuf cg3097 =7.15, an increase of approximately 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℃ for 24 hours, the heat-resistant element of Corynebacterium glutamicum OD obtained in Example 2 was... 600 The performance of Ptuf cg2611 heat-resistant element was significantly improved compared to Corynebacterium glutamicum ATCC 13032, and its growth at 42℃ was significantly better than that of Corynebacterium glutamicum ATCC 13032.
Claims
1. The application of a heat-resistant element in improving the heat resistance of Corynebacterium glutamicum, characterized in that, It is achieved by introducing the cg2611 gene or its degenerate form from Corynebacterium glutamicum under the control of the promoter Ptuf in Corynebacterium glutamicum. The nucleotide sequence of the cg2611 gene is shown in SEQ ID NO. 1; The nucleotide sequence of the promoter Ptuf is shown in SEQ ID No.
4.
2. The application as described in claim 1, characterized in that, The specific procedure for the introduction involves adding competent Corynebacterium glutamicum cells and a vector containing the cg2611 gene or its degenerate nucleic acid, controlled by the promoter Ptuf, to an electroporation jar. After ice bath, the cells are resuspended in BHIS, incubated at 46°C for 5-8 min, and then revived at 30-34°C and 200-240 rpm. The cells are then plated on BHI plates and cultured at 30-34°C to obtain single colonies for identification.
3. The application as described in claim 2, characterized in that, Further verification of the heat resistance of the obtained single-colony strains was conducted by culturing them at a high temperature of 40-44℃ and observing their growth.
4. A heat-resistant Corynebacterium glutamicum, characterized in that, The method involves introducing the cg2611 gene or its degenerate form, which is controlled by the promoter Ptuf, into Corynebacterium glutamicum to improve the heat resistance of Corynebacterium glutamicum. The nucleotide sequence of the cg2611 gene is shown in SEQ ID NO. 1; The nucleotide sequence of the promoter Ptuf is shown in SEQ ID No.
4.
5. The Corynebacterium glutamicum as described in claim 4, characterized in that, The method of introduction is to transform Corynebacterium glutamicum by combining a vector containing the cg2611 gene or its degenerate nucleic acid, which is controlled by the promoter Ptuf, with the nucleotide sequence of the cg2611 gene as shown in SEQ ID NO. 1; The nucleotide sequence of the promoter Ptuf is shown in SEQ ID No. 4.