Escherichia coli high-temperature tolerance related gene ychS and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a potential gene ychS and application. By systematically analyzing an escherichia coli high-temperature tolerance transcriptional regulation network, the potential biological function of coding speculated unrepresented protein gene ychS participating in escherichia coli high-temperature stress response is disclosed for the first time, and the expression quantity of the gene is in positive correlation with high-temperature tolerance; the high-temperature tolerance level of the strain is tested by respectively overexpressing and weakly expressing the ychS gene, which shows that under the high-temperature stress of 42 DEG C, the stable-phase biomass of the strain (p15-YchS) overexpressing the ychS gene is increased by 38.27% compared with that of a control strain, the maximum biomass is increased by 53.77%, the survival ability and metabolic activity of the strain at high temperature are remarkably enhanced, and the strain has a good application prospect. And a new strategy is provided for strain performance optimization in a high-temperature environment in industrial fermentation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to a potential gene. ychS and its applications. Background Technology

[0002] Microorganisms can sense changes in environmental conditions and regulate a series of physiological activities such as cellular stress and growth by modulating the expression of related genes through transcription factors. Temperature is a key environmental factor affecting cellular metabolism, protein structure, and gene expression. In industrial fermentation, *E. coli* often faces high temperatures, challenging its normal growth. High-temperature stress not only accelerates protein denaturation and disrupts membrane fluidity but also leads to metabolic imbalances and ROS (reactive oxygen species) bursts, thereby inhibiting cell growth and product synthesis. However, heat tolerance often involves multi-level dynamic regulation, and traditional methods struggle to capture these changes in real time. iModulons analysis, based on independent component analysis algorithms, can integrate transcriptome data to construct a comprehensive transcriptional regulatory network model, revealing potential regulatory targets related to heat tolerance. Analyzing the regulatory mechanisms of heat tolerance can provide a theoretical basis for optimizing strain performance, aiding in the design of synthetic biology components, and improving fermentation efficiency in industrial production processes.

[0003] The application of omics technologies has led to the discovery of numerous expressed but functionally unannotated proteins, often referred to as hypothetical proteins. Even in the most extensively studied *E. coli* K-12, the functions of proteins encoded by some genes remain uncharacterized. ychS The gene was first discovered in the complete genome sequence of *Escherichia coli* K-12 MG1655 in 1997 (Blattner et al., 1997). Located on the positive strand of *E. coli* K-12 MG1655, the 276 bp gene was predicted to encode a putative uncharacterized protein, YchS. To date, the specific function and regulatory relationships of this gene have not been systematically elucidated. Summary of the Invention

[0004] This invention constructs a comprehensive set of E. coli tolerance regulatory networks based on large-scale transcriptome data analysis, and screens and identifies related genes. ychS Further by constructing overexpression ychS Genetically engineered bacteria, identified ychS The role of genes in the regulation of heat tolerance in Escherichia coli.

[0005] The technical approach adopted in this invention is as follows: One of the technical solutions provided by this invention is the YchS protein or its encoding gene. ychSApplication of genes in regulating the heat tolerance of Escherichia coli; Specifically, the regulation of Escherichia coli's high-temperature tolerance is manifested in the following way: the expression level of YchS is positively correlated with the high-temperature tolerance of Escherichia coli, that is, the high-temperature tolerance of Escherichia coli increases with the increase of YchS expression level. Furthermore, the amino acid sequence of the YchS protein is shown in SEQ ID NO.1; Furthermore, the aforementioned ychS The gene, whose nucleotide sequence is shown in SEQ ID NO.2.

[0006] The second technical solution provided by the present invention is a method for improving the high-temperature tolerance of Escherichia coli, which is achieved by increasing the expression level of YchS protein in Escherichia coli; Furthermore, the methods for increasing expression levels include, but are not limited to: overexpressing YchS; the methods for overexpressing YchS include, but are not limited to, transcriptional overexpression or protein translational overexpression; the transcriptional overexpression includes, but is not limited to, using strong promoters or promoters with high copy numbers to regulate the expression of YchS; the protein translational overexpression includes, but is not limited to, using strong RBS (ribosome binding site) to improve translation efficiency, etc. Preferably, by introducing a recombinant expression vector containing the encoding gene of YchS into the target Escherichia coli, the expression level of YchS in the Escherichia coli is increased, thereby improving the high temperature tolerance of the recombinant Escherichia coli. The recombinant expression vector uses expression plasmids including, but not limited to, commonly used Escherichia coli expression vectors in the field, such as pBR322, pTK204, p15C-Kong plasmid, etc.

[0007] The third technical solution provided by this invention is a recombinant Escherichia coli with improved high-temperature tolerance. This recombinant Escherichia coli is constructed using the p15C-Kong plasmid as a backbone plasmid and contains… ychS The recombinant expression vector of the gene was introduced into the starting strain to obtain the gene; Preferably, the recombinant Escherichia coli uses MG1655 as the starting strain; Furthermore, the recombinant E. coli showed improved tolerance at temperatures of 37-42°C, and the survival ability of the strain was enhanced, especially at 42°C, where overexpression was observed. ychS The biomass of the gene strain was significantly higher than that of the control strain.

[0008] Beneficial effects: This invention, through systematic analysis of the transcriptional regulatory network of Escherichia coli's heat tolerance, reveals for the first time genes encoding inferred, uncharacterized proteins. ychSThe potential biological function of the gene involved in the high-temperature stress response of Escherichia coli was investigated, and its expression level was positively correlated with high-temperature tolerance. Further investigation was conducted by overexpression and weak expression of the gene. ychS Gene testing revealed that overexpression of the gene demonstrated high-temperature tolerance levels under 42°C high-temperature stress. ychS The biomass of the gene-producing strain (p15-YchS) during the stationary phase increased by 38.27% and the maximum biomass increased by 53.77% compared to the control strain, significantly enhancing the strain's survival ability and metabolic activity at high temperatures; the effect was weakened by sRNA technology. ychS The expressed strain (p20-sYchS) showed a 10.57% decrease in maximum biomass at 42℃ and exhibited a significant decline trend in the later stages, which conversely confirms... ychS The key regulatory role of genes in high-temperature tolerance further supports their reliability as targets for high-temperature tolerance, providing a new strategy for optimizing strain performance under high-temperature conditions in industrial fermentation. Attached Figure Description

[0009] Figure 1 p15C- ychS Plasmid map.

[0010] Figure 2 p20C-sRNA- ychS Plasmid map.

[0011] Figure 3 The growth level of strain p15-YchS at 37°C.

[0012] Figure 4 The growth level of strain p15-YchS under high temperature stress of 42℃.

[0013] Figure 5 The growth level of strain p20-sYchS under high temperature stress of 42℃. Detailed Implementation

[0014] The present invention will be further described below with reference to the embodiments. The following embodiments are provided to enable those skilled in the art to better understand the present invention, but do not limit the present invention in any way.

[0015] The original strain of Escherichia coli MG1655 used in this invention is a typical strain (ATCC®700926) preserved by ATCC.

[0016] The construction method of the p15C-Kong plasmid involved in the embodiments of the present invention has been disclosed in the following literature: Lin, Z., Xu, Z., Li, Y., Wang, Z., Chen, T., Zhao, X., 2014. Metabolic engineering of Escherichia coli for the production of riboflavin. Microb. Cell Fact. 13, 1-12; The nucleotide sequence of the p15C-Kong plasmid is shown in SEQ ID NO.3.

[0017] The construction methods of the p20C-MicC plasmid and p20C-Kong plasmid involved in the embodiments of the present invention have been disclosed in the following literature: Hu, W., Liu, S., Wang, Z., Chen, T., 2021. Improving riboflavin production by knocking down ribF, purA and guaC genes using synthetic regulatory small RNA. J. Biotechnol. 336, 25-29; wherein the p20C-Kong plasmid is an empty plasmid, which does not contain TBS and has no Mic; the nucleotide sequence of the p20C-MicC plasmid is shown in SEQ ID NO.4.

[0018] The Phanta DNA polymerase and related systems, 2×Rapid Taq Master Mix, 1kb DNA Ladder, 10×Loading Buffer, and other molecular biology reagents used in this invention were purchased from Nanjing Vazyme Co., Ltd. (https: / / www.vazyme.com / ). The 2×MultiF Seamless Assembly Mix, DpnI enzyme, and other molecular biology reagents used were purchased from Abclonal Co., Ltd. (https: / / www.abclonal.com / ). Other biochemical reagents used were purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0019] The present invention will be further explained and illustrated below through specific embodiments.

[0020] Example 1: Method for constructing an aggregate of Escherichia coli-resistant iModulons This invention discloses a method for constructing a comprehensive set of E. coli tolerance regulatory networks based on large-scale transcriptome data analysis. First, 977 E. coli RNA-seq datasets covering 11 types of environmental stresses, including antibiotics, extreme temperatures, acidity, and osmotic pressure, were collected from the NCBI SRA database system. Through a standardized quality control process (including sequence quality assessment, low-quality sample filtering, and expression level normalization), a gene expression matrix containing 597 high-quality transcriptome data was obtained. Using the optimized independent component analysis algorithm (OptICA), after removing empty iModulons with zero gene counts, 142 stable non-empty iModulons were obtained, defined as the tolerance iModulons set ZYH142. This set covers 4305 genes, is associated with multiple tolerance phenotypes, and is classified based on gene functional annotation and overlap with known regulators. One hundred and four regulatory iModulons were identified using a 30% gene overlap threshold. Functional iModulons (18) were defined through KEGG pathway enrichment analysis, and the remaining iModulons were divided into monogenotypes (2) and uncharacterized types (18). The construction of this dataset provides a global regulatory module resource library for the systematic analysis of E. coli environmental adaptation mechanisms.

[0021] Example 2: Screening process of PspF iModulon and its regulatory factors Based on the tolerance aggregate ZYH142 constructed in Example 1, this invention further screens potential key regulatory factors through unique tolerance subset analysis. 597 RNA-seq data were divided according to tolerance type, and the OptICA algorithm was used to generate an extreme temperature tolerance aggregate (TE) composed of 77 RNA-seq data under extreme temperature stress conditions. By comparing the iModulons of the ZYH142 aggregate and the TE subset, extreme temperature-specific regulatory PspFiModulon was found to exist in the TE set, which co-occurs only in the aggregate and the temperature subset. Among them, PspFi iModulon contains unknown functions. ychS Gene. Furthermore, PspF iModulon can regulate membrane stress response, phage infection stress, and other functions, and is highly associated with temperature tolerance set. This invention is the first discovery of an unknown functional gene. ychS Potential association with high temperature tolerance.

[0022] Example 3 Adjustment ychS Construction of gene expression strains (1) p15C- ychS plasmid construction ( ychS (overexpression plasmid) Using the genome of Escherichia coli K-12 MG1655 as a template, ychS -F andychS -R was used as upstream and downstream primers for PCR amplification to obtain linearized target gene fragment 1 ( ychS ); Using p15C-Kong as a template, and p15- ychS -F and p15- ychS -R was used as upstream and downstream primers for PCR amplification to obtain plasmid backbone fragment 1; The PCR-obtained fragments were purified, and the concentration of the DNA fragments was quantified using a nucleic acid protein quantification instrument (Bio-Rad). Seamless cloning technology was used to ligate gene fragment 1 and backbone fragment 1, and the results were verified by colony PCR to obtain p15C- ychS Plasmid (SEQ ID NO.5), image as shown Figure 1 As shown in Table 1, the primer sequences used in the procedure are detailed in the table.

[0023] PCR amplification system: 2 μL template, 2 μL each of upstream and downstream primers, 1 μL dNTP, 25 μL phanta buffer, 17 μL sterile double-distilled water, 1 μL DNA polymerase, total volume 50 μL.

[0024] The PCR reaction conditions were: 95 ℃ for 3 min, 30 cycles (95 ℃ for 20 s, 58 ℃ for 20 s, 72 ℃ for 1 min), 72 ℃ for 10 min, and 4 ℃ for 10 min. Transformation and culture were then performed, and single clones were selected for colony PCR verification.

[0025] (2) p20C-sRNA- ychS plasmid construction ( ychS (weak expression plasmid) Small regulatory RNA (sRNA) technology can inhibit the expression of target genes at the translational level. sRNAs are based on natural regulatory elements and consist of a target-binding sequence and a scaffold sequence. TBSs designed based on the target gene mRNA to be attenuated can target the synthesized sRNA element to that mRNA and recruit Hfq protein through a scaffold sequence containing an Hfq protein binding site and a terminator to stabilize the hybridization structure of sRNA and mRNA, thereby inhibiting ribosome-induced translation. This study used the general design guidelines for sRNA elements proposed by Sang et al., selecting the most efficient MicC as the scaffold sequence. The specific design process was as follows: First, TBSs were designed based on the target gene mRNA sequence; second, the TBSs were inserted into the 5' end of the MicC scaffold sequence, and the binding affinity between the TBSs and the target gene and the binding free energy (ΔG) between the sRNA and mRNA were evaluated using the TargetRNA2 and UNAfold online tools, respectively; finally, the designed TBSs were fused into the scaffold sequence using primers and overexpressed on a plasmid, with a plasmid vector of 20 copies selected to ensure the highest attenuation efficiency.

[0026] TBS was designed into the upstream and downstream primers s- ychS -F and s- ychS -R, via primer s- ychS -F / R uses p20C-MicC as a template for PCR amplification, which ligates TB-ychS (TBS) into the plasmid vector to obtain plasmid backbone 2; The PCR-obtained fragments were purified, and the concentration of the DNA fragments was quantified using a nucleic acid protein quantification instrument (Bio-Rad). The plasmid backbone was double-circularized using seamless cloning technology, and verification was performed by colony PCR to obtain p20C-sRNA- ychS Plasmid, map as follows Figure 2 As shown in Table 1, the primer sequences used in the procedure are detailed in the table.

[0027] PCR amplification system: 2 μL template, 2 μL each of upstream and downstream primers, 1 μL dNTP, 25 μL phanta buffer, 17 μL sterile double-distilled water, 1 μL DNA polymerase, total volume 50 μL.

[0028] The PCR reaction conditions were: 95 ℃ for 3 min, 30 cycles (95 ℃ for 20 s, 58 ℃ for 20 s, 72 ℃ for 1 min), 72 ℃ for 10 min, and 4 ℃ for 10 min. After transformation and culture, single clones were selected and cultured at s⁻¹. ychS -F was used as the upstream primer and SR as the downstream primer for colony PCR verification.

[0029] (3) Overexpression, weak expression ychS The procedure for constructing gene strains is as follows: p15C- ychS The plasmid was introduced into Escherichia coli MG1655 by electroporation to obtain strain p15-YchS.

[0030] The p15C-Kong plasmid was introduced into Escherichia coli MG1655 by electroporation to obtain strain p15-C as a control.

[0031] p20C-sRNA- ychS The plasmid was introduced into Escherichia coli MG1655 by electroporation to obtain strain p20-sYchS.

[0032] The p20C-Kong plasmid (p20C-Kong is an empty plasmid, without TBS and without MicC) was introduced into Escherichia coli MG1655 by electroporation to obtain strain p20-C as a control.

[0033] Table 1 Primer sequences used for strain construction

[0034] Example 4 ychS Regulation of heat tolerance by genes (1) Overexpression ychS High temperature tolerance verification of the strain Single colonies were picked from streaked LB agar plates of strains p15-YchS and p15-C, respectively, and inoculated into 5 mL LB liquid medium tubes containing ampicillin-resistant culture medium. The cultures were incubated overnight at 37°C and 220 rpm for 12 h. 1 mL of the bacterial culture was transferred to a sterile 2 mL EP tube and centrifuged at 4200 rpm for 3 min, discarding the supernatant. 1 mL of sterile M9 basic salt medium was added to the EP tube, mixed thoroughly by pipetting, and centrifuged at 4200 rpm for 3 min, discarding the supernatant. This step was repeated. 1 mL of sterile M9 basic salt medium was added to the EP tube, and the culture was resuspended by pipetting. The OD600 value of the bacterial culture was measured. Using an initial OD600 value of 0.1 after inoculation, the culture was transferred to 50 mL of M9 basic salt medium containing ampicillin (pH 7.0), and 1 mM IPTG inducer was added. The shake flasks were placed in shakers at 37℃ and 42℃ respectively, and incubated at 220 rpm for 48 hours. During the logarithmic growth phase, samples were taken every 2 hours to measure the logarithmic growth curve. After the stationary phase, samples were taken every 12 hours to measure and record the OD of the bacterial culture. 600 Value. The results of the high-temperature tolerance verification are shown in [reference needed]. Figure 3 , Figure 4 .

[0035] like Figure 3 As shown, at the optimal growth temperature of 37°C for E. coli, overexpression ychS The engineered strain p15-YchS showed inferior growth compared to the control strain p15-C, with a 13.06% reduction in biomass and a 12.70% reduction in maximum biomass at the stationary phase.

[0036] like Figure 4 As shown, under high temperature stress of 42℃, when the p15-YchS strain reached the stationary phase, the growth level was 38.27% higher than that of the control strain p15-C, and the maximum biomass increased to 1.966 after 48 hours, which was 53.77% higher than that of the control.

[0037] The above validation results show that, compared to the control strain p15-C, overexpression... ychS The engineered strain (p15-YchS) exhibited inferior growth compared to the control strain at 37°C, but showed improvement under high-temperature stress at 42°C. ychS The gene significantly improved the strain's tolerance to high-temperature stress.

[0038] (2) Weak expression ychS High temperature tolerance verification of the strain Single colonies were picked from streaked LB agar plates of strains p20-sYchS and p20-C, respectively, and inoculated into 5 mL LB liquid medium tubes containing *Bacillus thuringiensis*. The cultures were incubated overnight at 37°C and 220 rpm for 12 h. 1 mL of the bacterial culture was transferred to a sterile 2 mL EP tube and centrifuged at 4200 rpm for 3 min, discarding the supernatant. 1 mL of sterile M9 basic salt medium was added to the EP tube, mixed thoroughly by pipetting, and centrifuged at 4200 rpm for 3 min, discarding the supernatant. This step was repeated. 1 mL of sterile M9 basic salt medium was added to the EP tube, and the culture was resuspended by pipetting. The OD value of the bacterial culture was then measured. 600 Value, based on the initial OD after inoculation 600 The bacterial culture medium with a value of 0.1 was transferred to 50 mL of M9 basic salt medium (pH 7.0) containing zizomycin. The shake flasks were placed in a shaker at 42°C and incubated at 220 rpm for 48 hours. During the logarithmic growth phase, samples were taken every 2 hours to measure the logarithmic growth curve. After the stationary phase, samples were taken every 12 hours to measure and record the OD value of the bacterial culture. 600 Value. Growth curve as shown. Figure 5 As shown.

[0039] like Figure 5As shown, after 48 hours of shake-flask fermentation, the growth level of p20-sYchS at the end of the logarithmic growth phase under high temperature conditions of 42℃ was reduced by 10.57% compared with the control, and it continued to decline within 12 to 48 hours. This phenomenon indicates that... ychS Weak gene expression significantly impairs the heat tolerance of Escherichia coli.

[0040] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. YchS protein or its encoding gene ychS Application of genes in regulating the heat tolerance of Escherichia coli.

2. The application as described in claim 1, characterized in that, The regulation of Escherichia coli's high-temperature tolerance is specifically manifested in the fact that the expression level of YchS is positively correlated with the high-temperature tolerance of Escherichia coli.

3. The application as described in claim 1, characterized in that, The amino acid sequence of the YchS protein is shown in SEQ ID NO.1; ychS The gene, with its nucleotide sequence shown in SEQ ID NO.

2.

4. A method for improving the heat tolerance of Escherichia coli, characterized in that, The method is achieved by increasing the expression level of YchS protein in Escherichia coli.

5. The method for improving the high-temperature tolerance of Escherichia coli as described in claim 4, characterized in that, The methods for increasing expression levels include, but are not limited to, overexpressing YchS; the methods for overexpressing YchS include, but are not limited to, transcriptional overexpression or protein translational overexpression.

6. The method for improving the heat tolerance of Escherichia coli as described in claim 5, characterized in that, By introducing a recombinant expression vector containing the YchS encoding gene into target Escherichia coli, the expression level of YchS in Escherichia coli is increased, thereby enhancing the high-temperature tolerance of the recombinant Escherichia coli.

7. A recombinant Escherichia coli strain with enhanced heat tolerance, characterized in that, The recombinant Escherichia coli was constructed using the p15C-Kong plasmid as a backbone plasmid, containing... ychS The recombinant expression vector of the gene was obtained and introduced into the starting strain.

8. The recombinant Escherichia coli with improved heat resistance as described in claim 7, characterized in that, The recombinant Escherichia coli used MG1655 as the starting strain.

9. The application of the recombinant Escherichia coli according to claim 7 in improving heat tolerance.

10. The application as described in claim 9, characterized in that, The high temperature is 37-42℃.

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