Yersinia pseudotuberculosis temperature-sensitive promoter and application thereof

Abstract

The invention relates to the field of microbial genetic engineering and synthetic biology, and particularly discloses a yersinia pseudotuberculosis temperature-sensitive promoter and application thereof. The screened and identified temperature-sensitive promoter element fills the blank of efficient constitutive promoter resources of yersinia pseudotuberculosis. The promoters can be used as universal gene expression regulation modules to be applied to metabolic engineering and synthetic biology research of yersinia pseudotuberculosis and other prokaryotic hosts, and are beneficial to high-efficiency expression of target protein or biosynthesis of high-value products. According to the method, the expression of the target gene can be regulated and controlled by changing the culture temperature without adding chemical inducers, so that the operation is convenient, the cost is saved, and the method has outstanding advantages in large-scale fermentation production.

Description

Technical fields:

[0001] This invention relates to the fields of microbial genetic engineering and synthetic biology, and in particular to the thermosensitive promoter of Yersinia pseudotuberculosis and its applications. Background technology:

[0002] Yersinia pseudotuberculosis (Y.pstb) is a Gram-negative enteric pathogen and one of the zoonotic enteric pathogens. It belongs to the same genus of Yersinia as Yersinia pestis[1] and Yersinia enterocolitica[2]. The epidemiological characteristics of Yersinia pseudotuberculosis are similar to those of Yersinia enterocolitica, but it can infect a wider range of host animals, while its infection rate in humans is relatively low. In humans, Yersinia pseudotuberculosis is usually transmitted through contaminated routes such as food and water, and is a typical foodborne pathogen[3]. Infection with Yersinia pseudotuberculosis can cause clinical diseases such as sepsis and mesenteric lymphadenitis. Its symptoms are similar to those of appendicitis. Patients often experience right lower quadrant pain and fever. About half of the patients also have diarrhea. Some patients may also experience complications such as joint pain or back pain[4]. As a potential imported pathogen, Yersinia pseudotuberculosis poses a threat to human health. Studying the gene transcription profile of this bacterium can provide important information for elucidating the function of certain genes, operons and regulatory factors, thereby deepening our understanding of the pathogenic mechanism of Yersinia pseudotuberculosis and host-pathogen interaction[5].

[0003] Temperature is a unique external input signal with characteristics such as non-invasiveness, good permeability, reversibility, and low cost. In synthetic biology, the development of thermosensitive promoters is of great significance. The expression of the target gene can be controlled by simply changing the culture temperature. Compared with expression systems using chemical inducers, temperature control systems can simplify the operation steps and reduce costs, and are especially suitable for large-scale gene expression [6,7]. In recent years, high-throughput screening of highly expressed genes and cloning their promoter sequences by RNA sequencing (RNA-Seq) has become one of the effective strategies for finding strong promoter elements [8,9]. For example, Luo et al.

[10] discovered and identified 32 strong constitutive promoters from Streptomyces albus J1074 in 2015. Their activity intensity was about 2 to 13 times that of the commonly used strong promoter ermE*p in actinomycetes. Li et al.

[11] identified and evaluated a group of constitutive promoters with different intensities by RNA-Seq, which greatly enriched the promoter library available for Streptomyces.

[0004] Regarding temperature-regulating elements, Li et al.

[12] isolated two temperature-sensitive promoters from Bacillus subtilis in their 2007 study, which can serve as potential elements for constructing a temperature-induced expression system for Bacillus. Kong et al.

[13] obtained six high-intensity constitutive promoters in Streptococcus thermophilus through RNA-Seq combined with fluorescent reporter gene screening, and used these promoters to drive the expression of superoxide dismutase gene, which significantly improved the enzyme activity in Streptococcus thermophilus, Lactobacillus casei and Escherichia coli, showing good application prospects. Another study

[14] identified a highly efficient temperature-sensitive promoter regulated by a quorum sensing system through global transcriptome analysis, whose activity at 37℃ was about 2.2 times higher than that at 28℃. After multiple rounds of directional modification of this promoter and its mediated phenazine biosynthesis pathway, the researchers successfully constructed an engineered strain that can efficiently synthesize a new type of agricultural antibiotic—phenazine-1-carboxamide—at 37℃. The above studies demonstrate that utilizing transcriptome data to mine natural strong promoters, especially temperature-responsive promoters, is of great significance for developing cost-effective gene expression regulatory elements.

[0005] However, the number of strongly constitutive promoters available for regulating gene expression in *Yersinia pseudotuberculosis* is currently very limited, and no temperature-sensitive promoters have been reported. This, to some extent, restricts the metabolic engineering and related research of this bacterium. Therefore, there is an urgent need to develop efficient promoter elements suitable for *Yersinia pseudotuberculosis*, especially promoters that can respond to temperature changes, in order to achieve flexible regulation of target gene expression.

[0006] References:

[0007] 1.Chain, PS, et al., Insights into the evolution of Yersinia pestisthrough whole-genome comparison with Yersinia pseudotuberculosis. Proceedings of the National Academy of Sciences, 2004.101(38):p.13826-13831.

[0008] 2. Skurnik, M., A. Peippo, and E. Characterization of the O-antigengene clusters of Yersinia pseudotuberculosis and the cryptic O-antigen genecluster of Yersinia pestis shows that the plague bacillus is most closelyrelated to and has evolved from Y.pseudotuberculosis serotype O:1b.Molecularmicrobiology,2000.37(2):p.316-330.

[0009] 3.NAGANO,T.,et al.,Identification of pathogenic strains withinserogroups of Yersinia pseudotuberculosis and the presence of non-pathogenicstrains isolated from animals and the environment.Journal of veterinarymedical science,1997.59(3):p.153-158.

[0010] 4.Voskressenskaya,E.,et al.,Evaluation of ribotyping as a tool formolecular typing of Yersinia pseudotuberculosis strains of worldwideorigin.Journal of clinical microbiology,2005.43(12):p.6155-6160.

[0011] 5. Julio, S. M., et al., DNA adenine methylase is essential for viability and plays a role in the pathogenesis of Yersinia pseudotuberculosis and Vibrio cholerae. Infection and immunity, 2001. 69(12): p. 7610 - 7615.

[0012] 6. Zheng, Y., et al., A tight cold - inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts. Nucleic Acids Research, 2019. 47(21): p. e137 - e137.

[0013] 7. Keto - Timonen, R., et al., Cold shock proteins: a minireview with special emphasis on Csp - family of enteropathogenic Yersinia. Frontiers in microbiology, 2016. 7: p. 1151.

[0014] 8. Guo, Maozu, Yang, Shu, and Zhao, Lingling, Transcriptome analysis method based on RNA - Seq. Computer Science, 2020. 47(S2): p. 35 - 39.

[0015] 9. Nagalakshmi, U., et al., The transcriptional landscape of the yeast genome defined by RNA sequencing. Science, 2008. 320(5881): p. 1344 - 1349.

[0016] 10.Luo,Y.,et al.,Systematic identification of a panel of strongconstitutive promoters from Streptomyces albus.ACS synthetic biology,2015.4(9):p.1001-1010.

[0017] 11.Li,S.,et al.,Genome-wide identification and evaluation ofconstitutive promoters in streptomycetes.Microbial cell factories,2015.14(1):p.172.

[0018] 12.Li,W.,et al.,Characterization of two temperature-induciblepromoters newly isolated from B.subtilis.Biochemical and biophysical researchcommunications,2007.358(4):p.1148-1153.

[0019] 13.Kong,L.-H.,et al.,Characterization of a panel of strongconstitutive promoters from Streptococcus thermophilus for fine-tuning geneexpression.ACS Synthetic Biology,2019.8(6):p.1469-1472.

[0020] 14.Jin, Z.-J., et al., Identification of a strong quorum sensing-andthermo-regulated promoter for the biosynthesis of a new metabolite pesticidephenazine-1-carboxamide in Pseudomonas strain PA1201. ACS synthetic biology, 2020.9(7):p.1802-1812. Summary of the Invention:

[0021] The technical problem this invention aims to solve is the lack of effective strong promoter elements in the genetic modification of *Yersinia pseudotuberculosis*, especially the lack of temperature-sensitive promoters that can regulate gene expression through temperature changes. To address this problem, this invention provides a method for screening and identifying temperature-sensitive promoters for *Yersinia pseudotuberculosis*, such as... Figure 1 As shown in the figure, this method compares and analyzes the transcriptome of *Yersinia pseudotuberculosis* at different culture temperatures (25℃ and 37℃) to screen for genes expressed at high levels at each temperature and obtain their upstream promoter regions as candidate promoter elements. Then, these candidate promoters are cloned into a reporter vector to construct a "promoter-green fluorescent protein (GFP)-terminator" reporter gene circuit. The expression levels of the reporter gene under different temperature conditions are then detected to screen for promoter sequences with temperature-sensitive regulatory characteristics.

[0022] Using this method, this invention obtained a set of natural promoter elements from *Yersinia pseudotuberculosis* and classified them into three series: T25, T37, and TN, based on temperature. Each series includes a promoter sequence that significantly responds to temperature changes, named T25_6, T37_1, and TN_11, respectively, with T37_1 and TN_11 showing the most significant effects. The activity of these three promoters in driving reporter gene expression at 37°C was significantly higher than that at 25°C. Furthermore, the three series also include some relatively weak promoter elements screened in this invention, such as T25_4 in the T25 series, T37_ and T37_7 in the T37 series, and TN_7 and TN_8 in the TN series, thus constructing a library of elements with different promoter strengths for *Yersinia pseudotuberculosis*.

[0023] The present invention provides an expression cassette containing the thermosensitive promoter of Yersinia pseudotuberculosis.

[0024] Specifically, the Yersinia pseudotuberculosis thermosensitive promoter can be manipulated to link the target gene.

[0025] The present invention also provides a recombinant expression vector containing the aforementioned expression cassette.

[0026] Specifically, it is a prokaryotic expression vector, and more specifically, a recombinant expression vector for Yersinia pseudotuberculosis.

[0027] This invention also provides the application of the Yersinia pseudotuberculosis thermosensitive promoter, the expression cassette, or the recombinant expression vector described herein in regulating the expression of a target gene by altering the culture temperature. Specifically, it is used for the expression of a target protein or the biosynthesis of a target product.

[0028] The present invention also provides a method for expressing a target protein, which involves first culturing a recombinant bacterium containing the recombinant expression vector at approximately 25°C with a host bacterium containing the temperature-sensitive promoter to maintain low-level expression of the target gene, and then raising the culture temperature to 37°C to significantly induce high-level expression of the target gene.

[0029] Furthermore, after high-temperature induced expression is completed, the culture temperature is lowered back to approximately 25°C, thereby terminating or reducing the expression of the target gene and achieving reversible temperature regulation of the synthesis process of the target protein or target product.

[0030] Preferably, the recombinant bacteria is Yersinia pseudotuberculosis.

[0031] The advantages of this invention are: the expression of the target gene can be controlled simply by changing the culture temperature without the need for external chemical inducers, thus making the operation convenient and cost-effective, and giving it a significant advantage in large-scale fermentation production. The temperature-sensitive promoter elements screened and identified in this invention fill the gap in efficient constitutive promoter resources for *Yersinia pseudotuberculosis*. These promoters can serve as universal gene expression regulation modules in metabolic engineering and synthetic biology research on *Yersinia pseudotuberculosis* and other prokaryotic hosts, contributing to the efficient expression of target proteins or the biosynthesis of high-value products. Attached image description:

[0032] Figure 1 A method for screening and identifying thermosensitive promoters of Yersinia pseudotuberculosis.

[0033] Figure 2 Growth curves of Yersinia pseudotuberculosis at 25°C and 37°C.

[0034] Figure 3 Differential gene expression profiles in Yersinia pseudotuberculosis at 25℃ and 37℃.

[0035] Figure 4 KEGG enrichment analysis of genes.

[0036] Figure 5Construction of gene circuits containing thermosensitive promoters.

[0037] Figure 6 The relative fluorescence intensity of GFP expression by different candidate promoters. Detailed implementation method:

[0038] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

[0039] Example 1: Transcriptome analysis and candidate promoter screening of Yersinia pseudotuberculosis at different temperatures

[0040] The strain used in this invention is *Yersinia pseudotuberculosis* (Pfeiffer) Smith and Thal. The strain was preserved in our laboratory. The culture medium used was Luria-Bertani (LB) medium (containing 0.5% yeast extract, 1.0% tryptone, and 1.0% NaCl). First, *Yersinia pseudotuberculosis* was inoculated onto LB solid medium and incubated statically at 37°C for 24–36 hours. After obtaining single colonies, the culture was stored at 4°C for later use to prevent further bacterial growth at room temperature. Then, a single colony was picked up with an inoculation loop and inoculated into 5 mL of LB liquid medium, and cultured overnight at 37°C and 220 rpm with shaking. The next day, the overnight culture was inoculated into fresh LB liquid medium at a ratio of 1:100 and cultured at 37°C and 25°C for approximately 6 hours each, until the OD600 of the bacterial culture reached 0.5–0.9.

[0041] Observe the growth characteristics of Yersinia pseudotuberculosis under culture conditions of 25℃ and 37℃, such as Figure 2 As shown, the growth rate and final cell density of *Yersinia pseudotuberculosis* were significantly higher at 37℃ than at 25℃. After approximately one hour of incubation at 37℃, the bacteria entered the logarithmic growth phase, with a steep increase in viable cell count. After about 5-6 hours, the growth curve gradually plateaued, indicating a decrease in bacterial reproduction rate. In contrast, at 25℃, the bacterial growth lag phase was longer, entering the exponential growth phase after approximately two hours of incubation. While the cell count continued to increase during the 2-8 hour period, the growth rate was significantly lower than at 37℃, after which the growth curve plateaued. These results indicate that culture temperature significantly affects the growth of *Yersinia pseudotuberculosis*, and temperature variations may further lead to differences in intracellular gene expression levels within the bacteria.

[0042] Cultures were collected under the above conditions and rapidly cooled for subsequent RNA extraction and transcriptome analysis. The extracted bacterial total RNA was used for sequencing library construction after passing quality testing, and paired-end sequencing of Yersinia pseudotuberculosis transcripts under two temperature conditions was performed using the Illumina high-throughput sequencing platform. To ensure data quality, R software was used for quality control and preprocessing of the raw sequencing data, and then the FPKM value (Fragments Per Kilobase of transcript per Million mapped reads, representing the count per million total reads per kilobase of transcript fragment) of each gene was calculated using edgeR software

[20] . The expression profiles of the 25℃ group and the 37℃ group were compared, and the differentially expressed gene set in response to temperature was obtained by screening the criteria of |log2 (fold change in expression)|>1 and the corrected p value (padj)<0.05.

[0043] Next, transcriptome sequencing analysis was performed on the gene expression profiles of *Yersinia pseudotuberculosis* under two temperature conditions. The results showed that approximately 4375 gene transcripts were detected, of which 1736 genes exhibited significantly different expression levels between 37℃ and 25℃ conditions (judgment criteria: |log2FoldChange|>1 and padj<0.05). Specifically, 824 genes were upregulated and 912 genes were downregulated at 37℃. Figure 3 This is a heatmap of differentially expressed genes clustering. Red indicates genes whose transcription is upregulated at 37°C, and green indicates genes whose transcription is downregulated at 37°C. Figure 3 It can be seen that there are significant differences in the gene expression profiles of *Yersinia pseudotuberculosis* under different culture temperatures: increased temperature leads to the activation and upregulation of transcription of certain genes within the bacterial cells, while the expression of other genes is inhibited and reduced. Further KEGG pathway enrichment analysis was performed on the differentially expressed gene set, such as... Figure 4 As shown, these temperature-affected genes are mainly enriched in functional pathways such as secondary metabolite biosynthesis, complex environmental microbial metabolism, and two-component systems, indicating that temperature signals can regulate multiple metabolic pathways in bacteria.

[0044] Subsequently, functional annotation (Gene Ontology, GO) and KEGG pathway enrichment analysis were performed on differentially expressed genes to analyze the effects of temperature changes on various metabolic pathways in bacterial cells. Based on the transcriptome analysis results, this invention further screened and identified genes and their promoter sequences that were specifically highly expressed by Yersinia pseudotuberculosis at different temperatures. Specifically, a genomic fragment of about 100 genomic units upstream of the start codon of the above-mentioned highly expressed genes was extracted as a candidate promoter sequence. Bioinformatics analysis tools were used to predict the promoter characteristics of the candidate sequences, and fragments containing known transcription factor binding sites were removed

[21] , and finally 60 candidate promoter sequences were determined.

[0045] Based on the above analysis data, this embodiment screened out genes that were highly expressed under different temperature conditions to obtain strong promoter candidate sequences. For genes that were significantly highly expressed at 25℃ but lowly expressed at 37℃, 20 such genes were screened and their upstream 100 genomic sequences were extracted as candidate promoters (numbered T251 to T2520, see Table 1).

[0046] Similarly, 20 target genes and their promoter sequences (numbered T37_1~T37_20, see Table 2) were screened from genes that were highly expressed at 37℃ and lowly expressed at 25℃. In addition, 20 genes that showed high expression at both 25℃ and 37℃ and their promoter sequences (numbered TN_1~TN_20, see Table 3) were also selected. To predict whether these sequence fragments had promoter function, they were analyzed using bioinformatics software such as BPROM. The results showed that most of these candidate sequences contained typical promoter feature sequences such as conserved elements in the -35 region and -10 region

[21] , which indicates that they are likely to have promoter activity.

[0047] Table 1. Twenty promoter sequences (T25 series) corresponding to genes highly expressed at 25℃.

[0048]

[0049]

[0050] Table 2. Twenty promoter sequences (T37 series) corresponding to genes highly expressed at 37℃ (SEQ ID NO: 21 to 40 respectively)

[0051]

[0052]

[0053] Table 3. Twenty promoter sequences (TN series) corresponding to genes that are highly expressed under both 25℃ and 37℃ conditions (SEQ ID NO: 41 to 60 respectively)

[0054]

[0055]

[0056] Example 2: Cloning, transformation and thermosensitive activity characterization of candidate promoters

[0057] To verify the promoter activity and temperature response characteristics of the candidate sequences, this invention constructed a fluorescent reporter gene expression vector. Each candidate promoter sequence was cloned upstream of the green fluorescent protein reporter gene (GFP, component BBa_E0040), and a double terminator sequence (BBa_B0015) was ligated downstream of it. The aforementioned "promoter-GFP-terminator" fragment was inserted into the multiple cloning site region of the plasmid vector pSB1A3 via EcoRI and PstI restriction enzyme sites. In each recombinant vector, GFP gene expression is controlled by the candidate promoter, reporting the relative strength of that promoter. All constructed recombinant plasmids were sequenced to verify their correctness and stored at -20°C for later use. Figure 5 A schematic diagram of plasmid vectors and gene elements is shown.

[0058] The recombinant plasmid was transferred into *Yersinia pseudotuberculosis* via electroporation. The specific steps included: collecting 1 mL of *Yersinia pseudotuberculosis* logarithmic growth phase culture medium by centrifugation at 4500 rpm for 5 minutes on ice; resuspending the bacterial cells in pre-cooled sterile deionized water and washing them three times by centrifugation to remove extracellular salt ions; then resuspending the bacterial cells in 200 μL of ice-cold sterile deionized water. Approximately 100 ng of the constructed recombinant plasmid DNA was added to the bacterial suspension, gently mixed by pipetting, and incubated on ice for 15 minutes. The bacterial suspension was transferred to a pre-cooled electroporation cuvette, and electroporation was performed using a Bio-Rad Gene PulserXcell electroporator at the following conditions: voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω. Immediately after electroporation, 1 mL of LB antibiotic-free liquid medium was added to the cuvette and mixed well. The bacterial suspension was then transferred to a sterile centrifuge tube and incubated at 37°C on a shaker for 1.5 hours. Subsequently, the bacterial cells were centrifuged at 4500 rpm for 5 minutes to precipitate the bacterial cells. The supernatant was discarded, and the bacterial cells were resuspended in approximately 200 μL of fresh LB medium. The medium was then evenly spread onto LB agar plates containing 100 μg / mL ampicillin-resistant bacteria and incubated at 37°C for 14–18 hours. After single colonies grew on the plates, colonies were picked and inoculated onto LB slants containing 100 μg / mL ampicillin-resistant bacteria to obtain recombinant Yersinia pseudotuberculosis strains. The presence of exogenous plasmids in the strains was confirmed by resistance screening and fluorescence microscopy.

[0059] The obtained recombinant strains were used in in vitro reporter gene experiments to characterize the activity intensity of each promoter. The specific detection method was as follows: Recombinant *Yersinia pseudotuberculosis* strains carrying different promoter-GFP plasmids were compared with wild-type strains without plasmids as controls, and cultured overnight at 37°C in LB liquid medium (with added ampicillin, 100 μg / mL). The next day, each culture was diluted 100-fold in fresh antibiotic-containing LB medium, aliquoted into 96-well microplates, and cultured for 24 hours at 25°C and 37°C with continuous shaking (dual-track shaking at approximately 807 rpm during culture). The green fluorescence intensity (excitation wavelength 485 nm, emission wavelength 515 nm) and optical density (OD600) of each well were measured every 30 minutes. Fluorescence readings at different time points were recorded and normalized with OD600 (RFU / OD600) to obtain the relative fluorescence units (RFU) versus time curve. RFU / OD600 values ​​at representative time points within the logarithmic growth phase were selected for comparative analysis of promoter activity under different conditions. All experiments were performed in triplicate. GraphPad Prism software was used for statistical processing and plotting. The significance of differences in promoter-driven reporter gene expression under the two temperature conditions was assessed using t-tests (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001).

[0060] Figure 6 The results show a comparison of the relative fluorescence intensities of different candidate promoters driving GFP expression at two temperatures. The experimental results indicate that among the T25 series candidate promoters, two promoters (T25_4 and T25_6) drove higher GFP expression levels, but the fluorescence intensity of both at 25℃ was lower than that at 37℃, deviating from initial expectations.

[0061] Among the T37 series of candidate promoters, a strong promoter, T37_1, with a significant temperature response was selected. Its GFP fluorescence intensity driven at 37°C was significantly higher than that at 25°C. In addition, some relatively weak promoters in this series (such as T37_6, T37_7, T37_8, and T37_13) produced only low fluorescence signals at both temperatures, with little difference between high and low temperatures. Among the TN series of candidate promoters, two promoters (TN_4 and TN_11) exhibited strong activity at 37°C, significantly higher than their activity at 25°C, which differed from our initial expectation of high expression at both temperatures. Furthermore, several other promoters in this series (such as TN_7, TN_8, TN_9, TN_10, TN_14, and TN_18) showed weak activity at both temperatures.

[0062] Through the aforementioned fluorescence reporter experiments, this invention identified five strong promoter elements (T25_4, T25_6, T37_1, TN_4, and TN_11). Four of these (T25_6, T37_1, TN_4, and TN_11) showed an activity increase of approximately 1.4 to 5.8 times at 37°C compared to 25°C, demonstrating significant temperature-induced characteristics. In particular, promoters T37_1 and TN_11 exhibited activities at 37°C that were approximately 2.8 and 5.8 times, respectively, compared to 25°C, demonstrating excellent temperature-sensitive regulation. Furthermore, this invention also provides several medium- and weak promoter elements of varying strengths, further enriching the promoter library resources of *Yersinia pseudotuberculosis*.

Claims

1. A Yersinia pseudotuberculosisosensitive promoter, characterized in that, the nucleotide sequence of any one of T25_1 to T25_20, T37_1 to T37_20, TN_1 to TN-20.

2. The Y. pseudotuberculosis temperature-sensitive promoter of claim 1, wherein, any one of the nucleotide sequences, preferably T25_4 or T25_6 in the T25 series, T37_1, T37_6, T37_7, T37_8, T37_13 in the T37 series, TN_4, TN_11, TN_7, TN_8, TN_9, TN_10, TN_14, TN_18 in the TN series, more preferably T25_6, T37_1 and TN_11.

3. An expression cassette comprising the Y. pseudotuberculosisis temperature-sensitive promoter of claim 1 or 2.

4. The expression cassette of claim 3, wherein, The Y. pseudotuberculosisis temperature-sensitive promoter can be operably linked to a gene of interest.

5. A recombinant expression vector comprising the expression cassette of claim 3 or 4.

6. The recombinant expression vector of claim 5, wherein, It is a prokaryotic expression vector, more particularly a recombinant expression vector suitable for Y. pseudotuberculosisis.

7. Use of the Y. pseudotuberculosisis temperature-sensitive promoter of claim 1 or 2, the expression cassette of claim 3 or 4, or the recombinant expression vector of claim 5 or 6 in regulating the expression of a gene of interest by changing the culture temperature.

8. Use according to claim 7, wherein the compound is ###0002### It is used for the expression of a protein of interest or the biosynthesis of a target product.

9. A method for expressing a protein of interest, characterized by, The recombinant bacteria into which the recombinant expression vector of claim 5 or 6 is introduced are first cultured at about 25°C to maintain low-level expression of the gene of interest, and then the culture temperature is raised to 37°C to significantly induce high-level expression of the gene of interest.

10. The expression method of claim 9, wherein, After the completion of high-temperature induced expression, the culture temperature is lowered again to about 25°C, thereby terminating or reducing the expression of the gene of interest, achieving reversible temperature regulation of the synthesis process of the protein of interest or the target product. Preferably, the recombinant bacteria are Y. pseudotuberculosisis.

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