T7 expression system suitable for escherichia coli Nissle 1917 and application of T7 expression system

Abstract

The invention relates to a T7 expression system suitable for escherichia coli Nissle 1917 and application of the T7 expression system, and belongs to the technical field of microorganisms. A T7 expression system is constructed and optimized in probiotic Escherichia coli Nissle 1917 and comprises a first expression box and a second expression box, the first expression box uses an inducible promoter to drive expression of lactose operon lacO and T7RNA polymerase, the second expression box uses a T7 promoter to regulate expression of lacO and a target gene, and the T7 expression box is used for inducing expression of lacO and T7RNA polymerase. The concentration of the inducer and the integrated expression site of the second expression cassette in the probiotic genome are screened, so that the efficient expression of an exogenous gene is realized, and the problems of low expression efficiency of T7RNA polymerase, lack of applicable promoters, indefinite induction conditions and insufficient integration stability in the prior art are solved; the method has important significance on application of the escherichia coli Nissle 1917 in the fields of synthetic biology and the like.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a T7 expression system suitable for Escherichia coli Nissle 1917 and its application. Background Technology

[0002] With the continuous advancement of synthetic biology and metabolic engineering technologies, engineered microorganisms have demonstrated enormous application potential in the synthesis of functional sugars, bioactive substances, and pharmaceutical intermediates. *Escherichia coli* Nissle 1917 (EcN) is a non-pathogenic strain with excellent probiotic properties and has been widely used in the treatment of intestinal diseases, the development of probiotic preparations, and the construction of biotherapeutic vectors. Compared with traditional *E. coli* strains, EcN not only possesses a stable genetic background and strong environmental adaptability but also exhibits good host safety and intestinal colonization ability, thus being considered an important candidate strain for constructing novel synthetic biology chassis.

[0003] The T7 expression system is a commonly used tool for achieving high-level exogenous gene expression. Its core consists of T7 RNA polymerase and a strong transcription-driven module mediated by the T7 promoter. This system has been widely used in common *E. coli* strains, enabling rapid, efficient, and controllable protein expression. However, the application of the T7 expression system in *EcN* strains is still limited by several factors, including low T7 RNA polymerase expression efficiency, lack of suitable promoters, unclear induction conditions, and insufficient integration stability. These issues severely affect the expression level of exogenous genes and the system's generalizability. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a T7 expression system suitable for Escherichia coli Nissle 1917, comprising a first expression cassette and a second expression cassette. The first expression cassette uses an inducible promoter to drive the expression of the lactose operon lacO and T7 RNA polymerase, while the second expression cassette uses a T7 promoter to regulate the expression of lacO and the target gene. The promoter type, induction conditions, and genomic sites for integrated expression have been screened.

[0005] The first objective of this invention is to provide a T7 expression system suitable for Escherichia coli Nissle 1917, the T7 expression system comprising a first expression cassette and a second expression cassette, wherein:

[0006] The first expression box uses P tac P J23119 or P lacUV5 The promoter expresses the lactose operon lacO and T7 RNA polymerase;

[0007] The second expression cassette uses the T7 promoter to express the lactose operon lacO and the target gene.

[0008] Furthermore, the P tac The nucleotide sequence of the promoter is shown in SEQ ID NO.8, wherein P J23119 The nucleotide sequence of the promoter is shown in SEQ ID NO.9, wherein P lacUV5 The nucleotide sequence of the promoter is shown in SEQ ID NO.2.

[0009] Furthermore, the first expression box uses P tac Promoter.

[0010] Furthermore, the lactose operon lacO and T7 RNA polymerase also contain an RBS sequence.

[0011] Furthermore, the RBS sequence is shown in SEQ ID NO.6.

[0012] A second objective of this invention is to provide Escherichia coli Nissle1917 comprising the aforementioned T7 expression system.

[0013] A third objective of this invention is to provide the application of the above-described T7 expression system in expressing exogenous genes in Escherichia coli Nissle 1917.

[0014] The fourth objective of this invention is to provide a method for expressing a foreign gene in Escherichia coli Nissle 1917, wherein the above-mentioned T7 expression system is introduced into Escherichia coli Nissle 1917.

[0015] Furthermore, the method also includes the step of adding IPTG to induce expression, wherein the amount of IPTG added is 0.05-5 mM.

[0016] Preferably, the amount of IPTG added is 0.1 mM.

[0017] Furthermore, the first expression cassette is integrated and expressed at the motA site in the genome of Escherichia coli Nissle 1917.

[0018] Furthermore, the second expression cassette is integrated and expressed in the genome of the Escherichia coli Nissle 1917.

[0019] Furthermore, the integration expression is performed at the wecB, manA, nanATEK, manXYZ, nagABCE, pykA, chew, ptsG, nagK, or kfiABC sites in the Escherichia coli Nissle 1917 genome.

[0020] In one embodiment of the present invention, the nucleotide sequence of the wecB site is shown in SEQ ID NO.11, the nucleotide sequence of the manA site is shown in SEQ ID NO.12, the nucleotide sequence of the nanATEK site is shown in SEQ ID NO.13, the nucleotide sequence of the manXYZ site is shown in SEQ ID NO.14, the nucleotide sequence of the nagK site is shown in SEQ ID NO.15, the nucleotide sequence of the pykA site is shown in SEQ ID NO.16, the nucleotide sequence of the chew site is shown in SEQ ID NO.10, the nucleotide sequence of the nagABCE site is shown in SEQ ID NO.17, the nucleotide sequence of the ptsG site is shown in SEQ ID NO.18, and the nucleotide sequence of the kfiABC site is shown in SEQ ID NO.19.

[0021] Preferably, the integration expression is performed at the wecB or manA site in the genome of Escherichia coli Nissle 1917.

[0022] The beneficial effects of this invention are:

[0023] This invention successfully constructed and optimized a T7 expression system suitable for Escherichia coli Nissle 1917. 0.1 mM IPTG was the optimal induction concentration for this system. tac This study identified the optimal promoter for driving T7 RNA polymerase expression. The expression capacity of the target gene expression cassette at different sites on the *E. coli* Nissle 1917 genome was further validated. Results showed that all tested sites achieved high levels of exogenous gene expression, demonstrating good expression capacity and suitability as candidate integration sites in synthetic biology. This T7 expression system and its genome integration strategy have broad applicability and promotional value, providing a new technical means for the efficient expression of target genes in the field of synthetic biology. Attached Figure Description

[0024] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0025] Figure 1 This is a schematic diagram of the T7 expression system constructed in Embodiment 2 of the present invention.

[0026] Figure 2 P in Embodiment 2 of the present invention tac The expression results of promoter-mediated T7 RNA polymerase under different IPTG induction conditions;

[0027] Figure 3 P in Embodiment 2 of the present invention J23119The expression results of promoter-mediated T7 RNA polymerase under different IPTG induction conditions;

[0028] Figure 4 P in Embodiment 2 of the present invention lacUV5 The expression results of promoter-mediated T7 RNA polymerase under different IPTG induction conditions;

[0029] Figure 5 The results show the integration efficiency and expression intensity of sfGFP at different sites in the genome in Example 3 of this invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] (I) Strains and Vectors

[0032] The plasmid construction process was completed in *Escherichia coli* Top10. The constructed plasmid was then used with the inducible promoter P. T7 The superfolded green fluorescent protein (sfGFP) expression plasmid was transformed into different engineered bacterial strains. The growth curves and fluorescence intensity of each strain were measured using a microplate reader to evaluate the expression efficiency of the T7 system.

[0033] All T7 RNA polymerase-expressing strains underwent genome integration using CRISPR-Cas9 gene editing, and the integration accuracy was verified by first-generation sequencing (Sanger sequencing). Strains that integrated T7-sfGFP at different genomic loci were plated on LB agar containing 0.1 mM IPTG after genome integration via CRISPR-Cas9 gene editing. Successful sfGFP expression was further verified by observing whether the colonies produced green fluorescence. Growth curves and fluorescence intensity were measured using a microplate reader to assess the expression intensity of sfGFP at each locus.

[0034] (II) Culture medium

[0035] LB medium for Escherichia coli: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar powder 20 g / L.

[0036] SOC medium: yeast extract 5g / L, peptone 20g / L, sodium chloride 0.5g / L, glucose 20mM, magnesium chloride 10mM / L.

[0037] (III) Fluorescence Measurement

[0038] Fluorescence data for promoter characterization are expressed as fluorescence value / OD. 600 Based on this, the excitation wavelength of GFP is 488nm, the emission wavelength is 523nm, the gain is 60, and the OD... 600 Detected using an ELISA reader.

[0039] Example 1

[0040] BL21(DE3) is a common host for the E. coli T7 expression system and is often used for the expression of various recombinant proteins. Its T7 RNA polymerase expression cassette (nucleotide sequence shown in SEQ ID NO.1) is generated by the promoter P lacUV5 The expression cassette (nucleotide sequence shown in SEQ ID NO.2) and the lactose operon lacO (nucleotide sequence shown in SEQ ID NO.3) are combined. However, there is a 306 bp gap between lacO and the open reading frame (ORF) of T7 RNA polymerase (nucleotide sequence shown in SEQ ID NO.4) (nucleotide sequence shown in SEQ ID NO.5). Apart from the potential RBS sequence, there are a large number of redundant sequences. Directly amplifying the T7 expression cassette derived from E. coli BL21(DE3) and using it directly in E. coli Nissle 1917 will result in a serious decrease in the expression effect of the T7 expression system.

[0041] The redundant 306 bp sequence was replaced with an RBS sequence (nucleotide sequence shown in SEQ ID NO. 6). The modification effect was verified using GFP (green fluorescent protein). An expression plasmid carrying the T7 promoter-driven green fluorescent protein reporter gene (sfGFP) (nucleotide sequence shown in SEQ ID NO. 7) was introduced into the strain, and co-expression was achieved via electroporation. Before optimization, the average relative fluorescence was 4114 (4101, 4119, 4122 respectively), and after optimization, the average relative fluorescence was 39083 (39071, 39083, 39095 respectively), which is 9.5 times that of before optimization. Removing the 306 bp redundant sequence and replacing it with the RBS sequence significantly improved the expression intensity of the T7-driven gene.

[0042] Example 2

[0043] The T7 expression system mainly consists of two parts: a T7 RNA polymerase expression cassette and an expression regulation module. To improve the expression efficiency of exogenous genes in *Escherichia coli* Nissle 1917, the expression level of T7 RNA polymerase in the genome was optimized by changing different types of promoters to enhance its ability to drive the transcriptional activation of target genes by the T7 promoter. A schematic diagram of the T7 expression system is shown below. Figure 1 .

[0044] To construct an efficient T7 RNA polymerase expression system, we screened three commonly used inducible promoters in E. coli: P tac (nucleotide sequence as shown in SEQ ID NO.8), P J23119 (nucleotide sequence as shown in SEQ ID NO. 9) and P lacUV5 T7 RNA polymerase expression cassettes containing different promoters were constructed (i.e., the promoters of the T7 RNA polymerase expression cassettes constructed in Example 1 were replaced), and they were integrated into the motA site of the EcN genome using the CRISPR-Cas9 method.

[0045] Subsequently, the green fluorescent protein reporter gene expression plasmid carrying the T7 promoter, constructed in Example 1, was introduced into the bacterial strain and co-expressed via electroporation. To evaluate the expression performance of different promoters, the engineered bacterial strain carrying the reporter plasmid was directly inoculated into SOC medium containing different concentrations of IPTG (inducer) and fermented in 96-well shallow plates for 68 hours. The expression effects of different promoters under different induction conditions were compared and analyzed by measuring fluorescence intensity and growth curves. The results are shown below. Figure 2 , Figure 3 and Figure 4 As shown, the three promoters exhibited differentiated inducible expression levels at different IPTG concentrations. Among them, P tac The promoter exhibited the highest specific fluorescence intensity under 0.1 mM IPTG conditions.

[0046] Example 3

[0047] Eleven different expression sites were selected in the genome of *E. coli* Nissle 1917. The T7 promoter-driven green fluorescent protein (T7-sfGFP) expression cassette was in situ integrated into each site using the CRISPR-Cas9 system. The recombinant strains were inoculated into LB agar containing IPTG. Successful integration and expression of sfGFP were determined by observing whether the colonies produced green fluorescence. Subsequently, representative clones were screened from the fluorescently positive colonies and subjected to 96-well plate fermentation experiments, where the cell density (OD) was measured. 600 The fluorescence intensity was measured to further evaluate the expression capacity of exogenous genes at different genomic loci driven by the T7 promoter. Integration expression at different loci was shown in the figure. Figure 5 As shown.

[0048] Taking the cheW site (nucleotide sequence as shown in SEQ ID NO.10) as an example, the results were compared with those from the constitutive promoter P. tac With induced priming P T7 The expression intensity of sfGFP driven by the P-value was shown in the results. T7The intensity of sfGFP expression mediated by P is tac The expression intensity was 1.7 times higher than that of the T7 promoter, indicating that the T7 system has a stronger transcriptional activation capacity at this site. Further evaluation of the sfGFP expression intensity at the remaining 10 sites driven by the T7 promoter was conducted, using fluorescence intensity as a function of cell density (OD). 600 The ratio of nucleotides was used as a quantitative indicator of expression level. Among them, wecB (nucleotide sequence as shown in SEQ ID NO.11) (51921.67) and manA (nucleotide sequence as shown in SEQ ID NO.12) (51984.82) had the highest expression intensity, followed by nanATEK (nucleotide sequence as shown in SEQ ID NO.13) (45860.27), manXYZ (nucleotide sequence as shown in SEQ ID NO.14) (42827.00), nagK (nucleotide sequence as shown in SEQ ID NO.15) (46321.96) and pykA (nucleotide sequence as shown in SEQ ID NO.16) (48445.74), then nagABCE (nucleotide sequence as shown in SEQ ID NO.17) (37355.58), cheW (38539.96), ptsG (nucleotide sequence as shown in SEQ ID NO.18) (38452.47) and kfiABC (nucleotide sequence as shown in SEQ ID NO.18) (51984.82). As shown in NO.19)(39999.04), the expression intensity of the nagAB (nucleotide sequence as shown in SEQ ID NO.20)(29465.90) ​​site was the lowest, with expression intensity ranging from 29465.90 to 51984.82, and the highest expression intensity being 1.76 times that of the lowest.

[0049] The results indicate that most sites exhibit high exogenous gene expression capacity under the T7 system, further validating the feasibility and effectiveness of these sites as neutral integration sites for gene expression regulation in synthetic biology.

[0050] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A T7 expression system suitable for Escherichia coli Nissle 1917, characterized in that, The T7 expression system comprises a first expression cassette and a second expression cassette, wherein: The first expression cassette expresses the lactose operon lacO and T7 RNA polymerase using the P tac , P J23119 , or P lacUV5 promoter. The second expression cassette expresses a lactose operon lacO and a target gene using a T7 promoter.

2. The T7 expression system of claim 1, wherein: The first expression cassette uses P tac The promoter expresses the lactose operon lacO and the T7 RNA polymerase.

3. The T7 expression system of claim 1, wherein: The second expression cassette further comprises a RBS sequence between the lactose operon lacO and the T7 RNA polymerase.

4. Escherichia coli Nissle 1917 comprising the T7 expression system of any one of claims 1-3.

5. Use of the T7 expression system of any one of claims 1-3 for expressing an exogenous gene in Escherichia coli Nissle 1917.

6. A method of expressing a foreign gene in Escherichia coli Nissle 1917, characterized by:

6. A method for introducing the T7 expression system of any one of claims 1-3 into Escherichia coli Nissle 1917.

7. The method of claim 6, wherein: The method further comprises a step of adding IPTG to induce expression, and the amount of IPTG added is 0.05-5 mM.

8. The method of claim 6, wherein: The first expression cassette is integrated for expression at the motA site of the genome of the Escherichia coli Nissle 1917.

9. The method of claim 6, wherein: The second expression cassette is integrated for expression in the genome of the Escherichia coli Nissle 1917.

10. The method of claim 9, wherein: The integrated expression is at the wecB, manA, nanATEK, manXYZ, nagABCE, pykA, chew, ptsG, nagK or kfiABC site of the genome of the Escherichia coli Nissle 1917.

Images