Escherichia coli strong promoter and application thereof
The strong promoter designed by the three promoters PdnaK, Plpp and PmglB was solved by connecting the three promoters of PdnaK, Plpp and PmglB, and the problems of limited number of promoters and insufficient expression intensity in E. coli were solved, achieving efficient gene expression and multigene regulation.
Patent Information
- Application Number
- CN202311664042.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-06
- Publication Date
- 2025-06-06
AI Technical Summary
The existing E. coli promoters have limited number, low intensity and narrow expression intensity span, making it difficult to achieve precise regulation of efficient expression of exogenous genes and multigene expression pathways.
A strong promoter formed by three E. coli-derived promoters PdnaK, Plpp and PmglB were designed to achieve efficient gene expression and multigene regulation without the need for additional inducers in E. coli.
It has achieved efficient expression of exogenous genes in E. coli, significantly improved translation intensity, and can accurately regulate multigene expression pathways, solving the problems of insufficient expression intensity and multigene regulation in the prior art.
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Figure BDA0004591157040000041 
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Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a strong Escherichia coli promoter and application thereof. Background Art
[0002] In the field of genetic engineering, promoters are crucial elements used to regulate gene expression, and their performance directly affects the expression intensity of exogenous genes in organisms such as Escherichia coli. Existing Escherichia coli promoters have problems such as limited quantity, low intensity, and narrow expression intensity span. Currently, the commonly used promoter types are mainly a series of inducible promoters, and their application faces some challenges when expressing multi-gene metabolic pathways. Although these inducible promoters can achieve on-off control of gene expression, it is often difficult to achieve precise regulation of each gene due to the influence of other external factors. And because the regulation method usually relies on the addition of exogenous inducers, this will increase the complexity and cost of the operation.
[0003] In recent years, some researchers have tried to construct promoter libraries by mutating constitutive promoters to obtain diverse promoter sequences. However, these mutant promoters are highly similar and are prone to homologous recombination in multi-gene expression, leading to gene loss. In addition, it is not easy to obtain variants with higher expression strength than the original promoter in mutant promoters, which limits the application scope of this strategy. In addition, although the engineering method of modifying promoter strength by mutation can obtain promoters that meet the needs, modifying constitutive promoters often destroys their intrinsic functional regions and has a high risk of failure.
[0004] Therefore, existing technologies have some limitations in achieving high expression intensity, precise control, and simplified operation. Summary of the invention
[0005] In view of the limitations of the above-mentioned background technology, the present invention aims to solve the following technical problems: how to achieve efficient expression of exogenous genes in Escherichia coli while avoiding dependence on the addition of inducers, and realize precise regulation of multi-gene expression pathways, thereby controlling the metabolic flow of the bacteria.
[0006] The purpose of the invention is to provide a strong Escherichia coli promoter and application thereof.
[0007] In a first aspect, the present invention claims a DNA molecule.
[0008] The DNA molecule claimed in the present invention is composed of all or part of the following three Escherichia coli-derived promoters connected in series: PdnaK promoter, Plpp promoter and PmglB promoter.
[0009] The sequence of the PdnaK promoter is as shown in SEQ ID No. 1 or is a sequence having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined by SEQ ID No. 1 and having promoter function.
[0010] The sequence of the Plpp promoter is shown in SEQ ID No. 2 or is a sequence having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined by SEQ ID No. 2 and having promoter function.
[0011] The sequence of the PmglB promoter is shown in SEQ ID No. 3 or is a sequence having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined by SEQ ID No. 3 and having promoter function.
[0012] In a specific embodiment of the present invention, the DNA molecule is any of the following:
[0013] (A1) DNA molecule A, which is composed of the PdnaK promoter, the Plpp promoter and the PmglB promoter in series;
[0014] (A2) DNA molecule B, which is composed of the PdnaK promoter and the Plpp promoter in series;
[0015] (A3) DNA molecule C, which is composed of the PdnaK promoter and the PmglB promoter in series;
[0016] (A4) DNA molecule D, which is composed of the Plpp promoter and the PmglB promoter connected in series.
[0017] Specifically, the sequence of the DNA molecule A is shown as SEQ ID No.7; the sequence of the DNA molecule B is shown as SEQ ID No.4; the sequence of the DNA molecule C is shown as SEQ ID No.5; and the sequence of the DNA molecule D is shown as SEQ ID No.6.
[0018] In a second aspect, the present invention claims protection for the use of the DNA molecule described in the first aspect as a promoter.
[0019] Furthermore, the application is to use the DNA molecule as a promoter to initiate the expression of a target gene in Escherichia coli.
[0020] Furthermore, the initiation of target gene expression is initiation of multi-gene expression or initiation of single gene expression, wherein the multi-gene is two or more genes.
[0021] In the application, the DNA molecule performs promoter function without the need for additional addition of an inducer.
[0022] In a third aspect, the present invention claims protection for an expression cassette, a recombinant vector or a recombinant bacterium containing the DNA molecule described in the first aspect above.
[0023] The expression cassette contains the DNA molecule as a promoter, which initiates the transcription of the target gene, and a transcription termination sequence.
[0024] In the present invention, the target gene is a single gene or multiple genes, wherein the multiple genes are two or more genes.
[0025] Furthermore, the recombinant vector is an Escherichia coli expression vector; and the recombinant bacteria is recombinant Escherichia coli.
[0026] In a fourth aspect, the present invention claims to protect the use of the DNA molecule described in the first aspect as a promoter in controlling the metabolic flow of Escherichia coli.
[0027] In the application, the DNA molecule performs promoter function without the need for additional addition of an inducer.
[0028] In the above aspects, in a specific embodiment of the present invention, the Escherichia coli is Escherichia coli MG1655.
[0029] In the above aspects, in a specific embodiment of the present invention, the backbone vector of the recombinant vector is pCDFDuet-1.
[0030] In the above aspects, in the specific embodiments of the present invention, the EGFP encoding gene or the red fluorescent protein encoding gene or the β-galactosidase encoding gene is used as an example to illustrate the target gene. In practical applications, the target gene can be determined according to actual needs.
[0031] Beneficial effects of the present invention:
[0032] 1. High expression efficiency: The present invention obtains a series of tandem promoters by transforming the promoter of Escherichia coli, including PdnaK-Plpp, PdnaK-PmglB, Plpp-PmglB and PdnaK-Plpp-PmglB. Experimental data show that these tandem promoters can achieve efficient gene expression in Escherichia coli. Taking EGFP as the expression gene, the measurement results show that compared with the PBAD promoter with 10mM arabinose as the inducer, the translation intensity of these tandem promoters is increased by 2.01, 1.8, 1.1 and 3.2 times, respectively. This means that the tandem promoter of the present invention can achieve a high level of gene expression, thereby solving the problem of low gene expression intensity in the prior art.
[0033] 2. Multi-gene regulation: The application of tandem promoters is not limited to single gene expression, but can also be used for precise regulation of multi-gene expression pathways. Experimental data show that the translation intensity of the PdnaK-Plpp-PmglB tandem promoter fused with red fluorescent protein (RFP) and β-galactosidase (LacZ) is 3.62 times that of the PBAD promoter. This means that the tandem promoter of the present invention has high universality and can be used for the expression of different target genes to achieve multi-gene regulation, thereby solving the problem that multi-gene expression pathways are difficult to precisely regulate in the prior art.
[0034] In summary, the technical solution of the present invention has achieved remarkable results in solving the promoter performance limitations, insufficient expression intensity and multi-gene regulation problems existing in the prior art. Through the design and optimization of tandem promoters, the present invention provides an efficient, flexible and widely applicable gene expression regulation solution, which brings important technical breakthroughs and application prospects to the field of genetic engineering. DETAILED DESCRIPTION
[0035] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.
[0036] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.
[0037] The quantitative data in the following examples are all the mean values of the results of at least three repetitions.
[0038] The strains and plasmids used in the following examples are specifically shown in Table 1.
[0039] Table 1. Strains and plasmids used in the present invention
[0040]
[0041]
[0042] Example 1: Escherichia coli MG1655 was used to express proteins and to determine promoter strength
[0043] 1. Tandem promoter drives EGFP expression
[0044] 1. Construction of tandem promoter-related expression vector
[0045] The E. coli transcriptome data was analyzed, and a series of promoters with high-intensity expression effects were selected. Such as the E. coli constitutive promoters PdnaK (SEQ ID No. 1), Plpp (SEQ ID No. 2) and PmglB (SEQ ID No. 3). In Hongxun Biosynthesized plasmids: pCDFDuet-1-PdnaK-Plpp-EGFP, pCDFDuet-1-PdnaK-PmglB-EGFP, pCDFDuet-1-Plpp-PmglB-EGFP and pCDFDuet-1-PdnaK-Plpp-PmglB-EGFP. Among them, in order to determine the strength of these four promoters, the constitutive promoters of the above combinations were fused with the reporter gene EGFP.
[0046] Description of the structure of the recombinant plasmid pCDFDuet-1-PdnaK-Plpp-EGFP: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.8 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-PdnaK-Plpp-EGFP, the promoter composed of PdnaK and Plpp in series (referred to as PdnaK-Plpp promoter, as shown in SEQ ID No.4) drives the expression of the EGFP coding gene.
[0047] Description of the structure of the recombinant plasmid pCDFDuet-1-PdnaK-PmglB-EGFP: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.9 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-PdnaK-PmglB-EGFP, the promoter formed by the tandem connection of PdnaK and PmglB (referred to as PdnaK-PmglB promoter, as shown in SEQ ID No.5) drives the expression of the EGFP coding gene.
[0048] Description of the structure of the recombinant plasmid pCDFDuet-1-Plpp-PmglB-EGFP: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.10 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-Plpp-PmglB-EGFP, the promoter composed of Plpp and PmglB in series (referred to as Plpp-PmglB promoter, as shown in SEQ ID No.6) drives the expression of the EGFP coding gene.
[0049] Description of the structure of the recombinant plasmid pCDFDuet-1-PdnaK-Plpp-PmglB-EGFP: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.11 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-PdnaK-Plpp-PmglB-EGFP, the promoter composed of PdnaK, Plpp and PmglB in series (referred to as PdnaK-Plpp-PmglB promoter, as shown in SEQ ID No.7) drives the expression of the EGFP coding gene.
[0050] At the same time, the PdnaK, Plpp, PmglB promoter controls and the PBAD promoter control were set up. That is, the expression vectors containing the above four promoters were constructed at the same time, as follows:
[0051] Description of the structure of the recombinant plasmid pCDFDuet-1-PdnaK-EGFP: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.12 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-PdnaK-EGFP, the expression of the EGFP encoding gene is driven by the PdnaK promoter (shown in SEQ ID No.1).
[0052] Description of the structure of the recombinant plasmid pCDFDuet-1-Plpp-EGFP: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No. 13 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-Plpp-EGFP, the expression of the EGFP encoding gene is driven by the Plpp promoter (shown in SEQ ID No. 2).
[0053] Description of the structure of the recombinant plasmid pCDFDuet-1-PmglB-EGFP: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.14 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-PmglB-EGFP, the expression of the EGFP encoding gene is driven by the PmglB promoter (shown in SEQ ID No.3).
[0054] Description of the structure of the recombinant plasmid pCDFDuet-1-PBAD-EGFP: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No. 15 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-PmglB-EGFP, the expression of the EGFP encoding gene is driven by the PBAD promoter (shown in SEQ ID No. 16).
[0055] 2. Translation strength of tandem promoters
[0056] In order to determine the expression intensity of the tandem promoters PdnaK-Plpp, PdnaK-PmglB, Plpp-PmglB, PdnaK-Plpp-PmglB, PdnaK, Plpp, and PmglB after promoter engineering, the intracellular EGFP level was detected in the stable phase of Escherichia coli MG1655 using EGFP as the expression gene. The specific operation is as follows: Each recombinant vector constructed in step 1 above was transformed into the competent state of Escherichia coli MG1655, and the corresponding recombinant bacteria were obtained for each recombinant vector. Then, each recombinant bacteria was cultured in LB medium or MOPS at 37°C and 220rpm. After culturing to the stable phase (11h), the bacteria were collected by centrifugation at 4000rpm and 4°C, and the cells were washed twice with PBS buffer (pH=7.4) to remove dead bacteria, bacterial secretions, and culture medium precipitation components to reduce the fluorescence detection background. The washed bacteria were placed in a 96-well fluorescent plate and the fluorescence intensity and OD600 of the bacteria were detected by a multifunctional fluorescence microplate reader. The detection conditions were 488nm excitation and 520nm emission. The promoter translation intensity was the ratio of fluorescence intensity to OD600. The results are shown in Table 2.
[0057] Table 2. Promoter translation strength
[0058]
[0059]
[0060] The results of fluorescence microplate reader detection showed that the translation intensity of PdnaK-Plpp, PdnaK-PmglB, Plpp-PmglB, PdnaK-Plpp-PmglB, PdnaK, Plpp, and PmglB promoters were 2.01, 1.8, 1.1, 3.2, 0.2, 0.31, and 0.23 times that of the PBAD promoter induced by 10 mM arabinose, respectively.
[0061] The above results show that after promoter engineering, the four tandem promoters PdnaK-Plpp, PdnaK-PmglB, Plpp-PmglB and PdnaK-Plpp-PmglB obtained have high translation efficiency.
[0062] 2. Universality of tandem promoter-expressed genes
[0063] The PdnaK-Plpp-PmglB tandem promoter was used to express the red fluorescent protein (RFP) and β-galactosidase (LacZ) fusion respectively, and then the translation intensity was determined. The details are as follows:
[0064] 1. Construction of relevant recombinant vectors
[0065] Description of the structure of the recombinant plasmid pCDFDuet-1-PdnaK-Plpp-PmglB-RFP-LacZ: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.17 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-PdnaK-Plpp-PmglB-RFP-LacZ, the promoter formed by the tandem connection of PdnaK-Plpp-PmglB (referred to as PdnaK-Plpp-PmglB promoter, as shown in SEQ ID No.7) drives the expression of the RFP-LacZ coding gene.
[0066] Description of the structure of the recombinant plasmid pCDFDuet-1-PBAD-RFP-LacZ: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.18 between the restriction sites BamHI and SacI of the pCDFDuet-1 plasmid. In the recombinant plasmid pCDFDuet-1-PBAD-RFP-LacZ, the expression of the EGFP encoding gene is driven by the PBAD promoter (shown in SEQ ID No.16).
[0067] 2. Translation intensity determination
[0068] Each recombinant vector constructed in step 1 above was transformed into competent E. coli MG1655, and corresponding recombinant bacteria were obtained for each recombinant vector. Then each recombinant bacteria was cultured in LB medium or MOPS at 37°C and 220rpm. After culturing to the stable period (11h), the bacteria were collected by centrifugation at 4000rpm and 4°C, and the cells were washed twice with PBS buffer (pH=7.4) to remove dead bacteria, bacterial secretions and culture medium precipitation components to reduce the fluorescence detection background. The washed bacteria were placed in a 96-well fluorescent plate and the fluorescence intensity and OD600 of the bacteria were detected in a multifunctional fluorescent microplate reader. The detection conditions were 488nm excitation and 520nm emission. The promoter translation intensity is the ratio of fluorescence intensity to OD600. The results are shown in Table 3.
[0069] Table 3. Promoter translation strength
[0070] PdnaK-Plpp-PmglB PBAD Translation intensity (OD600) 296746 81974
[0071] The results showed that the PdnaK-Plpp-PmglB tandem promoter was fused with red fluorescent protein (RFP) and β-galactosidase (LacZ) and then its translation intensity was 3.62 times that of the PBAD promoter. The experiment proved that the tandem promoter also had a high expression intensity for different target genes.
[0072] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.
Claims
1. DNA molecules, Features: The DNA molecule is composed of all or part of the following three promoters derived from Escherichia coli connected in series: PdnaK promoter, Plpp promoter and PmglB promoter.
2. The DNA molecule according to claim 1, Features: The sequence of the PdnaK promoter is as shown in SEQ ID No. 1 or is a sequence having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in SEQ ID No. 1 and having promoter function; and / or The sequence of the Plpp promoter is as shown in SEQ ID No. 2 or is a sequence having 99% or more, 95% or more, 90% or more, 85% or more or 80% homology with the DNA sequence defined in SEQ ID No. 2 and having promoter function; and / or The sequence of the PmglB promoter is shown in SEQ ID No. 3 or is a sequence having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined by SEQ ID No. 3 and having promoter function.
3. The DNA molecule according to claim 1 or 2, Features: The DNA molecule is any of the following: (A1) DNA molecule A, which is composed of the PdnaK promoter, the Plpp promoter and the PmglB promoter in series; (A2) DNA molecule B, which is composed of the PdnaK promoter and the Plpp promoter in series; (A3) DNA molecule C, which is composed of the PdnaK promoter and the PmglB promoter in series; (A4) DNA molecule D, which is composed of the Plpp promoter and the PmglB promoter connected in series.
4. The DNA molecule according to claim 3, Features: The sequence of the DNA molecule A is shown in SEQ ID No.7; and / or The sequence of the DNA molecule B is shown in SEQ ID No. 4; and / or The sequence of the DNA molecule C is shown in SEQ ID No.5; and / or The sequence of the DNA molecule D is shown as SEQ ID No.
6.
5. Use of the DNA molecule described in any one of claims 1 to 4 as a promoter.
6. The use according to claim 5, Features: In the application, the DNA molecule is used as a promoter to initiate the expression of a target gene in Escherichia coli.
7. The use according to claim 6, Features: The initiating target gene expression is initiating multi-gene expression or initiating single gene expression.
8. An expression cassette, recombinant vector or recombinant bacterium containing the DNA molecule according to any one of claims 1 to 4.
9. The expression cassette, recombinant vector or recombinant bacterium according to claim 8, Features: The recombinant vector is an Escherichia coli expression vector; and / or The recombinant bacteria is recombinant Escherichia coli.
10. Use of the DNA molecule according to any one of claims 1 to 4 as a promoter in controlling the metabolic flux of Escherichia coli.