Method for optimizing performance of luxi / r quorum sensing system in escherichia coli on basis of alkl transport protein

Abstract

Disclosed in the present application is a method for optimizing the performance of a LuxI / R quorum sensing system in Escherichia coli on the basis of an AlkL transporter protein. The method comprises: cloning a fatty acid transporter protein gene AlkL, a repressor protein gene luxR of a quorum sensing system, a target protein gene mcherry, a promoter Plux and a promoter Ptet onto a pCOLADuet-1 vector to construct a first plasmid, wherein the promoter for controlling the expression of the target protein gene is selected from Plux, and the promoter for controlling the expression of the repressor protein gene and the fatty acid transporter protein gene is selected from Ptet; and transforming the first plasmid into an Escherichia coli MG1655 strain to construct engineered Escherichia coli containing a self-induction system.

Description

A method for optimizing the performance of the LUXI / R quorum sensing system in Escherichia coli based on the ALKL transporter Technical Field

[0001] The present invention relates to the technical field of quorum sensing system performance, and in particular to a method for optimizing the performance of a LuxI / R quorum sensing system in Escherichia coli based on an AlkL transporter protein. Background Art

[0002] Escherichia coli is widely used in fields such as enzyme engineering, metabolic engineering and pharmaceutical engineering, and is the most commonly used strain in genetic engineering. At present, the genetic modification strategy commonly used in Escherichia coli mainly carries out static regulation by gene overexpression and gene knockout. Although these strategies have improved the production performance of bacterial strain to a certain extent, they can not balance the growth of bacterial strain and the synthesis of target product usually. In addition, although the inducible promoter that is usually used in Escherichia coli can achieve the balance of growth and production to a certain extent, due to the expensive and toxic inducing agent, it is restricted in large-scale production process. Therefore, it is necessary to develop a dynamic control element with wide adaptability to adapt to different industrial production processes.

[0003] Quorum sensing is a system in which microorganisms regulate the expression of related genes. Its inducer is a signal molecule secreted by the microorganism itself. Therefore, as the microorganism grows, the population density continues to increase, and the concentration of the inducer also accumulates. When the concentration of the inducer reaches the threshold, the related genes are induced to express. The quorum sensing systems in different microorganisms are different. Take the LuxI / LuxR system in Vibrio fischeri as an example: as the cell density continues to increase, the signal molecule acyl homoserine lactone (AHL) synthesized by the signal molecule synthase LuxI continues to increase. AHL freely enters and exits the cell along the concentration gradient and continues to accumulate inside and outside the cell. When the AHL concentration reaches a certain threshold, AHL will bind to the signal molecule binding protein LuxR to form a LuxR-AHL dimer. This dimer can serve as a promoter P lux activator, thereby activating the lux Controls the transcription of genes.

[0004] With the development and demand of industrial production, how to optimize the performance of the LuxI / R quorum sensing system in Escherichia coli has become an urgent problem to be solved.

[0005] Summary of the Invention

[0006] Based on this, it is necessary to address the technical problem of low performance of the existing technology for optimizing the LuxI / R quorum sensing system in Escherichia coli, and propose a method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter protein.

[0007] Provided is a method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter, the method comprising:

[0008] The fatty acid transporter gene, the repressor protein gene of the quorum sensing system, the target protein gene, the promoter P lux and promoter P tet Clone into pCOLADuet-1 vector to form the first plasmid, wherein the promoter for controlling the expression of the target protein gene is selected from P lux The promoter for controlling the expression of the repressor protein gene and the fatty acid transporter gene is selected from P tet ;

[0009] The first plasmid was transformed into Escherichia coli MG1655 strain to construct an engineered Escherichia coli containing an autoinduction system.

[0010] The method for optimizing the performance of LuxI / R quorum sensing system in Escherichia coli based on AlkL transporter is proposed in the present invention. The fatty acid transporter gene, the repressor protein gene of the quorum sensing system, the target protein gene, the promoter P lux and promoter P tet The first plasmid was constructed by assembling and cloning into the pCOLADuet-1 vector, wherein the promoter for controlling the expression of the target protein gene was selected from P lux The promoter for controlling the expression of the repressor protein gene and the fatty acid transporter gene is selected from P tet The first plasmid is transformed into the Escherichia coli MG1655 strain to construct an engineered Escherichia coli containing an autoinduction system, which can use the fatty acid transport protein AlkL to optimize the performance of the LuxI / R quorum sensing system heterologously expressed in Escherichia coli, including the strength, sensitivity and expression uniformity of the quorum sensing system. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0012] in:

[0013] FIG1 is a flow chart of a method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter in one embodiment;

[0014] FIG2 is a first experimental data diagram of optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter in one embodiment;

[0015] FIG3 is a second experimental data diagram of optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter in one embodiment;

[0016] FIG4 is a third experimental data diagram of optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter in one embodiment. DETAILED DESCRIPTION

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The terms used in the specification of the application are for the purpose of describing specific embodiments only and are not intended to limit this application. The terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0018] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0020] Please refer to FIG1 , which is a flow chart of a method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter provided in one embodiment of the present invention, including the following steps:

[0021] Step S101: fatty acid transporter gene, quorum sensing system repressor protein gene, target protein gene, promoter P lux and promoter P tetClone into pCOLADuet-1 vector to form the first plasmid, wherein the promoter for controlling the expression of the target protein gene is selected from P lux The promoter for controlling the expression of the repressor protein gene and the fatty acid transporter gene is selected from P tet ;

[0022] Wherein, the target protein gene may be a red fluorescent protein gene.

[0023] Step S201: transforming the first plasmid into Escherichia coli MG1655 strain to construct an engineered Escherichia coli containing an autoinduction system.

[0024] Among them, the fatty acid transport protein gene is AlkL, the repressor protein gene of the quorum sensing system is luxR, the target protein gene is mcherry, P lux It is a promoter that responds to the quorum sensing system.

[0025] In one embodiment, the first plasmid is pCOLADuet-P tet _luxR_AlkL-P lux _mcherry.

[0026] Specifically, after step S201, by exogenously adding signal molecules for testing, the fluorescence intensity generated by the engineered Escherichia coli of the constructed quorum sensing system was 2-3 times that of the control group. Not only that, the sensitivity and expression uniformity of the expression system were also improved. As shown in Figure 2, AlkL enhanced the intensity, sensitivity, and expression uniformity of the Lux system.

[0027] P lux The nucleotide sequence is shown in SEQ ID NO 1 below;

[0028] The nucleotide sequence of AlkL is shown in SEQ ID NO 2;

[0029] The nucleotide sequence of mcherry is shown in SEQ ID NO 3;

[0030] The nucleotide sequence of luxR is shown in SEQ ID NO 4;

[0031] P tet The nucleotide sequence is shown in SEQ ID NO 5;

[0032] The method proposed in this example for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter protein is to transform the fatty acid transporter gene, the repressor protein gene of the quorum sensing system, the target protein gene, and the promoter P into a single gene. lux and promoter P tet Clone into pCOLADuet-1 vector to form the first plasmid, wherein the promoter for controlling the expression of the target protein gene is selected from P lux The promoter for controlling the expression of the repressor protein gene and the fatty acid transporter gene is selected from P tet The first plasmid is transformed into the Escherichia coli MG1655 strain to construct an engineered Escherichia coli containing an autoinduction system, which can use the fatty acid transport protein AlkL to optimize the performance of the LuxI / R quorum sensing system heterologously expressed in Escherichia coli, including the strength, sensitivity and expression uniformity of the quorum sensing system.

[0033] In one embodiment, the method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter further comprises:

[0034] Step S201: Repressor protein gene, target protein gene, promoter P apFAB65 , promoter P lux , cloned into the pCOLADuet-1 vector to construct a second plasmid, wherein the promoter controlling the expression of the repressor protein gene is selected from P apFAB65 , the promoter controlling the expression of the target protein gene is selected from P lux ;

[0035] Step S202: constructing a third plasmid using the fatty acid transporter and the pACYCDuet vector, wherein the promoter for controlling the expression of the fatty acid transporter gene is selected from an arabinose-inducible promoter;

[0036] Step S203: constructing a fourth plasmid by inducing molecular synthetase and pUC19 vector, wherein the promoter for inducing molecular synthetase expression is P tet ;

[0037] Step S204: co-transforming the second plasmid, the third plasmid, and the fourth plasmid into Escherichia coli.

[0038] Among them, the inducible molecule synthase is LuxI.

[0039] In one embodiment, the second plasmid is pCOLADuet-P apFAB65 _luxR-P lux _mcherry.

[0040] In one embodiment, the third plasmid is pACYCDuet-Para_BAD _AlkL.

[0041] In one embodiment, the fourth plasmid is pUC19-P tet _LuxI.

[0042] Specifically, as shown in FIG3 , it can be seen that AlkL can relieve the inhibitory effect of the repressor protein luxR on the system.

[0043] P apFAB65 The nucleotide sequence is shown in SEQ ID NO 6;

[0044] P ara_BAD The nucleotide sequence is shown in SEQ ID NO 7;

[0045] The nucleotide sequence of LuxI is shown in SEQ ID NO 8;

[0046] The method proposed in this example for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter can transform the repressor protein gene, target protein gene, promoter P apFAB65 , promoter P lux , cloned into the pCOLADuet-1 vector to construct a second plasmid, wherein the promoter controlling the expression of the repressor protein gene is selected from P apFAB65 , the promoter controlling the expression of the target protein gene is selected from P lux Then, the third plasmid is constructed by using the fatty acid transporter and the pACYCDuet vector, wherein the promoter for controlling the expression of the fatty acid transporter gene is selected from the arabinose inducible promoter, thereby constructing the fourth plasmid by inducing the molecular synthetase and the pUC19 vector, wherein the promoter for inducing the expression of the molecular synthetase is P tet Finally, the second plasmid, the third plasmid and the fourth plasmid are co-transformed into Escherichia coli, and the fatty acid transport protein AlkL can be used to weaken the inhibition of the LuxR repressor protein on the Lux system.

[0047] In one embodiment, the method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter further comprises:

[0048] Step 301: The repressor protein gene, fatty acid transporter gene, target protein gene, promoter P tet and promoter P lux , cloned into the pCOLADuet-1 vector to form the fifth plasmid, wherein the promoter controlling the expression of the target protein gene is selected from Plux The promoter for controlling the expression of repressor protein gene and fatty acid transporter gene is selected from P tet ;

[0049] Step 302: constructing a sixth plasmid using the inducible molecular synthetase and the vector pACYCDuet, wherein the RBS controlling the inducible molecular synthetase is selected as RBS1;

[0050] Step 303: constructing a seventh plasmid using the inducible molecule synthetase and the vector pACYCDuet, wherein the RBS controlling the inducible molecule synthetase is selected as RBS2;

[0051] Step 304: constructing an eighth plasmid using the inducible molecular synthetase and the vector pACYCDuet, wherein the RBS controlling the inducible molecular synthetase is selected as RBS3;

[0052] Step 305: The sixth plasmid, the seventh plasmid, and the eighth plasmid are respectively transformed into the chassis cells containing the fifth plasmid to obtain a quorum sensing system induced by different cell concentrations.

[0053] Specifically, the sixth plasmid was transformed into cells containing the fifth plasmid, the seventh plasmid was transformed into cells containing the fifth plasmid, and the eighth plasmid was transformed into cells containing the fifth plasmid, resulting in quorum sensing systems induced at three different cell concentrations. Figure 4 shows the effect diagram of the quorum sensing system induced at different cell concentrations, a flow chart, and a comparison diagram with a stationary phase promoter.

[0054] In one embodiment, the fifth plasmid is pCOLADuet-P tet _luxR_AlkL-P lux _mcherry.

[0055] In one embodiment, the sixth plasmid is pACYCDuet-P apFAB65 _RBS1_luxI, the seventh plasmid is pACYCDuet-P apFAB65 _RBS2_luxI.

[0056] In one embodiment, the eighth plasmid is pACYCDuet-P apFAB65 _RBS3_luxI.

[0057] The nucleotide sequence of the RBS1 is shown in SEQ ID NO 9;

[0058] The nucleotide sequence of RBS2 is shown in SEQ ID NO 10;

[0059] The nucleotide sequence of the RBS3 is shown in SEQ ID NO 11;

[0060] The method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter proposed in this embodiment is to transform the repressor protein gene, fatty acid transporter gene, target protein gene, promoter P tet and promoter P lux , cloned into the pCOLADuet-1 vector to form the fifth plasmid, wherein the promoter controlling the expression of the target protein gene is selected from P lux The promoter for controlling the expression of repressor protein gene and fatty acid transporter gene is selected from P tet , and then the sixth plasmid is constructed by inducing molecular synthetase and vector pACYCDuet, wherein the RBS controlling the inducible molecular synthetase is selected as RBS1, and the seventh plasmid is constructed by inducing molecular synthetase and vector pACYCDuet, wherein the RBS controlling the inducible molecular synthetase is selected as RBS2, and then the eighth plasmid is constructed by inducing molecular synthetase and vector pACYCDuet, wherein the RBS controlling the inducible molecular synthetase is selected as RBS3, and finally the sixth plasmid, the seventh plasmid and the eighth plasmid are respectively transformed into chassis cells containing the fifth plasmid to obtain quorum sensing systems induced by different cell concentrations, and the quorum sensing system activated by different cell concentrations can be constructed by adjusting the amount of LuxI in the LuxI / R quorum sensing system in Escherichia coli.

[0061] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention, and should all be included in the scope of protection of the present invention.

Claims

1. A method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter, characterized in that, the method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter includes: Clone the fatty acid transport protein gene, the repressor gene of the quorum sensing system, the target protein gene, promoter P lux and promoter P tet onto the pCOLADuet-1 vector to construct the first plasmid. Among them, the promoter controlling the expression of the target protein gene is selected from P lux , and the promoter controlling the expression of the repressor gene and the fatty acid transport protein gene is selected from P tet ; Transform the first plasmid into Escherichia coli MG1655 strain to construct an engineered Escherichia coli containing a self-induction system.

2. The method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter according to claim 1, characterized in that, the method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter further includes: Clone the repressor protein gene, the target protein gene, promoter P apFAB65 , promoter P lux onto the pCOLADuet-1 vector to construct the second plasmid. Among them, the promoter controlling the expression of the repressor protein gene is selected from P apFAB65 , and the promoter controlling the expression of the target protein gene is selected from P lux ; Construct a third plasmid through a fatty acid transporter and the pACYCDuet vector, wherein the promoter controlling the expression of the fatty acid transporter gene is selected from an arabinose-inducible promoter; Construct a fourth plasmid by inducing molecular synthetase and pUC19 vector, wherein the promoter for inducing the expression of molecular synthetase is P tet ; Co-transform the second plasmid, the third plasmid and the fourth plasmid into Escherichia coli.

3. The method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter according to claim 2, characterized in that, The third plasmid is pACYCDuet-P ara_BAD _AlkL.

4. The method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter according to claim 2, characterized in that, The fourth plasmid is pUC19-P tet _LuxI.

5. The method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter according to claim 1, characterized in that, the method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter further includes: Clone the repressor protein gene, fatty acid transporter protein gene, target protein gene, promoter P tet and promoter P lux onto the pCOLADuet-1 vector to construct the fifth plasmid. Among them, the promoter controlling the expression of the target protein gene is selected from P lux , and the promoter controlling the expression of the repressor protein gene and the fatty acid transporter protein gene is selected from P tet ; Construct a sixth plasmid through an inducer synthase and the vector pACYCDuet, wherein the RBS controlling the inducer synthase selects RBS1; Construct a seventh plasmid through an inducer synthase and the vector pACYCDuet, wherein the RBS controlling the inducer synthase selects RBS2; Construct an eighth plasmid through an inducer synthase and the vector pACYCDuet, wherein the RBS controlling the inducer synthase selects RBS3; Transform the sixth plasmid, the seventh plasmid and the eighth plasmid into the chassis cells containing the fifth plasmid respectively to obtain a quorum sensing system induced at different cell concentrations.

6. The method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter according to claim 5, characterized in that, The sixth plasmid is pACYCDuet-P apFAB65 _RBS1_luxI, and the seventh plasmid is pACYCDuet-P apFAB65 _RBS2_luxI.

7. The method for optimizing the performance of the LuxI / R quorum sensing system in Escherichia coli based on the AlkL transporter according to claim 5, characterized in that, The eighth plasmid is pACYCDuet-P apFAB65 _RBS3_luxI.

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