AUTO-INDUCTION REGULATORY SYSTEM BASED ON QUORUM SENSING AND APPLICATION THEREOF

Abstract

Disclosed is an auto-induction regulatory system based on quorum sensing, comprising luxI, luxR and egfp, wherein, the promoter for controlling the expression of luxI and luxR is selected from P.sub.luxI, P.sub.BB or P.sub.J23100; the promoter for controlling the expression of egfp is selected from P.sub.luxI, P.sub.luxI(T-38C) or P.sub.luxI(C-77T). Also disclosed are an application of the auto-induction regulatory system based on quorum sensing in the automatic regulation of expression of a target gene of engineered Escherichia coli, as well as an application thereof in the preparation of alginate lyase and esterase. Further disclosed are a recombinant expression vector and a recombinant engineered bacterium comprising the auto-induction regulatory system based on quorum sensing.

Claims

1. An auto-induction regulatory system based on quorum sensing, wherein: the system comprises luxI, luxR and egfp, wherein, a promoter for controlling the expression of luxI and luxR is selected from P.sub.luxI, P.sub.BB or P.sub.J23100; a promoter for controlling the expression of egfp is selected from P.sub.luxI(C-77T); the nucleotide sequence of said P.sub.luxI(C-77T) is as set forth in SEQ ID NO 5.

2. The auto-induction regulatory system based on quorum sensing according to claim 1, wherein: the nucleotide sequence of said P.sub.luxI is as set forth in SEQ ID NO 1; the nucleotide sequence of said P.sub.BB is as set forth in SEQ ID NO 2; the nucleotide sequence of said P.sub.J23100 is as set forth in SEQ ID NO 3; the nucleotide sequences of said luxI, luxR and egfp are as set forth in SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11.

3. An application of the auto-induction regulatory system based on quorum sensing according to claim 1 in the automatic regulation of expression of a target gene of Escherichia coli engineered bacteria.

4. The application according to claim 3, wherein: said target gene is selected from an alginate lyase gene, and an esterase gene.

5. An application of the auto-induction regulatory system based on quorum sensing according to claim 1 in the preparation of alginate lyase and esterase.

6. An engineered bacterium containing the auto-induction regulatory system based on quorum sensing according to claim 1.

7. A recombinant expression vector, whose structure is P.sub.J23100-luxIR-P.sub.luxI(C-77T)-est7, wherein, the nucleotide sequence of said P.sub.luxI is as set forth in SEQ ID NO 1; the nucleotide sequence of said P.sub.luxI(C-77T) is as set forth in SEQ ID NO 5; the nucleotide sequences of said luxI, luxR are as set forth in SEQ ID NO 9, SEQ ID NO 10; the nucleotide sequence of said est7 is as set forth in SEQ ID NO 13.

8. A recombinant engineered bacterium comprising the recombinant expression vector according to claim 7 in its genome.

9. An application of the recombinant expression vector according to claim 7 in the preparation of alginate lyase or esterase.

10. An application of the recombinant engineered bacterium according to claim 8 in the preparation of alginate lyase or esterase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1A-B are schematic diagrams showing the principle of a LuxI/LuxR quorum sensing system, wherein, FIG. 1A, low cell density; FIG. 1B, high cell density.

[0034] FIG. 2A-D are diagrams characterizing a quorum sensing system that regulates luxIR with different promoters, wherein, FIG. 2A, P.sub.hixi-egfp; FIG. 2B, P.sub.hixi-luxIR-P.sub.luxI-egfp; FIG. 2C, P.sub.BB-luxIR-P.sub.luxI-egfp; FIG. 2D, P.sub.J23100-luxIR-P.sub.luxI-egfp.

[0035] FIG. 3A-D are summarized schematic diagrams characterizing P.sub.luxI quorum sensing-mutated strains, wherein, FIG. 3A, initial OD600 at which fluorescence intensity starts to increase; FIG. 3B, OD600 at which fluorescence intensity reaches the highest; FIG. 3C, the highest fluorescence intensity; FIG. 3D, highest fluorescence intensity per cell density.

[0036] FIG. 4A-B are schematic diagrams showing the results of the expression of alginate lyase and esterase regulated by an auto-induction dynamic regulatory system and a modified system thereof, wherein, FIG. 4A, alginate lyaseAL493; FIG. 4B, esterase Est7.

[0037] FIG. 5A-B are schematic diagrams showing the results of high-density fermentation of alginate lyase, wherein, FIG. 5A, P.sub.luxI-luxIR-P.sub.luxI(T-38C)-al493; FIG. 5B, T7-al493.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] The present disclosure will be further illustrated in conjunction with the embodiments below. However, the scope of the present disclosure is not limited to the following embodiments. It should be understood by those skilled in the art that various changes and modifications can be made to the present disclosure without deviating from the spirit and scope of the present disclosure.

[0039] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. available in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are conventional experimental methods, detection methods, etc. available in the prior art.

Embodiment 1 Construction of an Auto-Induction Dynamic Regulatory System

[0040] With plasmid pET28a as the original plasmid, egfp, P.sub.luxI, luxI and luxR fragments were successively recombined with a plasmid vector by a seamless splicing method. Engineered strains were obtained with Escherichia coli BL21(DE3) as the host. After cultivation with P.sub.luxI-egfp strains as control, OD600 and fluorescence intensity (Ex: 488 nm; Em: 530 nm) were determined. As shown in the results of FIG. 2A and FIG. 2B, in the case of the promoter P.sub.luxI alone, the highest value of the unit fluorescence intensity was about 200 au, while in strains containing a complete quorum sensing system, the unit fluorescence intensity was about 7000 au, suggesting that the auto-induction dynamic regulatory system was constructed successfully, which could function in Escherichia coli, and luxI and luxR were key components of the auto-induction dynamic regulatory system.

Embodiment 2 Optimization of the Regulatory luxIR Promoter

[0041] The regulatory promoter P.sub.luxI of luxIR was replaced with constitutive promoters P.sub.BB and P.sub.J23100, cultured with E. coli BL21(DE3) as the expression host, and determined for OD600 and fluorescence intensity. By comparing the unit fluorescence intensity, with the results shown in FIG. 2A-D, it was indicated that the unit fluorescence intensity of strains with promoters PluxI and P.sub.BB were about 7000 au and 9500 au respectively, while the unit fluorescence intensity of the strain regulated by P.sub.J23100 could reach 38000 au, and fluorescent protein with high expression level could be obtained under the regulation of P.sub.J23100, so P.sub.J23100 had the best regulation effect.

Embodiment 3 Construction, Screening and Characterization of PluxI Mutant Library

[0042] With plasmid P.sub.J23100-luxIR-P.sub.luxI-egfp as the original plasmid, P.sub.luxI was randomly mutated using a controlled error-prone PCR kit in a manner of multiple rounds of error-prone PCR. Preliminary screening was performed by observing the fluorescence intensity of the strains and the time of fluorescence generation through plate culture, and the screened strains were cultured in liquid, and the strains that did not generate fluorescence were removed by further screening. The obtained strains were sequenced to identify the mutation sites. After screening and sequencing, 13 single-site mutants and 3 multi-site mutants were obtained (the 3 multi-site mutants were deletion of three bases in Q1, G-6T/A-105G, T-21C/C-35T/T-38C/T-46G, respectively, whose nucleotide sequences were as set forth in SEQ ID NOs 6-8), wherein, all the multi-site mutants did not generate fluorescence, and the single-site mutants were determined for growth curve and fluorescence intensity. With the initial OD600 at which the fluorescence expression was regulated, the OD600 at which FI reached the highest, the highest FI and the highest FI/OD600 as indicators, the data were summarized as shown in Table 1 and FIG. 3A-D, thereby identifying two representative promoters P.sub.luxI(T-38C) and P.sub.luxI(C-77T).

TABLE-US-00001 TABLE 1 Summary of characterization data of single-site mutants of P.sub.luxI, the promoter in quorum sensing Initial OD.sub.600 Promoter for regulation OD.sub.600 at the Final Mutation of expression highest FI Highest FI FI/OD.sub.600 Control 0.097 1.162 45024.2 38699.8 T-3C 0.196 1.434 8049.3 7908.0 T-8C 0.088 1.984 5616.5 27254.8 T-21C 0.099 0.874 18751.3 24369.8 C-34A 0.129 0.705 2275.4 3672.6 C-35T 0.243 0.779 769.4 987.7 T-36C 0.097 0.592 3839.4 7122.7 G-37A 0.105 0.750 3156.5 4889.4 T-38A 0.028 0.847 2580.5 6740.8 T-38C 0.195 0.868 48493.6 56115.2 T-38G 0.099 0.647 3771.8 6055.9 T-46G 0.187 0.820 25883.8 31579.7 C-77T 0.358 0.678 6933.5 10234.3 C-102T 0.135 0.796 2154.0 4089.9

Embodiment 4 Application of P.SUB.luxI .Mutants in the Expression of Alginate Lyase AL493

[0043] The quorum sensing system with three promoters P.sub.luxI, P.sub.luxI(T-38C) and P.sub.luxI(C-77T) was applied in the expression of alginate lyase AL493 (whose nucleotide sequence was set forth in SEQ ID NO 12, and whose amino acid sequence was set forth in SEQ ID NO 17), so as to construct strains PluxI-luxIR-PluxI-al493 (the complete sequence of the constructed related gene was set forth in SEQ ID NO 19), P.sub.J23100-luxIR-P.sub.luxI(T-38C)-al493 and P.sub.J23100-luxIR-P.sub.luxI(C-77T)-al493, while using the fermentation culture results of T7-al493 as control. The strains regulated by the quorum sensing system were cultured at 20° C., while the strains regulated by T7 promoter needed to be firstly cultured at 37° C. to an OD600 of 0.6 and induced by adding IPTG with a final concentration of 0.5%0, and then the culture temperature was changed to 20° C. Interval sampling was conducted to determine OD600 and enzymatic activity.

[0044] The enzymatic activity was characterized by employing a 200 μL reaction system with 0.3% (w/v) sodium alginate as substrate to react for a period of 20 min, inactivating by boiling for 2 min and then determining the content of reducing sugar in the product by the DNS method.

[0045] The results were shown in FIG. 4A and Table 2. The experimental results showed that: the enzymatic activity of P.sub.J23100-luxIR-P.sub.luxI(T-38C)-al493 strain and the enzymatic activity per cell density were relatively high, where the enzymatic activity was 5.090 U/mL, which was 96.37% that of the control group, and the enzymatic activity per cell density was 3.240 U/mL, which was 83.06% that of the control group.

TABLE-US-00002 TABLE 2 Result summary of regulation on the expression of alginate lyase and esterase by an auto-induction dynamic regulatory system and a modified system thereof Highest enzymatic activity Enzymatic Strains (U/mL) activity/OD.sub.600 OD.sub.600 T7-al493 5.282 3.901 1.354 P.sub.J23100-luxIR-P.sub.luxI-al493 4.914 2.752 1.786 P.sub.J23100-luxIR-P.sub.luxI(T-38C)-al493 5.090 3.240 1.563 P.sub.J23100-luxIR-P.sub.luxI(C-77T)-al493 4.689 2.622 1.789 T7-est7 3.284 2.443 1.341 P.sub.J23100-luxIR-P.sub.luxI-est7 1.380 0.757 1.824 P.sub.J23100-luxIR-P.sub.luxI(T-38C)-est7 1.497 1.079 1.387 P.sub.J23100-luxIR-P.sub.luxI(C-77T)-est7 3.499 2.454 1.427

Embodiment 5 Application of P.SUB.luxI .Mutants in the Expression of Esterase Est7

[0046] The quorum sensing system with three promoters P.sub.luxI, P.sub.luxI(T-38C) and P.sub.luxI(C-77T) was applied in the expression of esterase Est7 (whose nucleotide sequence was set forth in SEQ ID NO 13, and whose amino acid sequence was set forth in SEQ ID NO 18), so as to construct strains P.sub.J23100-luxIR-P.sub.luxI-est7 (the complete sequence of the constructed related gene was set forth in SEQ ID NO 20), P.sub.J23100-luxIR-P.sub.luxI(T-38C)-est7 and P.sub.J23100-luxIR-P.sub.luxI(C-77 T)-est7, while using the fermentation culture results of T7-est7 as control. The strains regulated by the quorum sensing system were cultured at 20° C., while the strains regulated by T7 promoter needed to be firstly cultured at 37° C. to an OD600 of 0.6 and induced by adding IPTG with a final concentration of 0.5%0, and then the culture temperature was changed to 20° C. Interval sampling was conducted to determine OD600 and enzymatic activity.

[0047] The reaction substrate for esterase Est7 was a mixed solution of 20 mM p-nitrophenol laurate (pNPL) dissolved in isopropanol and DMSO (3:1). During the reaction, 460 μL of 100 mM Tris-HCl at pH 7.5 was firstly added as the buffer of the reaction, mixed with 20 μL of crude enzyme fluid and then incubated at 37° C. for 5 min. Then, 20 μL of reaction substrate was added to react in a water bath at 37° C. for 5 min. Finally, 500 μL of 1% SDS was added to terminate the reaction, and absorbance was determined at a wavelength of 405 nm.

[0048] The results were shown in FIG. 4B and Table 2. The experimental results showed that: the enzymatic activity of P.sub.J23100-luxIR-P.sub.luxI(C-77T)-est7 and the enzymatic activity per cell density were relatively high, where the highest value of enzymatic activity was 3.499 U/mL, which was 106.55% that of the control group, and the enzymatic activity per cell density was 2.454 U/mL.

Embodiment 6 High-Density Fermentation of Alginate Lyase AL493

[0049] High-density fermentation was conducted in a 5 L fermenter, while controlling pH at 7.0 and the stirring speed at 300 rpm. The initial medium for fermentation culture was LB medium (10 g/L of peptone, 5 g/L of yeast powder, and 10 g/L of sodium chloride) and 10 g/L of glucose. Glucose dry powder was supplemented every 12 h to a final concentration of 10 g/L. P.sub.J23100-luxIR-P.sub.luxI(T-38C)-al493 strains were cultured at 20° C., while T7-al493 strains needed to be firstly cultured at 37° C. to an OD600 of 0.6 and induced by adding IPTG with a final concentration of 0.5%0, and then the culture temperature was changed to 20° C. Interval sampling was conducted every 12 h to determine OD600 and enzymatic activity.

[0050] The results were shown in FIG. 5A-B and Table 3. The experimental results showed that: P.sub.J23100-luxIR-P.sub.luxI(T-38C)-al493 strains could grow to higher bacterial density, which was 1.46 times that of the control group. At the same time, the highest enzymatic activity of P.sub.J23100-luxIR-P.sub.luxI(T-38C)-al493 was 31.878 U/mL, which was 4.33 times that of the control group.

TABLE-US-00003 TABLE 3 Summary of high-density fermentation results of alginate lyase Highest enzymatic Strains Highest OD.sub.600 activity (U/mL) T7-al493 9.067 7.367 P.sub.J23100-luxIR-P.sub.luxI(T-38C)-al493 13.227 31.878

[0051] The above embodiments are provided to those skilled in the art to completely disclose and describe how to implement and use the claimed embodiments, rather than limiting the scope disclosed herein. Modifications apparent to those skilled in the art shall be covered within the scope of the attached claims.