Novel butenolide signal molecule and application thereof in gene expression regulation

Abstract

The invention discloses a novel butenolide signal molecule and application thereof in gene expression regulation and control. The invention provides a novel butenolide signal molecule SFB1. The structure of the novel butenolide signal molecule SFB1 is shown as a formula I). An inducible gene expression regulation and control circuit composed of the SFB1, the receptor protein of the SFB1 and the heterozygous promoter can efficiently work in escherichia coli, that is, the signal molecule SFB1 with nanomolar concentration can effectively remove repression of the receptor protein TylP on the heterozygous promoter and induce expression of a target gene; other types of streptomycete signal molecules including butenolide and gamma-butyric acid lactone from other streptomycetes cannot remarkably remove repression of TylP on the hybrid promoter, that is, the regulation and control circuit disclosed by the invention is not interfered by other types of common signal molecules in the streptomycetes, and can be used in combination with the existing AHL or GBL regulation and control tools; and a synthetic biological tool library is enriched.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a novel butenolide signal molecule and its application in gene expression regulation. Background Art

[0002] Quorum sensing (QS) refers to the phenomenon that microorganisms monitor population density by producing and sensing diffusible small molecules and regulate group behavior when the population reaches a certain concentration (reference A. Jayaraman, TK Wood, Bacterial quorum sensing: Signals, circuits, and implications for biofilms and disease. Annual Review of Biomedical Engineering 10, 145-167(2008)). For example, the marine bacterium Vibrio fischeri can inhibit the production of acylhomoserine lactones ( N -acylhomoserine lactone (AHL) signaling molecules regulate their own bioluminescence, thereby promoting symbiosis with marine organisms such as squid (reference KA Egland, EP Greenberg, Quorum sensing in Vibrio fischeri : elements of the luxI promoter. Molecular Microbiology 31, 1197-1204(1999)); For example, the human pathogenic bacterium Pseudomonas aeruginosa can regulate the secretion of its own extracellular enzymes, the formation of biofilms and the pathogenicity to the host through two sets of AHL signaling systems (reference P. Moura-Alves et al., Host monitoring of quorum sensing during Pseudomonas aeruginosa infection. Science(New York, NY) 366, eaaw1629 (2019)). In addition, many microorganisms can also regulate a variety of different physiological behaviors through quorum sensing signaling molecules, including the production of antibiotics (reference J. Zhang, H. Tan, Microbial quorum sensing signaling molecules and their roles in the biosynthesis of natural products. Science China Life Sciences66, 2429-2432 (2023)). As one of the main sources of natural antibiotics, the Gram-positive filamentous bacteria Streptomyces can produce a variety of different signal molecules to regulate its own morphological differentiation and secondary metabolism (reference G. Niu, KF Chater, Y. Tian, ​​J. Zhang,H. Tan, Specialised metabolites regulating antibiotic biosynthesisin Streptomyces spp. FEMS Microbiology Reviews 40, 554-573 (2016)). For example, the anti-tuberculosis drug streptomycin, which won the Nobel Prize in Physiology or Medicine in 1952, is produced by Streptomyces griseus and is regulated by the γ-butyrolactone (GBL) signaling molecule A factor (reference JY Kato, I. Miyahisa, M. Mashiko, Y. Ohnishi, S. Horinouchi, A single target is sufficient to account for the biological effects of the A-factor receptor protein of Streptomyces griseus . Journal of Bacteriology 186, 2206-2211 (2004)); For example, the antiparasitic antibiotic avermectin, which won the Nobel Prize in Physiology or Medicine in 2015, is produced by Streptomyces avermitilis and is regulated by the butenolide signaling molecule avenomectin (reference S. Kitani et al., Avenolide, a Streptomyces controlling hormone antibiotic production in Streptomyces avermitilis . Proceedings of the National Academy of Sciences of the United States of America108, 16410-16415 (2011)). Although there are relatively few studies on butenolides compared to γ-butyrolactones, they play an important role. For example, in Streptomyces cyclochromogenes, butenolide signaling molecules SABs not only regulate the synthesis of the antifungal compound nikkomycin, but also affect the production of the anticancer compound oviedomycin (reference J. Li et al., A butenolide signaling systemsynergized with biosynthetic gene modules led to effective activation and enhancement of silent oviedomycin production in Streptomyces . Metabolic Engineering 72, 289-296 (2022)); For example, the butenolide signaling molecule produced by Streptomyces albus can also cross-species activate the production of avermectin in Streptomyces avermitilis (reference TB Nguyen, S. Kitani, S. Shimma, T. Nihira, Butenolides from Streptomyces albus J1074 act as external signals to stimulate avermectin production in Streptomyces avermitilis . Applied and Environmental Microbiology 84, (2018). Therefore, the discovery of new butenolide signaling molecules will not only help in the discovery of new antibiotics, but also provide new strategies for increasing the production of important medicinal antibiotics.

[0003] Research on the quorum sensing signaling system of microorganisms can not only provide ideas for the prevention and treatment of pathogens and the discovery of new antibiotics, but also provide important component resources for the development of synthetic biology control tools (reference N. Kylilis, ZA Tuza, GB Stan, KM Polizzi, Tools for engineering coordinated system behaviour in synthetic microbial consortia. Nature Communications9,2677 (2018). For example, researchers have constructed a new gene expression regulatory circuit based on the AHL quorum sensing effect of Gram-negative bacteria and successfully applied it to the efficient synthesis of salicylic acid and 4-hydroxycoumarin in Escherichia coli (reference C. Ge et al., Redesigning regulatory components of quorum-sensing systemfor diverse metabolic control. Nature Communications 13, 2182 (2022)); For another example, the researchers also redesigned the AHL quorum sensing signal system to enable the relevant regulatory circuit to have a function that it did not originally have - the oscillatory expression of the target gene, and successfully applied it to control the cyclic release of anticancer drugs in engineered bacteria (reference MODin et al., Synchronized cycles of bacterial lysis for in vivo delivery. Nature 536, 81-85 (2016). It can be seen that discovering and studying the quorum sensing signaling system of microorganisms will contribute to the development of various disciplines such as synthetic biology and medicine.

[0004] However, unlike the common AHL quorum sensing signaling molecules in Gram-negative bacteria, there are relatively few studies on the quorum sensing signaling molecules of Streptomyces, especially the butenolide signaling molecules, due to their low content and difficulty in separation. So far, only 10 different butenolide signaling molecules have been identified from 4 different Streptomyces (reference D. Kong, X. Wang, J. Nie, G. Niu, Regulation of antibiotic production bysignaling molecules in Streptomyces . Frontiers in Microbiology 10, 2927 (2019). However, given the important role of butenolide signaling molecules in the regulation of secondary metabolism in Streptomyces, it is necessary to conduct more in-depth research on them and discover more new butenolide signaling molecules. At the same time, although there are many studies on the construction of synthetic biology gene expression regulation circuits based on microbial quorum sensing signaling systems, there are relatively few studies focusing on the quorum sensing effect of Streptomyces. One of the reasons is that there are relatively few quorum sensing signaling molecules found in Streptomyces. Therefore, the discovery of new Streptomyces signaling molecules is of great significance. Summary of the invention

[0005] The purpose of the present invention is to provide a novel butenolide signal molecule and its application in gene expression regulation.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a novel butenolide signal molecule SFB1, the structure of which is shown in Formula I): Formula I) In a second aspect, the present invention provides a method for preparing the signal molecule SFB1, comprising: Streptomyces fradiae ) The fermentation broth of ATCC 19609 was centrifuged to obtain the supernatant, which was extracted with ethyl acetate and then concentrated and dried to obtain an oily crude extract; the oily crude extract was dissolved with methanol and eluted through Sephadex LH-20 gel column chromatography, the eluate was collected, and the target molecule was separated by HPLC.

[0007] Furthermore, the HPLC separation method includes: separating and purifying the active ingredients through a chromatographic column Zorbax SB-C18 with a specification of 4.6 mm × 250 mm, 5 μm, and the elution conditions are: mobile phase A is methanol, and mobile phase B is H2O; 0-50 min, the proportion of mobile phase A is maintained at 45%; 50-60 min, the proportion of mobile phase A gradually increases from 45% to 95%; 60-70 min, the proportion of mobile phase A is maintained at 95%; 70-75 min, the proportion of mobile phase A gradually decreases from 95% to 45%; 75-80 min, the proportion of mobile phase A is maintained at 45%; the flow rate is 3 mL / min, the detection wavelength is 210 nm, and the compound peak eluted at 42.8 min is the target molecule.

[0008] In a third aspect, the present invention provides the use of the signal molecule SFB1 in the regulation of gene expression in synthetic biology.

[0009] In a fourth aspect, the present invention provides an engineered Escherichia coli, the construction method of which comprises the following steps: 1) Escherichia coli lacUV5 The LacI-binding sequence on the promoter is replaced with a TylP-binding sequence (such as a sequence shown in any one of SEQ ID NOs: 1-7) to obtain a hybrid promoter TylP-P (SEQ ID NO: 8), and the hybrid promoter is used to drive the expression of the target gene to obtain a hybrid promoter plasmid; 2) Use a constitutive promoter to drive tylP The expression of the gene (SEQ ID NO: 9) was used to obtain the signal molecule receptor plasmid; 3) Use the above two plasmids to introduce E. coli together to obtain; Wherein, the TylP is the receptor protein TylP of the signal molecule in Streptomyces freundii,tylP The gene encodes the receptor protein TylP.

[0010] Furthermore, the target gene includes a reporter gene, such as lux Luminescent gene.

[0011] Furthermore, the Streptomyces flexneri is Streptomyces flexneri ATCC 19609.

[0012] Furthermore, the constitutive promoter is the constitutive promoter P of the tetracycline resistance gene. con. , whose nucleotide sequence is shown in SEQ ID NO: 18.

[0013] In a specific embodiment of the present invention, the method for constructing the Escherichia coli engineered bacteria specifically comprises the following steps: S1. Using primers P15F (SEQ ID NO: 10) and P15R (SEQ ID NO: 11), the genomic DNA of Escherichia coli BL21 (DE3) (purchased from Tiangen) was used as a template to amplify the obtained DNA fragment (SEQ ID NO: 12), which is lacUV5 Promoter, through Xho I and Bam After double digestion with HI Xho I and Bam pCS26-Pac plasmid after HI double digestion (reference K. Tahlan et al., Initiation of actinorhodin export in Streptomyces coelicolor . Molecular Microbiology 63, 951-961 (2007)) to drive the bioluminescent gene cluster lux (GenBank: M90093.1), plasmid pLacUV5 was obtained; then primers Pl5F and Pl5TylPPR (SEQ ID NO: 13) were used to amplify the DNA fragment (SEQ ID NO: 14) with plasmid pLacUV5 as a template, which was the hybrid promoter TylP-P. Xho I and Bam After double digestion with HI Xho I and Bam The pCS26-Pac plasmids were ligated after HI double restriction digestion to obtain the hybrid promoter plasmid pTylPP; S2, using primers TylPF (SEQ ID NO: 15) and TylPR (SEQ ID NO: 16), with genomic DNA of Streptomyces freundii ATCC19609 as template, amplified the obtained DNA fragment (SEQ ID NO: 17), Bam After HI digestion, Eco RV and Bam pACYC184 plasmid after HI double digestion (reference K. Tahlan et al., Initiation of actinorhodin export in Streptomyces coelicolor . Molecular Microbiology 63, 951-961(2007)) so that it is linked to the constitutive promoter P of the tetracycline resistance gene on the plasmid con. The signal molecule receptor plasmid pRTylP (i.e., the potential butenolide signal molecule receptor protein expression plasmid in Streptomyces freundii) was obtained under the driving force of S3. The hybrid promoter plasmid pTylPP and the signal molecule receptor plasmid pRTylP were co-transformed into Escherichia coli JM109 (purchased from Invitrogen).

[0014] In a fifth aspect, the present invention provides the use of the engineered bacteria in the detection of the TylP ligand of Streptomyces freundii.

[0015] Furthermore, the application includes: adding the butenolide signal molecule to be tested to the bacterial liquid of the engineered bacteria, and determining its derepression ability on the TylP receptor protein according to the recovery of the bioluminescence of the engineered bacteria.

[0016] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The signal molecule SFB1 provided by the present invention is a novel butenolide signal molecule; the inducible gene expression control circuit composed of SFB1, its receptor protein and hybrid promoter can work efficiently in Escherichia coli, that is, the signal molecule SFB1 at a nanomolar concentration can effectively remove the repression of the hybrid promoter by the receptor protein TylP, that is, induce the expression of the target gene; other types of Streptomyces signal molecules including butenolide and γ-butyrolactone from other Streptomyces cannot significantly remove the repression of TylP on the hybrid promoter, that is, the control circuit of the present invention is not subject to crosstalk from other types of signal molecules commonly found in Streptomyces, and can be used in combination with existing AHL or GBL-type control tools, enriching the synthetic biology tool library. In summary, the novel butenolide signal molecule of the present invention can provide tools and resources for the study of secondary metabolism of Streptomyces and the construction of synthetic biology control circuits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The following is the construction and verification of the butenolide signal molecule detection strain in Streptomyces freundii in the preferred embodiment of the present invention. A. Working principle of the butenolide signal molecule detection strain. B. Detection of hybrid promoter activity and signal molecule receptor repression activity of hybrid promoter. RLU: relative luminescence intensity.

[0018] Figure 2 The gel column separation and detection of butenolide signal molecules in Streptomyces freundii in the preferred embodiment of the present invention. MeOH is a methanol solvent control for elution and dissolution of the extraction product, Crude extract is the ethyl acetate extraction component of Streptomyces freundii fermentation, and Fr. 1-30 is the fraction collected by Sephadex LH-20 gel column chromatography of the crude extract of Streptomyces freundii fermentation.

[0019] Figure 3 The HPLC separation and detection of the butenolide signal molecule SFB1 in Streptomyces freundii in the preferred embodiment of the present invention. A. HPLC detection of separation and purification of signal molecules, the detection wavelength is 210nm. B. Detection of SFB1 signal molecules by reporter strains. MeOH is the solvent methanol control for the dissolved product, Crude extract is the Fr. 5-8 mixture with signal molecule activity after separation by gel chromatography column, and SFB1 is the pure signal molecule separated by HPLC.

[0020] Figure 4 The mass spectrometry identification of SFB1 in the preferred embodiment of the present invention is shown in Figure 1. A. The retention time of SFB1 in the total ion current of the mass spectrometer is 3.79 min. B. The characteristic ion fragments of SFB1 in the positive ion mode are m / z 181.0843, [M-H2O] + 251.1641, [M+H] + 269.1755 and [M+Na] + 291.1561.

[0021] Figure 5 The SFB1 in the preferred embodiment of the present invention is 1 H-NMR (CDCl3, 500 MHz) spectrum.

[0022] Figure 6 The SFB1 in the preferred embodiment of the present invention is 13 C-NMR (CDCl3, 125 MHz) spectrum.

[0023] Figure 7 This is the DEPT spectrum of SFB1 in a preferred embodiment of the present invention.

[0024] Figure 8The SFB1 in the preferred embodiment of the present invention is 1 H- 1 H COSY spectrum.

[0025] Figure 9 This is the HSQC spectrum of SFB1 in a preferred embodiment of the present invention.

[0026] Figure 10 This is the HMBC spectrum of SFB1 in a preferred embodiment of the present invention.

[0027] Figure 11 The molecular structure and NMR data of SFB1 in the preferred embodiment of the present invention are shown in bold. 1 H- 1 The H COSY key correlation signal and the arrows indicate the HMBC key correlation signal.

[0028] Figure 12 The application of the SFB1 signal system in gene expression regulation in a preferred embodiment of the present invention is to detect the derepression activity of the signal molecule receptor protein TylP by different concentrations of signal molecules SFB1 (A), SAB1 (B) and SCB2 (C). DETAILED DESCRIPTION

[0029] Previous studies have shown that the production of tylosin, an important antibiotic in Streptomyces freundii, is regulated by quorum sensing signaling molecules (reference DRD Bignell, N. Bate, E. Cundliffe, Regulation of tylosin production: role of a TyIP-interactive ligand. Molecular Microbiology 63, 838-847 (2007)), and the results of related sequence comparisons showed that the signal molecule controlling the synthesis of tylosin may be butenolide (reference S. Kitani et al., Avenolide, a Streptomyces controlling hormone antibiotic production in Streptomyces avermitilis . Proceedings of the National Academy of Sciences of the United States of America 108, 16410-16415 (2011)). Therefore, the purpose of the present invention is to identify signal molecules in Streptomyces freundii and provide an application method thereof in the regulation of gene expression related to synthetic biology.

[0030] The present invention adopts the following technical solution: The overall technical solution of the present invention is to first target Streptomyces freundii ( Streptomyces fradiae) ATCC19609 (reference YQ Tian et al., An efficient method for targeted cloning oflarge DNA fragments from Streptomyces . Applied Microbiology and Biotechnology 107, 5749-5760(2023).) to construct an efficient detection method for potential butenolide signaling molecules in Streptomyces freundii; then, the potential butenolide signaling molecules in Streptomyces freundii were fermented, prepared, isolated, purified and structurally identified; finally, the application of the identified signaling molecules in synthetic biology gene expression regulation was characterized.

[0031] The specific plan is as follows: 1. Establishment of a method for detecting signal molecules in Streptomyces freundii: Since Escherichia coli has the characteristics of fast growth and simple genetic manipulation, it is often constructed as a reporter strain for different small molecule compounds. Therefore, the present invention uses Escherichia coli as the host and selects lux The luminescent gene was used as a reporter gene to construct a detection strain for the butenolide signal molecule in Streptomyces freundii ATCC 19609. The specific principle and method are as follows: Based on literature reports, the signal molecule that regulates the synthesis of tylosin in Streptomyces freundii ATCC 19609 may function through the receptor protein TylP, and TylP binds to tylQ The promoter region of the gene plays a regulatory role (reference DRD Bignell, N. Bate, E. Cundliffe, Regulation of tylosin production: role of a TylP-interactive ligand. Molecular Microbiology 63, 838-847 (2007)). The present invention first overcomes the problem that many Streptomyces promoters do not work in Escherichia coli due to genetic background differences. First, a promoter that can work in Escherichia coli and is regulated by TylP is constructed. Specifically, the Escherichia coli lacUV5 The LacI-binding sequence on the promoter was replaced with a TylP-binding sequence (such as a sequence shown in any one of SEQ ID NOs: 1-7) to obtain a hybrid promoter TylP-P (SEQ ID NO: 8), which was used to drive lux The expression of the luminescent gene was driven by a constitutive promoter. tylP The expression of the gene (SEQ ID NO: 9) was used to obtain a signal molecule receptor plasmid; finally, the two plasmids were co-transformed into Escherichia coli as a detection strain for potential butenolide signal molecules that can be recognized by TylP in Streptomyces freundii (seeFigure 1 ).

[0032] 2. Identification of signal molecules in Streptomyces flexneri: The present invention uses a suitable culture medium to perform a large-scale fermentation of Streptomyces flexneri ATCC19609 and extract the product; then the crude fermentation extract is separated and purified by gel chromatography column and high performance liquid chromatography (HPLC), and the components with TylP ligand activity (which can dissociate the repression of TylP on the hybrid promoter and restore the bioluminescence of the reporter strain) are collected by using an Escherichia coli reporter strain (see Figure 2 , Figure 3 ); Finally, the structure of the final isolated compound monomer SFB1 was identified by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy (see Figures 4 - 11 ).

[0033] 3. Application of Streptomyces freundii signal molecules in synthetic biology gene expression regulation: The present invention uses the above-constructed E. coli reporter strain to detect the application potential of the isolated novel butenolide signal molecule in synthetic biology gene expression regulation. Specifically, different concentrations of SFB1 are added to the above-mentioned E. coli reporter strain containing both the hybrid promoter-driven reporter gene plasmid and the constitutive promoter-driven signal molecule receptor expression plasmid to detect its derepression ability of the receptor protein TylP, that is, its ability to activate the expression of the target gene (see Figure 12 ).

[0034] The signal molecule receptor involved in the present invention can also be a receptor protein with equivalent function formed by replacing, deleting, or adding one or more amino acids in the TylP amino acid sequence, or other types of regulatory proteins that can recognize the signal molecule SFB1; the use method involved in the present invention can also be other regulatory circuits or usage scenarios that can respond to SFB1 and thus regulate the expression of the target gene; the host involved in the present invention can also be other microorganisms or higher organisms other than Escherichia coli.

[0035] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0036] The pCS26-Pac plasmid used in the following examples was obtained from the State Key Laboratory of Microbial Resources Preliminary Development, Institute of Microbiology, Chinese Academy of Sciences (reference K. Tahlan et al., Initiation of actinorhodinexport in Streptomyces coelicolor . Molecular Microbiology 63, 951-961 (2007)).

[0037] pACYC184 plasmid was obtained from the State Key Laboratory of Microbial Resources Development, Institute of Microbiology, Chinese Academy of Sciences (reference K. Tahlan et al., Initiation of actinorhodin export in Streptomyces coelicolor . Molecular Microbiology 63, 951-961 (2007)).

[0038] Example 1 Establishment of a method for detecting signal molecules in Streptomyces freundii Using primers Pl5F (SEQ ID NO: 10) and Pl5R (SEQ ID NO: 11), genomic DNA of Escherichia coli BL21 (DE3) (purchased from Tiangen) was used as a template to amplify a DNA fragment of approximately 0.13 kb (SEQ ID NO: 12). lacUV5 Promoter, through Xho I and Bam After double digestion with HI Xho I and Bam pCS26-Pac plasmid after HI double restriction digestion (reference K. Tahlan et al., Initiation of actinorhodin export in Streptomyces coelicolor . Molecular Microbiology 63, 951-961 (2007)) to drive the bioluminescent gene cluster lux , and obtained plasmid pLacUV5; then, primer pair Pl5F / Pl5TylPPR (SEQ ID NO: 13) was used to amplify a DNA fragment of about 0.13 kb (SEQ ID NO: 14) with plasmid pLacUV5 as template, and the hybrid promoter TylP-P was obtained. Xho I and Bam After double digestion with HI Xho I and Bam The pCS26-Pac plasmids were ligated after HI double restriction digestion to obtain the hybrid promoter characterization plasmid pTylPP.

[0039] Using primers TylPF (SEQ ID NO: 15) and TylPR (SEQ ID NO: 16), the genomic DNA of Streptomyces freundii ATCC19609 was used as a template to amplify a DNA fragment of about 0.77 kb (SEQ ID NO: 17). Bam After HI digestion, Eco RV and BampACYC184 plasmid after HI double digestion (reference K. Tahlan et al., Initiation of actinorhodin export in Streptomyces coelicolor . Mol Microbiol 63, 951-961(2007)) so that it is linked to the constitutive promoter P of the tetracycline resistance gene on the plasmid con. (SEQ ID NO: 18) was driven to express, and the expression plasmid pRTylP of potential butenolide signal molecule receptor protein in Streptomyces freundii was obtained.

[0040] Plasmids pCS26-Pac, pLacUV5 and pTylPP or plasmid combinations pTylPP + pACYC184 and pTylPP + pRTylP were introduced into Escherichia coli JM109 (purchased from Invitrogen) by chemical transformation to obtain five different engineered bacteria. Finally, the bioluminescence of the above engineered bacteria was measured by a plate reader (Gen5 1.10). The specific operation is as follows: pick a single colony of the above-mentioned engineered bacteria into a small test tube containing 3 mL of liquid LB medium containing kanamycin (single-plasmid engineered bacteria) or kanamycin and chloramphenicol (double-plasmid engineered bacteria), shake and culture at 28°C, 220 rpm for 8-12 h, then add it to a 96-well plate at a ratio of 1:100 (each well has 200 μL of LB medium containing the corresponding antibiotics), place it in a constant temperature shaker at 220 rpm, and culture it at 28°C for 8-12 hours. Detect the luminescence of the sample wells with a plate reader, and compare the relative activity of the hybrid promoter and the repression of the hybrid promoter by the receptor protein based on the luminescence value (reference X. Liu et al., A widespread response of Gram-negative bacterial acyl-homoserine lactone receptors to Gram-positive Streptomyces gamma-butyrolactone signaling molecules. Sci China Life Sci64, 1575-1589 (2021)). The results showed that compared with the engineered bacteria containing the empty plasmid pCS26-Pac, the engineered bacteria containing the pLacUV5 plasmid only showed weak bioluminescence, mainly because it was repressed by the Escherichia coli's own repressor protein LacI; compared with pLacUV5, the pTylPP transformed bacteria showed significant bioluminescence, indicating that the LacI protein binding sequence was successfully replaced and the repression was removed; and when the TylP receptor protein expression plasmid and the hybrid promoter plasmid pTylPP were co-transformed into Escherichia coli, the bioluminescence was significantly inhibited. In contrast, the luminescence value of the co-transformed bacteria containing the hybrid promoter plasmid and the pACYC184 empty plasmid was not inhibited, indicating that the TylP receptor protein was successfully expressed and could have a significant repressive effect on the corresponding hybrid promoter (see Figure 1 ). In summary, the present invention successfully constructed a strain for detecting the TylP ligand (potential butenolide signal molecule) in Streptomyces freundii ATCC 19609.

[0041] The formula of LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and 1.5% agar added to the solid medium.

[0042] The conditions of the PCR reaction were as follows: DNA polymerase KOD FX (1U / μL) 1 μL, KOD FX PCR buffer 2×PCRBuffer 25 μL, dNTPs (2 mM each) 10 μL (the above reagents were purchased from TOYOBO, Japan), DMSO (dimethylsulfoxide) 2.5 μL, 10 μM primers 1.5 μL each, template DNA 2.5 μL (10-50 ng), ddH2O 6 μL, total reaction volume 50 μL. PCR cycle conditions: 94℃ pre-denaturation for 5 min; 30 cycles of amplification reaction (94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 68℃ extension, 1 kb / min, the specific extension time is determined according to the length of the amplified fragment); full extension at 68℃ for 5 min; 4℃, storage.

[0043] Example 2 Fermentation preparation, separation and purification, and structural identification of butenolide signal molecules from Streptomyces freundii 1. Fermentation and preparation of quorum-sensing signal molecules of Streptomyces freundii (1) Seed culture: Use a cotton swab to collect mature spores grown in Gao's medium No. 1 and store them in 1 ml of 10% glycerol milk powder. Take 100 μL of Streptomyces freundii spores and inoculate them into sterilized composite seed culture medium at 220 rpm / min and 28°C. After 3 days of culture, obtain the seed solution.

[0044] The composite seed culture medium was prepared as follows: 6 g corn syrup, 5 g hot fried bean cake powder, 5 g yeast powder, 3 g CaCO3, add water to make up to 1 L, adjust the pH to 6.7, dispense 50 mL of each bottle into 500 ml shake flasks, add 200 μL soybean oil, and sterilize at 120°C for 30 minutes.

[0045] (2) Fermentation culture: The fermented seed culture solution was inoculated into the composite culture medium at a rate of 5% for 17 L fermentation at 220 rpm / min and 28°C for 24 h.

[0046] The ingredients of the composite medium include CaCO31.9 g, fish meal 10.5 g, corn flour 17 g, corn protein 10.5 g, betaine hydrochloride 0.37 g, NaCl 1 g, KCl 0.9 g, (NH4)2HPO40.37 g, nickel sulfate 0.004 g, cobalt chloride 0.003 g, MgSO4·7H2O 0.1 g. Add water to make up to 1 L, adjust the pH to 7.2, dispense 60 mL per bottle into 500 mL shake flasks, add 2.5 mL soybean oil, and sterilize at 120°C for 30 minutes.

[0047] (3) Extraction and concentration of signal molecules from Streptomyces freundii: The fermentation broth obtained from Streptomyces freundii was centrifuged at 12,000 rpm / min for 10 min to obtain the fermentation supernatant. The supernatant was extracted twice with an equal volume of ethyl acetate and concentrated and dried using a rotary evaporator to obtain 1.368 g of an oily crude extract.

[0048] 2. Isolation and purification of quorum sensing signal molecules of Streptomyces freundii (1) Gel chromatography column separation and activity detection: Sephadex LH-20 (GE Healthcare) gel chromatography was activated with methanol overnight and equilibrated with methanol; the crude oil extract was dissolved with 1 mL of methanol and eluted through Sephadex LH-20 gel column chromatography, with methanol as the eluent, and one tube was collected for every 10 mL, with a total of 30 tubes (Fr. 1-30); the signal molecule detection strain in Example 1 was cultured and used to detect the activity of signal molecules in different segments, and the specific operation was as follows: a single colony of the above-mentioned engineered bacteria was picked into a small test tube of 3 mL liquid LB culture medium containing kanamycin and chloramphenicol, and cultured at 28°C and 220 rpm for 8-12 h, and then added to a 96-well plate at a ratio of 1:100 (each well had 200 μL of LB culture medium containing kanamycin and chloramphenicol, and the above-mentioned segmented collection was added to the pTylPP + pRTylP co-transformation strain culture system at a ratio of 1% (v / v)), and placed in a constant temperature shaker at 220 rpm, and then cultured at 28°C for 8–12 hours. The luminescence of the sample wells was detected by a plate reader. The activation of the bioluminescence of the engineered bacteria by different segments was compared to determine the segment containing the signal molecule. The results showed that segments 5–8 (Fr. 5–8) had signal molecule activity (see Figure 2 ), Fr. 5-8 were combined into Fr. B, and 0.901 g of active mixture was obtained after concentration under reduced pressure and drying.

[0049] (2) HPLC separation and activity detection: The combined fragmented product Fr. B was separated and prepared by HPLC, and the signal molecule activity was detected by the reporter strain method, and finally the pure signal molecule was obtained. The specific operation is as follows: the active ingredients were separated and purified by chromatographic column Zorbax SB-C18 (4.6mm× 250 mm, 5 μm), and the elution conditions were as follows: mobile phase A was methanol, mobile phase B was H2O; 0-50min, the proportion of mobile phase A was maintained at 45%; 50-60min, the proportion of mobile phase A gradually increased from 45% to 95%; 60-70min, the proportion of mobile phase A was maintained at 95%; 70-75min, the proportion of mobile phase A gradually decreased from 95% to 45%; 75-80min, the proportion of mobile phase A was maintained at 45%. The flow rate was 3 mL / min, the detection wavelength was 210 nm, and the compound peak eluted at 42.8 min was detected by the reporter system to effectively activate the reporter system to emit light. After collecting and drying the active product peak under reduced pressure, 5.8 mg of the quorum sensing effector signal molecule of Streptomyces freundii was finally obtained, which was named SFB1 (see Figure 3 ).

[0050] 3. Structural identification of the quorum-sensing signal molecule SFB1 of Streptomyces freundii The quasi-molecular ion peak of the quorum sensing signal molecule was determined by Waters Xevo G2 XS QToF (Manchester, UK). m / z 291.1561 [M+Na] + (C 15 H 24 O4Na, calculated value is 291.1567) (see Figure 4 ). Combined with a 500MHz Bruker NMR spectrometer to measure the one-dimensional 1 H-NMR, 13 C-NMR, DEPT, 2D 1 H- 1 H COSY, 1 H- 13 CHMBC and 1 H- 13 C HSQC data infer that the molecular formula of the quorum sensing effector molecule is C 15 H 24 O4 (see Figure 5~Figure 10 ). 1 H-NMR (500 MHz, CDCl3) and 13 C-NMR (125 MHz, CDCl3) showed that H-2 ( δ H 6.11), H-3 ( δ H 7.45)、H-4( δ H 5.04) signal and C-1 ( δ C 173.2), C-9 ( δ C 216.1) signals are basically consistent, which shows that SFB1 has the same γ-butene lactone skeleton as Avenolide, and C-9 is a carbonyl group. In Avenolide, the C-10 position does not contain a hydrogen proton signal connected to the hydroxyl, methyl and ethyl groups, while the C-10 position of SFB1 contains a hydrogen proton signal, and the DEPT spectrum also shows the tertiary carbon signal. The HMBC spectrum shows that C-10 has related signals with H-15, and H-10 has related signals with C-9 and C-14, proving that the hydroxyl group is lost at C-10. At the same time, H-8 ( δ H 4.28) The signal shifted from low field to high field and was a double peak, which was speculated to be the addition of a hydroxyl group at the C-8 position. By analyzing the MS and NMR spectra, we determined the planar structure of SFB1 and assigned the carbon and hydrogen signals of SFB1 in the NMR spectrum (see Fig.11 ).

[0051] The above results indicate that the present invention successfully separated and identified the signal molecule recognized by TylP in Streptomyces freundii, which is a butenolide with a new structure.

[0052] Example 3 Application of the Streptomyces freundii signal molecule SFB1 in gene expression regulation in synthetic biology The engineered bacteria containing the TylP receptor protein expression plasmid pRTylP and the corresponding hybrid promoter plasmid pTylPP in Example 1 were selected to detect different concentrations of SFB1 and other types of butenolide signaling molecules SAB1 (reference W. Wang et al., Identification of a butenolide signaling system that regulatesnikkomycin biosynthesis in Streptomyces . J Biol Chem 293, 20029-20040 (2018)) and the GBL-like signaling molecule SCB2 (as a control) (reference X. Liu et al., A widespread response of Gram-negative bacterial acyl-homoserine lactone receptors to Gram-positive Streptomyces gamma-butyrolactone signaling molecules. Sci China Life Sci 64, 1575-1589 (2021)) on its bioluminescence, the specific operation is as follows: pick the above-mentioned engineering bacteria single colony into a small test tube of 3 mL liquid LB medium containing kanamycin and chloramphenicol, shake and culture at 28°C, 220 rpm for 8-12 h, then add it to a 96-well plate at a ratio of 1:100 (each well has 200 μL LB medium containing kanamycin and chloramphenicol and different concentrations of SFB1, SAB1 or SCB2), place it in a constant temperature shaker at 220 rpm, 28°C for 8-12 hours, and then detect the luminescence of the sample wells by a plate reader. By comparing the recovery of bioluminescence of the engineering bacteria by different signal molecules, its derepression ability of the TylP receptor protein is determined. The results show that only SFB1 has a significant activation effect on the bioluminescence of the above-mentioned engineering bacteria, and the nanomolar (100 nanomolar) concentration of SFB1 can exert the effect. In contrast, SAB1 and SCB2 had no activating effect on the bioluminescence of the engineered bacteria within the concentration range tested (see Fig.12 ).

[0053] The above results indicate that the present invention has successfully constructed an inducible gene expression regulatory circuit based on the butenolide signal SFB1 signal system of Streptomyces freundii, and has good orthogonality with the reported butenolide and GBL signal systems, providing a new type of component resource for synthetic biology-related construction.

[0054] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A novel butenoic acid lactone signal molecule SFB1, the structure of which is shown in Formula I): Formula I).

2. The method for preparing the signal molecule according to claim 1, characterized in that: Streptomyces freundii ( Streptomyces fradiae ) The fermentation broth of ATCC 19609 was centrifuged to obtain the supernatant, which was extracted with ethyl acetate and then concentrated and dried to obtain an oily crude extract; the oily crude extract was dissolved with methanol and eluted through Sephadex LH-20 gel column chromatography, the eluate was collected, and the target molecule was separated by HPLC.

3. The method according to claim 2, characterized in that The HPLC separation method includes: separating and purifying the active ingredients through a chromatographic column ZorbaxSB-C18 with a specification of 4.6 mm × 250 mm, 5 μm, and the elution conditions are: mobile phase A is methanol, and mobile phase B is H2O; 0-50 min, the proportion of mobile phase A is maintained at 45%; 50-60 min, the proportion of mobile phase A gradually increases from 45% to 95%; 60-70 min, the proportion of mobile phase A is maintained at 95%; 70-75 min, the proportion of mobile phase A gradually decreases from 95% to 45%; 75-80 min, the proportion of mobile phase A is maintained at 45%; the flow rate is 3 mL / min, the detection wavelength is 210 nm, and the compound peak eluted at 42.8 min is the target molecule.

4. Use of the signal molecule according to claim 1 in regulating gene expression in synthetic biology.

5. An engineered Escherichia coli, characterized in that: The construction method includes the following steps: 1) Escherichia coli lacUV5 The LacI-binding sequence on the promoter is replaced with the TylP-binding sequence to obtain the hybrid promoter TylP-P, which is used to drive the expression of the target gene to obtain a hybrid promoter plasmid; 2) Use a constitutive promoter to drive tYP Gene expression, obtaining signal molecule receptor plasmid; 3) Use the above two plasmids to introduce E. coli together to obtain; Wherein, the TylP is the receptor protein TylP of the signal molecule in Streptomyces freundii, tYP The gene encodes the receptor protein TylP.

6. The engineered bacteria according to claim 5, characterized in that The target gene includes a reporter gene, preferably lux Luminescent gene; The Streptomyces flexneri is Streptomyces flexneri ATCC 19609; The constitutive promoter is the constitutive promoter P of the tetracycline resistance gene. con. .

7. The engineered bacteria according to claim 6, characterized in that The sequence bound by TylP is selected from any one of SEQ ID NOs: 1-7; The nucleotide sequence of the hybrid promoter is shown in SEQ ID NO: 8; Said tYP The nucleotide sequence of the gene is shown in SEQ ID NO:

9.

8. The engineered bacteria according to claim 5, characterized in that The construction method specifically includes the following steps: S1. Using primers P15F and P15R, the genomic DNA of Escherichia coli BL21 (DE3) was used as a template to amplify the obtained DNA fragment, namely lacUV5 Promoter, through XOt I and Bam After double digestion with HI XOt I and Bam The pCS26-Pac plasmid after HI double restriction digestion was connected to drive the bioluminescent gene cluster lux , obtaining plasmid pLacUV5; then using primers Pl5F and Pl5TylPPR, with plasmid pLacUV5 as template, amplifying the resulting DNA fragment, namely the hybrid promoter TylP-P, XOt I and Bam After double digestion with HI XOt I and Bam The pCS26-Pac plasmids were ligated after HI double restriction digestion to obtain the hybrid promoter plasmid pTylPP; S2, using primers TylPF and TylPR, the genomic DNA of Streptomyces freundii ATCC 19609 was used as a template to amplify the obtained DNA fragment. Bam After HI digestion, Eco RV and Bam HI double-digested pACYC184 plasmid, so that the constitutive promoter P of the tetracycline resistance gene on the plasmid con. The signal molecule receptor plasmid pRTylP was obtained under the driving force of , i.e., the expression plasmid of potential butenolide signal molecule receptor protein in Streptomyces freundii; S3, transforming the hybrid promoter plasmid pTylPP and the signal molecule receptor plasmid pRTylP into Escherichia coli, to obtain; Among them, the sequences of primers P15F and P15R are shown in SEQ ID NOs: 10 and 11, the sequence of primer P15TylPPR is shown in SEQ ID NO: 13, and the sequences of primers TylPF and TylPR are shown in SEQ ID NOs: 15 and 16.

9. Use of the engineered bacteria according to any one of claims 5 to 8 in detecting the TylP ligand of Streptomyces freundii.

10. The use according to claim 9, characterized in that: The application comprises: adding the butenolide signal molecule to be tested into the bacterial liquid of the engineering bacteria, and determining the derepression ability of the engineering bacteria on the TylP receptor protein according to the recovery of the bioluminescence of the engineering bacteria.

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