Butenolide signal molecules and their use in the regulation of gene expression

Abstract

The application discloses a novel butenolide signal molecule and application thereof in gene expression regulation. The application provides a novel butenolide signal molecule SFB1, and the structure is shown as formula I). The inducible gene expression regulation circuit composed of the SFB1, a receptor protein and a hybrid promoter can work efficiently in Escherichia coli, that is, nanomolar concentration of the signal molecule SFB1 can effectively remove the repression of the hybrid promoter by the receptor protein TylP, and induce the expression of a target gene; other types of streptomyces signal molecules including butenolide and gamma-butyrolactone from other streptomyces cannot significantly remove the repression of the hybrid promoter by the TylP, that is, the regulation circuit of the application is not interfered by other common types of signal molecules in streptomyces, and can be combined with existing AHL or GBL type regulation tools, so that the synthetic biology tool library is enriched.

Description

Technical Field

[0001] The present 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 in which 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 produce and respond to acylhomoserine lactones ( N -acylhomoserine lactone (AHL) signaling molecules regulate their own bioluminescence, thereby promoting symbiosis with marine organisms such as squid (References 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, biofilm formation and 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 regulate a variety of 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 bacterium Streptomyces can produce a variety of different signal molecules to regulate its own morphological differentiation and secondary metabolism (References 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) type 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 avemectin (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 relatively few studies have been conducted on butenolides compared to γ-butyrolactone, they play an important role. For example, in Streptomyces convolutus, 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 novel butenolide signaling molecules will not only contribute to the discovery of new antibiotics, but also provide new strategies for increasing the production of important medicinal antibiotics.

[0003] Research on microbial quorum sensing signaling systems not only provides insights into pathogen control and the discovery of new antibiotics, but also provides important component resources for the development of synthetic biology control tools (references N. Kylilis, Z.A. Tuza, G.B. Stan, K.M. Polizzi, Tools for engineering coordinated system behaviour in synthetic microbial consortia. Nature Communications9,2677 (2018). For example, researchers have constructed a novel 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 system for diverse metabolic control. Nature Communications 13, 2182 (2022)); For example, researchers have also redesigned the AHL quorum sensing signaling system to enable the relevant regulatory circuit to have a function that it did not originally have - oscillatory expression of target genes, 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 butenolide signaling molecules, due to low content and difficulty in isolation. To date, 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 regulating secondary metabolism in Streptomyces, further research is necessary to discover more novel butenolide signaling molecules. Furthermore, while there are many studies on constructing synthetic biology gene expression regulatory circuits based on microbial quorum sensing signaling systems, relatively few focus on quorum sensing in Streptomyces. One reason for this is that relatively few quorum sensing signaling molecules have been discovered in Streptomyces. Therefore, the discovery of novel 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 a first aspect, the present invention provides a novel butenolide signaling molecule SFB1, the structure of which is shown in Formula I):

[0007] Formula I)

[0008] 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 in methanol and eluted through Sephadex LH-20 gel column chromatography, the eluate was collected, and the target molecule was separated by HPLC.

[0009] Furthermore, the HPLC separation method includes: separating and purifying the active ingredient through a Zorbax SB-C18 chromatographic column with a column 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; from 0 to 50 minutes, the proportion of mobile phase A is maintained at 45%; from 50 to 60 minutes, the proportion of mobile phase A gradually increases from 45% to 95%; from 60 to 70 minutes, the proportion of mobile phase A is maintained at 95%; from 70 to 75 minutes, the proportion of mobile phase A gradually decreases from 95% to 45%; from 75 to 80 minutes, 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 minutes is the target molecule.

[0010] In a third aspect, the present invention provides the use of the signal molecule SFB1 in regulating gene expression in synthetic biology.

[0011] In a fourth aspect, the present invention provides an engineered Escherichia coli, the construction method of which comprises the following steps:

[0012] 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), which is used to drive the expression of the target gene to obtain a hybrid promoter plasmid;

[0013] 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;

[0014] 3) Use the above two plasmids to introduce E. coli together to obtain;

[0015] Wherein, the TylP is the receptor protein TylP of the signal molecule in Streptomyces flexneri, tylP The gene encodes the receptor protein TylP.

[0016] Furthermore, the target gene includes a reporter gene, such as lux Luminescent gene.

[0017] Furthermore, the Streptomyces flexneri is Streptomyces flexneri ATCC 19609.

[0018] 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.

[0019] In one embodiment of the present invention, the method for constructing the engineered Escherichia coli bacteria specifically comprises the following steps:

[0020] S1. Using primers P15F (SEQ ID NO: 10) and P15R (SEQ ID NO: 11), genomic DNA of Escherichia coli BL21 (DE3) (purchased from Tiangen) was used as a template to amplify the resulting DNA fragment (SEQ ID NO: 12). lacUV5 Promoter, through Xho I and Bam After HI double enzyme digestion, Xho I and Bam pCS26-Pac plasmid after HI double enzyme 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 is the hybrid promoter TylP-P. Xho I and Bam After HI double enzyme digestion, Xho I and Bam The pCS26-Pac plasmids digested with HI were connected to obtain the hybrid promoter plasmid pTylPP;

[0021] S2, using primers TylPF (SEQ ID NO: 15) and TylPR (SEQ ID NO: 16), the genomic DNA of Streptomyces flexneri ATCC19609 was used as a template to amplify the obtained DNA fragment (SEQ ID NO: 17). Bam After HI digestion, Eco RV and Bam pACYC184 plasmid after HI double enzyme digestion (reference K. Tahlan et al., Initiation of actinorhodin export in Streptomyces coelicolor . Molecular Microbiology 63, 951-961(2007)) so that 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 flexneri) was obtained under the driving force of .

[0022] S3. The hybrid promoter plasmid pTylPP and the signal molecule receptor plasmid pRTylP were co-transformed into Escherichia coli JM109 (purchased from Invitrogen).

[0023] In a fifth aspect, the present invention provides the use of the engineered bacteria in the detection of Streptomyces flexneri TylP ligand.

[0024] 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 based on the recovery of the bioluminescence of the engineered bacteria.

[0025] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0026] 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 Streptomyces secondary metabolism and the construction of synthetic biology control circuits. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This section describes the construction and validation of a strain for detecting butenolide signaling molecules in Streptomyces flexneri, a preferred embodiment of the present invention. A. Working principle of the butenolide signaling molecule detection strain. B. Detection of hybrid promoter activity and signaling molecule receptor repression activity on the hybrid promoter. RLU: relative luminescence intensity.

[0028] Figure 2 This figure illustrates the gel column separation and detection of butenolide signaling molecules from Streptomyces freundii in a preferred embodiment of the present invention. MeOH serves as the methanol solvent used for elution and dissolution of the extract. The crude extract is the ethyl acetate extract fraction from the S. freundii fermentation. Fr. 1-30 is the fraction collected from the crude extract of the S. freundii fermentation using Sephadex LH-20 gel column chromatography.

[0029] Figure 3 This is the HPLC separation and detection of the butenolide signaling molecule SFB1 from Streptomyces freundii, a preferred embodiment of the present invention. A. HPLC detection of the signaling molecule separation and purification, with a detection wavelength of 210 nm. B. Detection of the SFB1 signaling molecule using a reporter strain. MeOH is the methanol control solvent for the lysate. The crude extract is the Fr. 5–8 mixture with signaling activity after gel chromatography separation. SFB1 is the pure signaling molecule isolated by HPLC.

[0030] Figure 4 The mass spectrometric identification of SFB1 in the preferred embodiment of the present invention is as follows: 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.

[0031] Figure 5 The SFB1 in the preferred embodiment of the present invention is 1 H-NMR (CDCl3, 500 MHz) spectrum.

[0032] Figure 6 The SFB1 in the preferred embodiment of the present invention is 13 C-NMR (CDCl3, 125 MHz) spectrum.

[0033] Figure 7 This is the DEPT spectrum of SFB1 in a preferred embodiment of the present invention.

[0034] Figure 8 The SFB1 in the preferred embodiment of the present invention is 1 H- 1 H COSY spectrum.

[0035] Figure 9 HSQC spectrum of SFB1 in a preferred embodiment of the present invention.

[0036] Figure 10 This is the HMBC spectrum of SFB1 in a preferred embodiment of the present invention.

[0037] 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 arrows indicate the key related signals of H COSY and HMBC.

[0038] Figure 12 The application of the SFB1 signaling system in gene expression regulation in a preferred embodiment of the present invention is to detect the derepression activity of the signaling molecule receptor protein TylP by different concentrations of the signaling molecules SFB1 (A), SAB1 (B) and SCB2 (C). DETAILED DESCRIPTION

[0039] Previous studies have shown that the production of tylosin, an important antibiotic in Streptomyces freundii, is regulated by quorum sensing signaling molecules (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 molecules controlling the synthesis of tylosin may be butenolides (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 flexneri and provide an application method for regulating gene expression related to synthetic biology.

[0040] The present invention adopts the following technical solutions:

[0041] 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.

[0042] The specific plan is as follows:

[0043] 1. Establishment of a method for detecting signal molecules in Streptomyces flexneri: Due to the characteristics of fast growth and simple genetic manipulation, Escherichia coli 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 flexneri 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 flexneri 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 Ty1P is constructed. Specifically, the Escherichia coli promoter is transformed into lacUV5 The LacI-binding sequence on the promoter was replaced with a TylP-binding sequence (such as 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 To express the luminescent gene, a hybrid promoter plasmid was obtained; secondly, a constitutive promoter was used to drive tylPThe 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 test strain for potential butenolide signal molecules that can be recognized by TylP in Streptomyces flexneri (see Figure 1 ).

[0044] 2. Identification of signal molecules in Streptomyces flexneri: The present invention uses a suitable culture medium to perform large-scale fermentation of Streptomyces flexneri ATCC19609 and extract the product; the crude fermentation extract is then separated and purified using gel chromatography columns and high-performance liquid chromatography (HPLC), and the components with TylP ligand activity (capable of dissociating TylP from the repression of the hybrid promoter and restoring the bioluminescence of the reporter strain) are collected using an Escherichia coli reporter strain (see Figure 、 ​ Finally, the structure of the isolated compound SFB1 was identified by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy (see ​ ).

[0045] III. Application of Streptomyces flexneri signal molecules in synthetic biology gene expression regulation: This invention uses the above-constructed E. coli reporter strain to test 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 a hybrid promoter-driven reporter gene plasmid and a constitutive promoter-driven signal molecule receptor expression plasmid to test its ability to derepress the receptor protein TylP, that is, its ability to activate the expression of the target gene (see ​ ).

[0046] 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 usage 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.

[0047] 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.

[0048] The pCS26-Pac plasmid used in the following examples 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 actinorhodinexport in ​ . ​ 63, 951-961 (2007)).

[0049] The pACYC184 plasmid was obtained from the State Key Laboratory of Microbial Resources Development, Institute of Microbiology, Chinese Academy of Sciences (ref. K. Tahlan et al., Initiation of actinorhodin export in ​ ​ . ​ 63, 951-961 (2007)).

[0050] Example 1 Establishment of a Signal Molecule Detection Method in Streptomyces flexneri

[0051] Using primers P15F (SEQ ID NO: 10) and P15R (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). ​ Promoter, through ​ I and ​ After HI double enzyme digestion, ​ I and ​ pCS26-Pac plasmid after HI double enzyme digestion (reference K. Tahlan et al., Initiation of actinorhodin export in ​ ​ . ​ 63, 951-961 (2007)) to drive the bioluminescent gene cluster ​ , obtaining plasmid pLacUV5; then using primer pair Pl5F / Pl5TylPPR (SEQ ID NO: 13), with plasmid pLacUV5 as template, amplifying a DNA fragment of approximately 0.13 kb (SEQ ID NO: 14), obtaining the hybrid promoter TylP-P, ​ I and ​ After HI double enzyme digestion, ​ I and ​ The pCS26-Pac plasmids digested with HI were connected to obtain the hybrid promoter characterization plasmid pTylPP.

[0052] Using primers TylPF (SEQ ID NO: 15) and TylPR (SEQ ID NO: 16), a DNA fragment of approximately 0.77 kb (SEQ ID NO: 17) was amplified using genomic DNA of Streptomyces flexneri ATCC19609 as a template. ​ After HI digestion, ​ RV and ​ pACYC184 plasmid after HI double enzyme digestion (reference K. Tahlan et al., Initiation of actinorhodin export in Streptomyces coelicolor . Mol Microbiol 63, 951-961(2007)) so that 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 flexneri was obtained.

[0053] Five different engineered strains were obtained by chemically transforming the plasmids pCS26-Pac, pLacUV5, and pTylPP, or the plasmid combinations pTylPP + pACYC184 and pTylPP + pRTylP, into Escherichia coli JM109 (purchased from Invitrogen). Bioluminescence was measured using a plate reader (Gen5 1.10). The specific operation is as follows: a single colony of the above-mentioned engineered bacteria was picked and placed in 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), and cultured at 28°C, 220 rpm, and shaking for 8-12 h. Then, the colony was added to a 96-well plate at a ratio of 1:100 (200 μL of LB medium containing the corresponding antibiotics per well). After incubation at 28°C in a constant temperature shaker at 220 rpm for 8-12 h, the luminescence of the sample wells was detected by a plate reader. The luminescence value was used to compare the relative activity of the hybrid promoter and the repression of the hybrid promoter by the receptor protein (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 showed only weak bioluminescence, mainly because it was repressed by the E. coli self-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 E. 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 produce 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 flexneri ATCC 19609.

[0054] The formula of LB medium is as follows: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and 1.5% agar is added to the solid medium.

[0055] PCR reaction conditions were as follows: 1 μL of KOD FX DNA polymerase (1 U / μL), 25 μL of KOD FX 2× PCR buffer, 10 μL of dNTPs (2 mM each) (all reagents purchased from TOYOBO, Japan), 2.5 μL of DMSO (dimethylsulfoxide), 1.5 μL of each 10 μM primer, 2.5 μL of template DNA (10–50 ng), 6 μL of ddH₂O, and a total reaction volume of 50 μL. PCR cycling conditions included initial denaturation at 94°C for 5 min, 30 cycles of amplification (denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 68°C at 1 kb / min; the specific extension time depends on the length of the amplified fragment), full extension at 68°C for 5 min, and storage at 4°C.

[0056] Example 2 Fermentation Preparation, Isolation, Purification and Structural Identification of Butenolide Signaling Molecule from Streptomyces freundii

[0057] 1. Fermentation and preparation of quorum sensing signal molecules of Streptomyces freundii

[0058] (1) Seed culture: Use a cotton swab to collect mature spores grown on 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 seed solution.

[0059] The composite seed culture medium was prepared as follows: 6 g corn steep liquor, 5 g hot-fried bean cake powder, 5 g yeast powder, 3 g CaCO3, and water was added to make up to 1 L. The pH was adjusted to 6.7, and 50 mL of each bottle was dispensed into 500 ml shake flasks. 200 μL of soybean oil was added and sterilized at 120°C for 30 min.

[0060] (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.

[0061] The complex medium consists of 1.9 g of CaCO₃, 10.5 g of fish meal, 17 g of corn flour, 10.5 g of zein, 0.37 g of betaine hydrochloride, 1 g of NaCl, 0.9 g of KCl, 0.37 g of (NH₄)₂HPO₄, 0.004 g of nickel sulfate, 0.003 g of cobalt chloride, and 0.1 g of MgSO₄·7H₂O. Add water to 1 L, adjust the pH to 7.2, and aliquot 60 mL of the solution into 500 mL shake flasks. Add 2.5 mL of soybean oil and sterilize at 120°C for 30 minutes.

[0062] (3) Extraction and concentration of signal molecules from Streptomyces flexneri: The fermentation broth obtained from Streptomyces flexneri 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 on a rotary evaporator to obtain 1.368 g of crude oil.

[0063] 2. Isolation and purification of quorum sensing signal molecules of Streptomyces freundii

[0064] (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 in 1 mL of methanol and eluted through Sephadex LH-20 gel column chromatography, with methanol as the eluent. One tube was collected for every 10 mL, and a total of 30 tubes (Fr. 1-30) were collected; the signal molecule detection strain in Example 1 was cultured and used to detect the activity of signal molecules in different segments. The specific operation was as follows: a single colony of the above-mentioned engineered bacteria was picked up and placed in 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 After 8–12 hours of incubation at 28°C and 8 rpm, the luminescence of the sample wells was detected by a plate reader. By comparing the activation of the engineered bacteria bioluminescence by different segments, the segment containing the signal molecule was determined. The results showed that segments 5–8 (Fr. 5–8) had signal molecule activity (see Figure 2 ), Fr. 5-8 were combined to form Fr. B, which was concentrated under reduced pressure and dried to obtain 0.901 g of an active mixture.

[0065] (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 mentioned above, and finally the pure signal molecule was obtained. The specific operation was as follows: the active component was separated and purified by chromatographic column Zorbax SB-C18 (4.6mm×250mm, 5μm), 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, and the detection wavelength was 210 nm. The compound peak eluted at 42.8 min was detected by the reporter system and could effectively activate the reporter system to emit light. The active product peak was collected and dried under reduced pressure to finally obtain 5.8 mg of the quorum sensing effector signal molecule of Streptomyces freundii, which was named SFB1 (see Figure 3 ).

[0066] 3. Structural identification of the quorum sensing signal molecule SFB1 of Streptomyces freundii

[0067] 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 one-dimensional 1 H-NMR, 13 C-NMR, DEPT, two-dimensional 1 H- 1 H COSY, 1 H- 13 CHMBC and 1 H- 13 C HSQC data suggest that the molecular formula of the quorum sensing molecule is C 15 H 24 O4 (see Figures 5 to 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 indicates that SFB1 has the same γ-butenolide skeleton as Avenolide, with C-9 as 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 C-10 position has lost the hydroxyl group. At the same time, H-8 ( δ HThe signal at 4.28 shifted from low field to high field and was a doublet, 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 Figure 11 ).

[0068] The above results indicate that the present invention successfully separated and identified the signal molecule recognized by TylP in Streptomyces flexneri, which is a butenolide with a new structure.

[0069] Example 3 Application of the Streptomyces flexneri signal molecule SFB1 in synthetic biology gene expression regulation

[0070] 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 regulates snikkomycin biosynthesis in Streptomyces . J Biol Chem 293, 20029-20040 (2018)) and GBL-like signaling molecule SCB2 (as a control) (Reference X. Liu et al., A widespread response ofGram-negative bacterial acyl-homoserine lactone receptors to Gram-positive Streptomyces gamma-butyrolactone signaling molecules. Sci China Life Sci64, 1575-1589 (2021)) on their bioluminescence. The specific operation was as follows: a single colony of the above-mentioned engineered bacteria was picked and placed in a small test tube of 3 mL liquid LB medium containing kanamycin and chloramphenicol. The culture was shaken at 28°C and 220 rpm for 8–12 hours. Then, the colony was added to a 96-well plate at a ratio of 1:100 (200 μL of LB medium containing kanamycin, chloramphenicol and different concentrations of SFB1, SAB1, or SCB2 per well). After incubation at 28°C in a constant temperature shaker at 220 rpm for 8–12 hours, the luminescence of the sample wells was measured using a plate reader. By comparing the recovery of bioluminescence of the engineered bacteria by different signal molecules, the derepression ability of the TylP receptor protein was determined. The results showed that only SFB1 had a significant activation effect on the bioluminescence of the above-mentioned engineered bacteria, and the effect was achieved at a nanomolar (100 nanomolar) concentration of SFB1. In contrast, SAB1 and SCB2 had no activating effect on the bioluminescence of the engineered bacteria within the concentration range tested (see Figure 12 ).

[0071] The above results indicate that the present invention has successfully constructed an inducible gene expression regulatory circuit based on the butenolide signal SFB1 signaling system of Streptomyces freundii, and has good orthogonality with the reported butenolide and GBL signaling systems, providing a new component resource for synthetic biology-related construction.

[0072] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Butenolide signaling 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 flexneri ( 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 in 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 ingredient using a ZorbaxSB-C18 column with a column size of 4.6 mm × 250 mm, 5 μm. The elution conditions are: mobile phase A is methanol, and mobile phase B is H2O; from 0 to 50 minutes, the proportion of mobile phase A is maintained at 45%; from 50 to 60 minutes, the proportion of mobile phase A gradually increases from 45% to 95%; from 60 to 70 minutes, the proportion of mobile phase A is maintained at 95%; from 70 to 75 minutes, the proportion of mobile phase A gradually decreases from 95% to 45%; from 75 to 80 minutes, 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 eluting at 42.8 minutes is the target molecule.

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

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