Trimethylamine N-oxide biosensor, genetically engineered bacterium and application of genetically engineered bacterium

Abstract

The invention belongs to the technical field of synthetic biology and biological detection and analysis, and particularly relates to a trimethylamine N-oxide biosensor, a genetically engineered bacterium and application of the genetically engineered bacterium. Specifically, a TMAO response type biosensor based on a TorRST atypical two-component system (TCS) is developed in escherichia coli, and the TMAO response type biosensor is optimized. Meanwhile, a TMAO degradation pathway regulated and controlled by a TMAO biosensor is introduced into escherichia coli EcN, so that an engineering probiotic capable of sensitively detecting, regulating and controlling degradation and balance of TMAO is successfully developed. Therefore, the compound can be used for preventing and treating diseases caused by TMAO, and has good practical application value and prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic biology and biological detection and analysis, and specifically relates to a trimethylamine N-oxide biosensor, genetically engineered bacteria and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Trimethylamine N-oxide (TMAO) is widely present in nature and is a natural endogenous substance of aquatic organisms. The human intestinal microbiota decomposes carnitine and choline-containing foods to produce trimethylamine (TMA), which circulates to the liver and is oxidized by flavin monooxygenase (FMO) to produce TMAO. Many studies have shown that TMAO is associated with the occurrence of many diseases, including endothelial dysfunction, acute heart failure, foam cell formation, coronary artery infarction, inflammatory activation, and vascular aging. Therefore, TMAO is considered a biomarker for many diseases and plays an important role in early diagnosis and treatment. In addition, as an endogenous natural component of aquatic organisms, TMAO is also involved in protein stabilization, osmotic pressure regulation, and buoyancy regulation. TMAO and TMA content can be used to indicate the freshness of aquatic products. Due to its wide range of effects, it is very important to develop a simple, rapid, and accurate method for TMAO detection.

[0004] Currently, methods for detecting TMAO include gas chromatography, high-performance liquid chromatography (HPLC), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS / MS), nuclear magnetic resonance (NMR), mass spectrometry-based methods9, and enzyme-based sensors. These methods are expensive and time-consuming. Biosensors can capture target signals and convert them into specific outputs; for example, biosensors can combine TMAO concentration with fluorescent protein intensity, thereby greatly reducing detection time and cost. Therefore, biosensors are one of the most promising methods for disease and biomarker detection. Clinical studies have used genetically encoded biosensors for disease diagnosis and detection of disease-related biomarkers. By engineering Escherichia coli Nissle 1917 (EcN), a diagnostic method for liver cancer and colorectal cancer was developed that can detect the presence of metastatic tumors by urine luminescence within 24 hours. To date, there are no reports on transcriptional regulation biosensors for TMAO, and research on the detection and metabolic regulation of TMAO is also limited. Therefore, there is an urgent need to develop biosensors that can respond to TMAO. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a trimethylamine N-oxide biosensor, a genetically engineered bacterium and its application. Specifically, the present invention develops a TMAO-responsive biosensor based on the TorRST atypical two-component system (TCS) in Escherichia coli, and optimizes it. At the same time, the present invention also introduces the TMAO degradation pathway regulated by the TMAO biosensor into Escherichia coli EcN, thereby successfully developing an engineered probiotic that can sensitively detect and regulate TMAO degradation and balance. Based on the above research results, the present invention is completed.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] In a first aspect of the present invention, a trimethylamine N-oxide biosensor is provided, wherein the trimethylamine N-oxide biosensor is specifically a TMAO-responsive biosensor based on TorRST TCS, which includes a genetically engineered microorganism, wherein the genetically engineered microorganism at least contains TorRST TCS and a recombinant expression vector, wherein the recombinant expression vector contains a reporter gene and is functionally coupled to the TorRST TCS.

[0008] Wherein, the genetically engineered microorganism can be a genetically engineered bacterium, and the bacterium (starting strain) can be Escherichia coli.

[0009] The TorRST is an atypical two-component system. Unlike typical TCS, TorRST contains three proteins: TorS (NCBI-GeneID: 945595), TorT (NCBI-GeneID: 946289) and TorR (NCBI-GeneID: 946182). TorT and TorS form a complex that acts as a transmembrane histidine kinase to detect extracellular TMAO, and TorR is a regulatory protein. When TMAO is present, the TorST complex itself phosphorylates and transfers the phosphate group to TorR; once phosphorylated, it binds to a specific DNA binding site in the promoter and recruits RNA polymerase to activate gene expression.

[0010] Therefore, the recombinant expression vector further comprises a promoter of a gene regulated by torRST, and the reporter gene is located downstream of the promoter and is regulated by the promoter. In the present invention, the gene regulated by torRST may be torR (NCBI-Gene ID: 946182), tnaC (NCBI-Gene ID: 948223), torC (NCBI-Gene ID: 946252), and therefore, the promoter is any one of torR, tnaC and torC; the reporter gene may be a fluorescent protein gene, and further a green fluorescent protein gene (such as sfGFP);

[0011] Furthermore, in order to express and regulate the promoter and reporter gene, the recombinant expression vector also contains a replicon, and the replicon can be any one of P15A, ColEI and Psc101; wherein, the combination of p15A replicon and PtorC promoter has the most ideal regulatory tightness and responsiveness.

[0012] At the same time, the present invention selects a cascade amplifier circuit to further improve the dynamic range of the biosensor. The cascade amplifier circuit can be LuxRmut-PluxI or HrpRS-PhrpL. In the present invention, the cascade amplifier circuit is located on the above-mentioned recombinant expression vector.

[0013] In the LuxRmut-PluxI circuit, there can be two LuxRmuts, the first LuxRmut is located downstream of the promoter of the gene regulated by torRST (such as PtorC promoter) and is regulated by it. At the same time, the second LuxRmut and the reporter gene are also regulated by the PluxI promoter; when TMAO is present, TorRST activates the expression of the first LuxRmut, and the second LuxRmut activates the expression of the reporter gene and self-activates the second LuxRmut, thereby amplifying the activation of the biosensor.

[0014] In the HrpRS-PhrpL circuit, the HrpR and HrpS are located downstream of the promoter of the gene regulated by torRST (such as the PtorC promoter) and are regulated and expressed therein; at the same time, the reporter gene is regulated by the PhrpL promoter; when TMAO is present, TMAO activates the PtorC promoter, HrpR and HrpS are expressed, and the HrpRS complex strongly activates the PhrpL promoter, resulting in increased expression of the reporter gene, thereby achieving cascade amplification.

[0015] Furthermore, in order to make the biosensor more conducive to practical application, its substrate detection threshold is optimized. The ligand binding affinity of TCS or transcription factors can determine the sensitivity of the biosensor to the ligand response, thereby affecting the detection threshold. However, TorT in Escherichia coli has no structure, and its binding with TMAO requires the synergistic effect of TorS, so it is difficult to change its affinity with TMAO. The ability of TCS to bind to DNA will also change the activation level of regulatory proteins at the same phosphorylation level on the promoter. Therefore, we chose to change the affinity between TorR and DNA to change the TMAO reaction threshold. Specifically, by changing the affinity between TorR and PtorC, we achieved gene expression when the phosphorylation level of TorR was low. The nucleotide sequence of the PtorC promoter is as follows: cagagggttttactcattctgttcatatctgttcatattctgccgtaagccgttcatcctgaccagtgccgctgttcatatttgctcattaagatcgcttca (the four color-marked sequences are O1, O2, O3 and O4, respectively)

[0016] As shown above, the PtorC promoter contains four TorR regulatory regions: O1, O2, O3, and O4. The three different bases in O3 and O1 are key sites that affect the binding of the weak binding region to torR. Therefore, three different nucleotides in O3 were mutated, and the original unique cytosine nucleotides (C...CC) at positions 2, 9, and 10 were changed to T...CC, C...AC, C...CT, T...AC, T...CT, C...AT, and T...AT. These mutations in the O3 sequence reduced the EC of the TMAO biosensor. 50 When the O3 sequence was completely replaced by the O1 sequence, the EC50 dropped to 0.141 mM; compared with the wild-type sensor, the EC50 was reduced by 6.14 times, and the effect was optimal. At this time, the nucleotide sequence of the PtorC promoter was as follows: cagagggttttactcattctgttcatatctgttcatattctgccgtaagctgttcatattgaccagtgccgctgttcatatttgctcat taagatcgcttca (SEQ ID NO.1)

[0017] In a second aspect of the present invention, a genetically engineered bacterium is provided, wherein the genetically engineered bacterium can be the above-mentioned trimethylamine N-oxide biosensor;

[0018] Alternatively, the genetically engineered bacteria contain HrpR and HrpS, and the HrpR and HrpS are located downstream of the promoter of the gene regulated by torRST (such as PtorC promoter) and are expressed under its regulation; thereby achieving cascade amplification.

[0019] The genetically engineered bacteria contains overexpressed yehWXY;

[0020] The genetically engineered bacteria further comprises any one or more of overexpressed TMAO reductase, trimethylamine / dimethylamine dehydrogenase and amine dehydrogenase.

[0021] Wherein, the yehWXY is derived from Escherichia coli, and further, its GeneID: 949028, 946659, 946660; further, the yehWXY is regulated by the PhrpL promoter.

[0022] The TMAO reductase is derived from Escherichia coli and is encoded by five genes, torC / A / D / Z / Y, and the GeneIDs of torC / A / D / Z / Y are: 946252, 946267, 945625, 946389, 946490. Furthermore, the torC / A / D / Z / Y is regulated by the PhrpL promoter.

[0023] The trimethylamine / dimethylamine dehydrogenase is derived from Pseudomonas resinosa, and is encoded by the dmd gene. The dmd is regulated by the PhrpL promoter. Further, the dmd is located downstream of the torC / A / D / Z / Y. The amino acid sequence of the dmd encoded protein is as follows:

[0024] *(SEQ ID NO.2)

[0025] The amine dehydrogenase is derived from Pseudomonas prausnitzii KT2440, and is encoded by peaA / B / C genes. The peaA / B / C is regulated by the PhrpL promoter. Furthermore, the yehWXY is located downstream of peaA / B / C and is also regulated by the PhrpL promoter.

[0026] Wherein, the amino acid sequence of the peaA / B / C encoded protein is as follows:

[0027] peaA

[0028] *(SEQ ID NO.3)

[0029] headB

[0030] MGVLLNLVERNLHEVQVDADRMLFHIPSSSLFTADAVTGGIIDTLRQQGCSAEELMQRLGQQFAGEDIQDTLRELIALELVSDGSPLTPEIALRQVERTALNTVVLNVNTGCNLSCTY CYKEDLDKPSAGKKMSTTTAEASVEMLLKESPNEQRYSVVFFGGEPLSNRPLIEHMVAYCERRFAEAGKQVEFIMTTNATLLTEEIIDWLNAHRFGLSISIDGPKTVHDRNRITVGGQ GTYDVVRRKADLLLSRYTSRPVGARVTLTRGITDVETIWNHLFNEMGFAEVGFAPVTSGDMADFNLTGEELVQVFANMKALGRRYLEAALEHRNIGFSNLHQLITDIHEGHKKALPCG AGLKMLAVDHEGELNLCHRFTGSSLPTFGNVHQGVKQAQLNDFLSQRLDRSNTGCESCRIRNLCSGGCYHESYARYGDPQHPTYHYCELMRDWVDFGIEVYSRIMAVNPAFIDRYITPR

[0031] KAH* (SEQ ID NO. 4)

[0032] peaC

[0033] MKHLKPLNNKARILEQAAAEDRVEEVMAMSAVAGCTATTDPGWEVDAFGGVSSLCQPMEADLYGCSDPCWWPAQVPDMMSTYQDWNAQASNSAEDWRNLGTVFPKDK*(SEQ ID NO.5)

[0034] The starting strain of the genetically engineered bacteria may be Escherichia coli, and further may be Escherichia coli EcN.

[0035] The genetically engineered bacteria of the present invention can realize the detection, transportation and degradation of TMAO, and thus can be used for the prevention and / or treatment of TMAO-mediated diseases.

[0036] Among them, the TMAO-mediated diseases include but are not limited to endothelial dysfunction, acute heart failure, foam cell formation, coronary artery infarction, inflammatory activation and vascular aging.

[0037] The third aspect of the present invention provides the use of the above trimethylamine N-oxide biosensor and / or genetically engineered bacteria in any one or more of the following:

[0038] (a) detecting trimethylamine N-oxide or preparing a product for detecting trimethylamine N-oxide;

[0039] (b) preparing products for diagnosis or auxiliary diagnosis of trimethylamine N-oxide mediated diseases;

[0040] (c) preparing a medicament for treating trimethylamine N-oxide mediated diseases.

[0041] The product may be a detection kit, a detection device or a detection equipment, which is not specifically limited here.

[0042] The TMAO-mediated diseases include, but are not limited to, endothelial dysfunction, acute heart failure, foam cell formation, coronary artery infarction, inflammatory activation, and vascular aging.

[0043] Beneficial technical effects of one or more of the above technical solutions:

[0044] The above technical solution developed a TMAO-responsive biosensor based on TorRST TCS in Escherichia coli. By optimizing the expression intensity of the biosensor and using a cascade amplification circuit, the dynamic range of the biosensor was increased by 6.2 times compared to the original. At the same time, by analyzing the regulatory mechanism, the above technical solution improved the dynamic range of the reaction, and improved the detection sensitivity by optimizing the affinity between TorR and its DNA motif, making the detection threshold more suitable for monitoring needs. In addition, the above technical solution also introduced the TMAO degradation pathway regulated by the TMAO biosensor into EcN, and successfully developed an engineered probiotic that can sensitively detect and regulate TMAO degradation and balance, which can be used for the prevention and treatment of diseases caused by TMAO, and therefore has good practical application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0046] Figure 1 A: Schematic diagram of trimethylamine N-oxide (TMAO) synthesis and TorRST regulation in the embodiments of the present invention; B: Schematic diagram of the natural regulation circuit of TorRST; C: Schematic diagram of constructing a plasmid for replication and regulation promoter screening; D: Green fluorescence dynamic range of different replicons under different regulated promoters; E: Dose-response curve of TMAO with p15A replicon and regulated PtorC promoter.

[0047] Figure 2A: Schematic diagram of LuxRmut-PluxI cascade amplification in the embodiment of the present invention; B: Schematic diagram of HrpRS-PhrpL cascade amplification; C: TMAO dose-response curve after LuxRmut-Plux amplification; D: TMAO dose-response curve after HrpRS-PhrpL amplification; E: Dynamic range regulated by LuxRmut-PluxI and HrpRS-PhrpL: E: Dynamic range regulated by LuxRmut-PluxI and HrpRS-PhrpL.

[0048] Figure 3 A: Schematic diagram of TorR binding to PtorC in a non-phosphorylated state in an embodiment of the present invention; B: Schematic diagram of TorR binding to PtorC in a phosphorylated state; C: Affinity of TorR for O1 / O2 fragments in phosphorylated and non-phosphorylated states; D. Affinity of TorR for O3 / O4 fragments in phosphorylated and non-phosphorylated states; E: Schematic diagram of different bases in O3 and O1 sequences in PtorC; F. Schematic diagram of TorR binding to PtorC in phosphorylated and non-phosphorylated states: E: Schematic diagram of different bases in O3 and O1 sequences in PtorC; F: Dose-response curve after changing the key base of O1; G: Dose-response curve after changing the key base of O3; H: EC50 after changing the key base of O3.

[0049] Figure 4 A: TorRST reaction specificity verification in the embodiment of the present invention; B: TMAO reaction standard curve; C: determination of TMAO in water samples.

[0050] Figure 5 A: Functional verification of the "TMAO detection-transport-degradation" system in the embodiment of the present invention; B: TMAO dose-response curve of strain SERSL; C: Functional verification of the transporter YehWXY; DC: Functional verification of the transporter YehWXY; D: Functional verification of the transporter ProVWX; E: Functional verification of the transporter YeaV.

[0051] Figure 6 A: Schematic diagram of degradation pathway in the embodiment of the present invention; B: Functional verification of SEdegr1; C: Functional verification of SEdegr2; D: Schematic diagram of “TMAO detection-transport-degradation”; E: Functional verification of “TMAO detection-transport-degradation” system. DETAILED DESCRIPTION

[0052] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0053] It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology in the art are usually followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0054] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0055] Example

[0056] method

[0057] Plasmid construction

[0058] Plasmids were constructed in E. coli DH5α and transferred into EcN by electroporation. All strains were cultured in LB medium at 37 °C and 220 rpm. TMAO and TMA were purchased from Shanghai Mailin Biochemical Technology Co., Ltd.

[0059] Statistics

[0060] Data were processed using GraphPad 8.3.0 and Microsoft Excel 2019. Error bars represent the standard deviation of three parallel experiments.

[0061] Plasmid construction

[0062] Phanta Max ultra-fidelity DNA polymerase P505 (Vazyme Biotechnology Co., Ltd.) was used for all PCR reactions. The CYC fragment of the pACYCDuet-1 vector was cloned using CYC-F / R as primers using the pACYCDuet-1 vector as a template. The PtorC fragment was cloned using the E. coli MG1655 genome as a template using PtorC-F / R as primers. The sfGFP fragment was cloned using GFP-F / R as primers, and the p15AC plasmid was constructed using the Gibson assembly method. p15AA, p15AR, pColC, pColA, pColR, pSCC, pSCA, and pSCR were also constructed using the same method. The hrpR, hrpS, luxRmut, PhrpL, and PluxI fragments were synthesized. The CYC-gj fragment was cloned using the pACYCDuet-1 vector as a template using CYC-F1 / R1 as primers. The CYC-gj fragment, PtorC fragment, hrpR fragment, and hrpS fragment were assembled using the Gibson method to construct the p15AhrpRS plasmid. The p15AluxR plasmid was constructed in the same way. Using the pTrc-99a vector as a template and 99a-F1 / R1 as primers, the 99a-gj fragment was cloned and assembled with the PhrpL fragment and sfGFP fragment to construct the p99ahrpL plasmid. The p99aluxI plasmid was constructed using the same method. Using the pET-28a plasmid as a template, the pET fragment was amplified using primers pET28a-F / R. Using the Escherichia coli MG1655 genome as a template, the torR fragment was amplified using TorR-F / R as primers. The pET fragment and torR fragment were assembled using the Gibson method to construct the pET28a-torR plasmid. Using p15AhrpRS as template, TAT fragment was cloned with TAT-F / R primers, and pO3TAT plasmid was constructed by Gibson assembly method. pO3TCC, pO3CAC, pO3CCT, pO3TAC, pO3TCT, pO3CAT and pO1CCC were constructed by the same method.

[0063] Using plasmid pRSFDuet-1 as a template, RSF fragment was cloned using RSF-F / R primers. Using Escherichia coli MG1655 genomic DNA as a template, YehW-F and YehY R were used as primers to clone YehWX Y fragment. The RSF fragment and YehWXY fragment were assembled using the Gibson method to construct the pRSFyehWXY plasmid. Using Escherichia coli MG1655 genomic DNA as a template, ProV-F and ProX-R were used as primers to clone the ProVWX fragment. Using the Gibson method, the RSF fragment and ProVWX were assembled to construct the pRSFproVWX plasmid. Using Escherichia coli MG1655 genomic DNA as a template, yeaV fragment was cloned using yeaV-F / R primers. The RSF fragment and yeaV fragment were assembled using the Gibson method to construct the pRSFyeaV plasmid. Using pCDFDuet-1 plasmid as a template, the CDF fragment was cloned using primers pCDFDuet-F / R. Using Escherichia coli MG1655 genomic DNA as a template, torCAD fragment and torZY fragment were cloned using torC-F / torD-R and torZ-F / torY-R primers, respectively. The dmd fragment was synthesized by Beijing Qingke Biotechnology Co., Ltd. The CDF fragment, torCAD fragment, torZY fragment were assembled using the Gibson method, and the Pdegr1 plasmid was constructed. The CDF fragment, PtorC fragment, torCAD fragment, torZY fragment and dmd fragment were assembled using the Gibson method to construct the phrpLdegr1 plasmid. Using P. putidis KT2440 genomic DNA as a template, the peaABC fragment was cloned using peaA-F and peaC-R as primers. The CDF fragment, peaABC fragment and dmd fragment were assembled using the Gibson method to construct the Pdegr2 plasmid. The CDF fragment, PhrpL fragment, peaABC fragment, YehWXY fragment were assembled using the Gibson method to construct the phrpLdegr2 plasmid.

[0064] Gene knockout

[0065] The pTKRED plasmid was transformed into E. coli DH5α, and a fragment with kanamycin resistance was cloned. Using the PKD4 plasmid as a template, a fragment with kanamycin resistance was cloned. The cloned fragment contained 50 bp of homology arms upstream and downstream of the genomic torRST gene. The homology arms of 50 bp upstream and downstream of the genomic torRST gene. The obtained fragment was electroporated into E. coli DH5α. After recovery culture at 30°C for 1 hour, the cells were cultured for 12 hours. Gene knockout was verified by colony PCR. The resistance marker was subsequently removed with the pcp20 plasmid.

[0066] TorR protein purification

[0067] Based on the pET-28a vector, the pET28a-torR protein purification vector was constructed. The pET28a-torR vector was transformed into Escherichia coli BL21 (DE3) and cultured at 37°C and 220rpm for 12 hours. The culture was then transferred to 1L LB medium and induced with IPTG when the optical density at a wavelength of 600 nm reached 0.6. After induction, the culture was continued at 18°C ​​and 160rpm for 18 hours. The bacterial cells were harvested by centrifugation at 13000rpm, and the cell pellets were resuspended and lysed. The supernatant was then purified with a nickel column to isolate the target protein.

[0068] Fluorescence polarization experiments

[0069] Two reverse complementary single-stranded DNA chains were synthesized according to the O1 / O2 sequence in the PtorC promoter, and a FAM label was added to the 5' end of one of the single-stranded DNA chains. The two single strands were dissolved in annealing buffer (10mM Tris-HCl, 150mMNaCl), mixed in equal volumes (final concentration: 20μM), heated to 95℃ in a water bath, and then naturally cooled to room temperature to obtain DNA samples for detection. The wild-type and mutant O3 / O4 sequences were also obtained in the same way. The affinity of O1 / O2, O3 / O4 and mutant O3 / O4 sequences to phosphorylated and non-phosphorylated TorR was determined by fluorescence anisotropy.

[0070] In the fluorescence anisotropy experiment, the annealed double-stranded DNA was diluted to 2 nM using FP buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.05% Tween 20, 150 μM MnCl2). The diluted DNA fragments were mixed with equal volumes of 200 nM phosphorylated and non-phosphorylated TorR and incubated at 37°C in the dark for 15 minutes. Fluorescence was then measured using a 384-well plate reader 18 at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

[0071] Plasmids used in this experiment

[0072]

[0073]

[0074]

[0075]

[0076] The strains used in this experiment

[0077]

[0078]

[0079]

[0080] Primers used in this experiment

[0081]

[0082] Results and Analysis Construction of a Basic TMAO Biosensor TorRST is an atypical two-component system. Unlike typical TCS, TorRST contains three proteins: TorS (NCBI-GeneID: 945595), TorT (NCBI-GeneID: 946289), and TorR (NCBI-GeneID: 946182). TorT and TorS form a complex that acts as a transmembrane histidine kinase to detect extracellular TMAO, and TorR is a regulatory protein. In the presence of TMAO, the TorST complex itself phosphorylates and transfers a phosphate group to TorR; once phosphorylated, it binds to a specific DNA binding site in the promoter and recruits RNA polymerase to activate gene expression. Based on torRST, we designed and constructed a biosensor that responds to TMAO.

[0083] Natural torRST regulates the expression of multiple genes, and the wild-type promoters of these genes have become candidate promoters for biosensors. We selected the wild-type promoters of three genes, torR (NCBI-Gene ID: 946182), tnaC (NCBI-Gene ID: 948223), and torC (NCBI-Gene ID: 946252), to regulate the expression of green fluorescent protein sfgfp. At the same time, three plasmid replicons of different strengths (P15A, ColEI, Psc101) were selected to express the regulatory promoter and sfgfp. (The information of the three replicons has been reported before). A total of 9 combinations were generated (schematic diagram Figure 1 C, the result is Figure 1 D). When the high-copy ColE1 replicon was used, the basal leakage level was high and visible fluorescence intensity was observed even without the addition of TMAO. In contrast, when the low-copy pSC101 replicon was used, the fluorescence intensity remained low even when the promoter was fully activated. The combination of the p15A replicon and PtorC was selected for future studies because it had the most ideal regulatory tightness and responsiveness. sfGFP was activated when the TMAO concentration reached 1mM, and the fluorescence intensity reached a maximum at 30mM; the dynamic range of the sensor was 4.1-fold, making it ideal for detecting TMAO ( Figure 1 E).

[0084] Enhancing the dynamic range of TMAO biosensor using cascade amplifier circuits

[0085] The dynamic range used is an important parameter in developing biosensors. The more obvious the difference in GFP expression intensity before and after TMAO induction, the better the sensor is for detection.

[0086] To solve this problem, we designed a cascade amplifier circuit to optimize the dynamic range of the TMAO biosensor. We chose two cascade amplifier circuits: LuxRmut-PluxI (Nistala GJ, Wu K, Rao CV, et al. A modular positive feedback-based gene amplifier. Journal of Biological Engineering, 2010, 4 (1): 4.) and HrpRS-PhrpL (Baojun W, Mauricio B, Martin B. Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks. Nucleic Acids Research, 2014 (14): 9484.). In the LuxRmut-PluxI circuit, the PtorC promoter is used to regulate the expression of LuxRmut, which is a mutant variant of LuxR that can activate the PluxI promoter in the absence of an inducer; in this circuit, GFP and LuxRmut are regulated by the PluxI promoter. In the presence of TMAO, TorRST activates the expression of LuxRmut, which in turn activates the expression of GFP and self-activates LuxRmut, thereby amplifying the activation of the biosensor. The use of LuxRmut-PluxI significantly increased the fluorescence intensity after TMAO activation ( Figure 2 A), but the dynamic range is limited, with the maximum dynamic range being only 5.6 times ( Figure 2 C).

[0087] The HrpRS-PhrpL cascade amplification circuit requires the synergistic action of two proteins, HrpR and HrpS. In the amplification loop, the expression of HrpR and HrpS is regulated by the PtorC promoter. When HrpR and HrpS are expressed simultaneously, the activated proteins HrpR and HrpS form an ultra-sensitive high-order co-complex, which binds to the upstream activation sequence of the PhrpL promoter and remodels the closed σ54-RNAP-hrpL transcription complex into an open complex. The expression of GFP is controlled by the PhrpL promoter. When TMAO is present, TMAO activates the PtorC promoter, HrpR and HrpS are expressed, and the HrpRS complex strongly activates the PhrpL promoter, resulting in increased GFP expression, i.e., cascade amplification ( Figure 2 B). HrpRS-PhrpL increased some background compared to WT, but increased maximal activation to a greater extent. This resulted in a wider dynamic range under TMAO activation. Compared to LuxRmut-PluxI, the use of HrpRS-PhrpL resulted in stronger fluorescence activation and lower background leakage, and the dynamic range of the biosensor was increased by 6.2-fold compared to the original ( Figure 2 D, E). Therefore, in subsequent optimization modifications, the HrpRS-PhrpL circuit was used to amplify the TMAO response.

[0088] Threshold optimization of TMAO biosensor

[0089] Previous reports have shown that the TMAO content in the blood of healthy people is less than 30μM, while the TMAO content in patients with cardiovascular disease is between 60 and 150μM. In addition, the TMAO content in most aquatic products is between 20 and 200μM. Therefore, the substrate detection threshold of the developed biosensor needs to adapt to this concentration range. When wild-type TorRST activates PtorC, the EC50 is 1.008mM, which is far from the target detection range. To correct this situation, we analyzed the PtorC promoter sequence. The PtorC promoter contains four TorR regulatory regions: O1, O2, O3 and O4. When TMAO is not present, TorR is non-phosphorylated and can bind to O1 / O2, causing a slight bend in the DNA double helix. When TMAO is present, TorR is phosphorylated and binds to O1 / O2 and O3 / O4, causing complete bending of the DNA, thereby recruiting RNA polymerase and activating gene expression ( Figure 3 A, B). The nucleotide sequences of O1, O2, and O4 are identical (CTGTTCATAT), while O3 has cytosine residues at positions 2, 9, and 10 (CCGTTCATCC), suggesting that these three positions may directly affect TorR binding. It is hypothesized that the O1 / O2 regulatory region binds more strongly to TorR, while O3 / O4 forms a weaker binding.

[0090] To further explore the specific regulatory mechanism of TorR, we verified the effect of changes in the regulatory sequence on its binding affinity. We used nickel ion affinity chromatography to obtain highly pure TorR protein and phosphorylated it in vitro using acetyl phosphate. Fluorescence polarization was used to measure the binding ability of phosphorylated and non-phosphorylated TorR to the strong binding region (O1 / O2) and the weak binding region (O3 / O4). The results showed that non-phosphorylated TorR had a higher affinity with the O1 / O2 fragment, and phosphorylation did not significantly enhance its affinity ( Figure 3 C). In contrast, non-phosphorylated TorR has a low affinity for the O3 / O4 region, while phosphorylation significantly enhances its affinity ( Figure 3 D). This is consistent with our inference that O1O2 is a strong binding sequence and its binding to TorR does not require phosphorylation. In contrast, O3O4 can only bind to TorR in a phosphorylated state. This indicates the influence of the PtorC promoter sequence on TorR affinity and further reveals the activation mechanism of this promoter.

[0091] The experimental results were obtained by fluorescence polarization and sequence conservation analysis. We concluded that the three different bases in O3 and O1 are key sites that affect the binding of the weak binding region to torR. We mutated three different nucleotides in O3, changing the original unique cytosine nucleotides (C...CC) at positions 2, 9, and 10 to T...CC, C...AC, C...CT, T...AC, T...CT, C...AT, and T...AT. These mutations in the O3 sequence reduced the EC50 value of the TMAO biosensor ( Figure 3 E, G). When the O3 sequence was completely replaced by the O1 sequence, the EC50 dropped to 0.141 mM; compared with the wild-type sensor, the EC50 was reduced by 6.14 times ( Figure 3 G, H). This range meets the application requirements and can be used for subsequent sample testing. The results show that changes in the PtorC regulatory sequence greatly affect the detection threshold of TMAO. Replacing the O1 sequence in PtorC with O3 reduces the affinity between TorR and PtorC, resulting in an increase in the TMAO detection threshold ( Figure 3 F).

[0092] Detection of TMAO in aquatic samples using biosensors

[0093] We successfully designed and constructed a TMAO biosensor with excellent dynamic range and substrate detection threshold. To verify its application in actual detection, we used it to detect aquatic biological samples to verify its accuracy. When seafood dies, TMAO in the organism is naturally decomposed into TMA by anaerobic bacteria, resulting in a "rotten" odor. Therefore, the concentration of TMAO is considered an indicator of seafood freshness. We first verified the substrate specificity of the developed TMAO biosensor to avoid interference from TMA; the addition of TMA does not activate the expression of GFP ( Figure 4 A), indicating the specificity of the TMAO biosensor.

[0094] TMAO samples of different concentrations were prepared and added to the culture medium at a volume ratio of 10%. After culturing for 16 hours, the fluorescence intensity was measured, and a TMAO detection standard curve was drawn based on the green fluorescence intensity and concentration ( Figure 4 B). Eight different common seafood species were selected: squid legs, squid shells, yellowtail sea bream, bigeye sea bream, flounder, turbot, shrimp, and sea bass. These species were selected to cover a wide range of seafood types, including crustaceans, molluscs, cephalopods, and bony fish. The filtered and sterilized sample extracts were added to the culture medium at a volume ratio of 10%. The TMAO concentration in the sample was calculated based on the dose-response curve and accurately quantified using a TMAO concentration detection kit. The TMAO concentration detected by the biosensor was consistent with the standard concentration ( Figure 4 C), demonstrating the accuracy and reliability of the biosensor.

[0095] Engineering the probiotic Escherichia coli Nissle 1917 to detect, transport, and degrade TMAO

[0096] Genetically encoded biosensors can be connected to gene circuits to dynamically control metabolic fluxes of biosynthetic pathways or more complex gene networks, and can also be used for cancer therapy and metabolic disease treatment of different host cells. By using these gene circuit systems, engineered cells can be endowed with a variety of customized functions. Since its discovery, EcN has been used to treat various gastrointestinal diseases, including inflammatory bowel disease and irritable bowel syndrome, due to its long-term use, high safety and genetic manipulation. We designed and constructed an EcN probiotic strain that can detect, transport and degrade TMAO. When the strain detects extracellular TMAO, the engineered EcN activates the TMAO transporter to transport TMAO into the cell; it also simultaneously activates the TMAO degradation system to degrade it into CO2( Figure 5A). We first co-expressed HrpRS regulated by PtorC (the key base of O3 was mutated to TAT) and GFP regulated by PhrpL in the plasmid pRSL. The plasmid was then transformed into EcN to form the strain SERSL. Different concentrations of TMAO were added to the culture medium of SERSL, and the green fluorescence intensity was measured after 16 hours of culture. The results showed that the TorRST regulatory system amplified by the HrpRS-PhrpL cascade can still achieve an ideal dose response to TMAO in EcN ( Figure 5 B). At the same time, we measured the fluorescence intensity of single cells activated by TMAO. 0μM, 10μM, 20μM, 30μM, 150μM, 500μM, 1500μM, 2000μM, 3000μM, 4000μM, 5000μM and 6000μM TMAO were added to the SERSL culture medium, and after 16 hours of culture, the single-cell fluorescence intensity at the cellular level was measured using a flow cytometer. The results showed that the fluorescence intensity at the single-cell level showed a similar dose response, and the fluorescence intensity increased significantly with the increase in TMAO concentration.

[0097] The transporter of TMAO in E. coli has not been reported. We observed that when different concentrations of TMAO were added to the culture medium of the EcN strain, the extracellular TMAO concentration decreased after 24 hours. We speculated that EcN naturally has some ability to transport TMAO. Studies have shown that choline transporters can also transport TMAO due to structural similarities31. Based on this, we selected three transporters to test their ability to transport TMAO32-34: ProVWX (responsible for transporting betaine in E. coli) (NCBI-GeneID: 947148, 947145, 947165), YehWXY (presumed to be involved in choline transport) (NCBI-GeneID: 949028, 946659, 946660) and the predicted transporter yeaV (responsible for transporting betaine in E. coli) (NCBI-GeneID: 947326). We overexpressed these three transporters in EcN and added 140 μM TMAO to the culture medium and measured the residual TMAO in the culture medium every 3 hours. Compared with wild-type EcN, overexpression of proVWX and yeaV did not lead to a decrease in extracellular TMAO concentration, indicating that they cannot transport TMAO into the cell ( Figure 5 D, E). In contrast, overexpression of yehWXY led to a rapid decrease in extracellular TMAO; after 12 hours of culture, the extracellular TMAO concentration of the engineered strain (SEyeh) decreased by 50%, indicating that yehWXY can effectively import TMAO ( Figure 5 C).

[0098] Subsequently, the TMAO degradation pathway was designed and constructed. TMAO is converted into TMA under the action of TMAO reductase, and then undergoes a two-step reaction to generate methylamine (MA) under the catalysis of trimethylamine / dimethylamine dehydrogenase; methylamine is then converted into formaldehyde under the action of methylamine dehydrogenase, and formaldehyde is then converted into formic acid and carbon dioxide under the action of formaldehyde dehydrogenase ( Figure 6 A). The first reaction selected the TMAO reductase encoded by five genes (torC / A / D / Z / Y) in Escherichia coli (NCBI-GeneID: 946252, 946267, 945625, 946389, 946490). The second step reaction used the trimethylamine / dimethylamine dehydrogenase encoded by the dmd gene of Pseudomonas resinosa. The third step reaction used the amine dehydrogenase encoded by peaA / B / C of Pseudomonas prausnitzii KT2440. The fourth enzymatic reaction used the dehydrogenase encoded by the original fdhA of Escherichia coli (this gene was not overexpressed). EcN was transformed into the strain SEdegr1 overexpressing torC / A / D / Z / Y. When this strain was cultured in an environment of 150μM TMAO, it had the ability to degrade TMAO. Extracellular TMAO dropped to 48.39μM within 12 hours and to 28.42μM within 24 hours ( Figure 6 B), which shows that overexpression of torC / A / D / Z / Y has a strong ability to degrade TMAO. Next, dmd and peaA / B / C were overexpressed in EcN (SEdegr2) to verify the ability to degrade TMA. When the strain was cultured in 150μM TMA, it had the ability to degrade TMA. The extracellular TMA dropped to 68.75μM ( Figure 6 C).

[0099] Finally, a "TMAO detection-transport-degradation" system was constructed based on the screened transporter and degradation genes. The SEdegrT strain ( Figure 6 D). TMAO (300 μM) was added to the culture medium and samples were taken every 3 hours to measure its concentration. SEdegrT can rapidly degrade TMAO (less than 50 μM) Figure 6 E). This demonstrates the effectiveness of the “TMAO detection-transport-degradation” system and provides a probiotic chassis for the application of microbial therapy to treat TMAO-related diseases.

[0100] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A trimethylamine N-oxide biosensor, characterized in that The trimethylamine N-oxide biosensor is specifically a TMAO-responsive biosensor based on TorRST TCS, which includes a genetically engineered microorganism, wherein the genetically engineered microorganism at least contains TorRST TCS and a recombinant expression vector, wherein the recombinant expression vector contains a reporter gene and is functionally coupled to the TorRST TCS.

2. The trimethylamine N-oxide biosensor according to claim 1, characterized in that The genetically engineered microorganism is a genetically engineered bacterium, and the bacterium is Escherichia coli.

3. The trimethylamine N-oxide biosensor according to claim 1, characterized in that The recombinant expression vector further comprises a promoter of a gene regulated by torRST, and the reporter gene is located downstream of the promoter and is regulated by the promoter; further, the genes regulated by torRST are torR (NCBI-Gene ID: 946182), tnaC (NCBI-Gene ID: 948223) and torC (NCBI-Gene ID: 946252); the reporter gene is a fluorescent protein gene, further a green fluorescent protein gene; Furthermore, the recombinant expression vector also contains a replicon, and the replicon is any one of P15A, ColEI and Psc101.

4. The trimethylamine N-oxide biosensor according to claim 1, characterized in that The trimethylamine N-oxide biosensor further comprises a cascade amplifier circuit; further, the cascade amplifier circuit is LuxRmut-PluxI or HrpRS-PhrpL; the cascade amplifier circuit is located on the recombinant expression vector; In the LuxRmut-PluxI circuit, LuxRmut is located downstream of the promoter of the gene regulated by torRST (including the PtorC promoter) and is regulated by it. At the same time, the LuxRmut and the reporter gene are also regulated by the PluxI promoter. When TMAO is present, TorRST activates the expression of LuxRmut, and LuxRmut in turn activates the expression of the reporter gene and self-activates LuxRmut, thereby amplifying the activation of the biosensor. In the HrpRS-PhrpL circuit, the HrpR and HrpS are located downstream of the promoter (including the PtorC promoter) of the gene regulated by torRST and are regulated and expressed therein; at the same time, the reporter gene is regulated by the PhrpL promoter; When TMAO is present, TMAO activates the PtorC promoter, HrpR and HrpS expression, and the complex of HrpRS strongly activates the PhrpL promoter, leading to increased reporter gene expression, i.e., cascade amplification.

5. The trimethylamine N-oxide biosensor according to claim 3, characterized in that The nucleotide sequence of the PtorC promoter is shown in SEQ ID NO.

1.

6. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria is the trimethylamine N-oxide biosensor according to any one of claims 1 to 5.

7. The genetically engineered bacterium according to claim 6, characterized in that: The genetically engineered bacteria contains overexpressed yehWXY; further, the genetically engineered bacteria also contains any one or more of overexpressed TMAO reductase, trimethylamine / dimethylamine dehydrogenase and amine dehydrogenase.

8. The genetically engineered bacterium according to claim 7, characterized in that: The starting strain of the genetically engineered bacteria is Escherichia coli, further Escherichia coli EcN.

9. Use of the trimethylamine N-oxide biosensor according to any one of claims 1 to 5 and / or the genetically engineered bacteria according to any one of claims 6 to 8 in any one or more of the following: (a) detecting trimethylamine N-oxide or preparing a product for detecting trimethylamine N-oxide; (b) preparing products for diagnosis or auxiliary diagnosis of trimethylamine N-oxide mediated diseases; (c) preparing a medicament for treating trimethylamine N-oxide mediated diseases.

10. The use according to claim 9, characterized in that: The product is a test kit, a test device or a test equipment; The TMAO-mediated diseases include endothelial dysfunction, acute heart failure, foam cell formation, coronary artery infarction, inflammatory activation and vascular aging.

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