Transcription factor-based phosphoenolpyruvate biosensor and application thereof

Abstract

The present application relates to a kind of transcription factor-based phosphoenolpyruvate biosensor and its application, belong to the field of biotechnology.The present application utilizes the natural response mechanism of phosphoenolpyruvate and LysR type transcription factor CbbR, and the biosensor of phosphoenolpyruvate response is constructed, its main element includes CbbR protein, CbbR protein specific promoter and fluorescent reporter gene, using fluorescence signal intensity to characterize phosphoenolpyruvate concentration, combined with RBS sequence screening, sigma factor regulation and promoter engineering, the sensitivity and dynamic range of biosensor are significantly improved, so that biosensor shows excellent time resolution, can monitor the dynamic change of intracellular phosphoenolpyruvate level in real time, provide new ideas and means for the construction and optimization of biological dynamic monitoring tool.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a transcription factor-based phosphoenolpyruvate biosensor and its applications. Background Technology

[0002] Biosensors, as analytical devices that integrate biometric elements and physicochemical transducers, can specifically detect target analytes. Due to their high sensitivity, high specificity and rapid response characteristics, they have been widely used in clinical diagnosis, environmental monitoring, food safety and biotechnology.

[0003] In recent years, transcription factor (TF)-based biosensors, utilizing endogenous or engineered transcriptional regulatory mechanisms, have become a research hotspot in synthetic biology and biosensing due to their programmability, controllability, and ease of construction and integration into living cell systems. Compared to traditional biosensors that rely on enzymes, antibodies, or nucleic acids as recognition elements, transcription factor-based design strategies exhibit unique advantages. Their core working mechanism involves the binding of a target analyte ligand to a specific transcription factor, inducing a conformational change or activity alteration in the transcription factor, thereby regulating its binding affinity to a specific DNA promoter sequence. This change in binding state directly affects the transcriptional level of downstream reporter genes, such as those encoding fluorescent proteins, luminescent enzymes, or selectable markers, ultimately generating a quantifiable output signal related to the analyte concentration.

[0004] Phosphoenolpyruvate (PEP), a key pivot molecule in cellular carbon metabolism, not only reflects and regulates metabolic flux distribution and energy state but also serves as a core node connecting glycolysis, the tricarboxylic acid cycle, and various important biosynthetic processes. Therefore, accurate, real-time quantitative monitoring of PEP is of crucial scientific significance and practical value for in-depth analysis of metabolic regulatory networks, guiding rational metabolic engineering, and optimizing industrial bioprocesses. However, current PEP concentration monitoring primarily relies on offline analytical methods, such as high-performance liquid chromatography (HPLC) or mass spectrometry (MS). While accurate, these methods are complex, time-consuming, and cannot achieve dynamic monitoring. Furthermore, traditional chemical inducers such as IPTG require the addition of exogenous chemicals when regulating metabolic pathways, increasing production costs and potentially introducing complexities in separation and purification. Therefore, developing a biosensor capable of real-time monitoring of PEP concentration and responding to its changes is of significant application value for the dynamic regulation of metabolic pathways. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention utilizes the natural response mechanism of phosphoenolpyruvate and LysR-type transcription factor CbbR to construct a phosphoenolpyruvate-responsive biosensor. Its main components include CbbR protein, a CbbR protein-specific promoter, and a fluorescent reporter gene. The concentration of phosphoenolpyruvate is characterized by the intensity of the fluorescence signal.

[0006] The first objective of this invention is to provide a CbbR protein-specific promoter, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0007] A second object of the present invention is to provide a phosphoenolpyruvate responsive element, wherein the phosphoenolpyruvate responsive element expresses a CbbR protein by a first promoter and expresses a fluorescent reporter gene by a second promoter, the second promoter being as described in claim 1.

[0008] Furthermore, the fluorescent protein is expressed under the regulation of an RBS sequence, as shown in SEQ ID NO.4.

[0009] Furthermore, the first promoter is shown in SEQ ID NO.14.

[0010] A second objective of this invention is to provide a polynucleotide encoding the aforementioned phosphoenolpyruvate responsive element.

[0011] In one embodiment of the present invention, the nucleotide sequence of the phosphoenolpyruvate responsive element is shown in SEQ ID NO.12.

[0012] A third objective of the present invention is to provide a phosphoenolpyruvate-responsive biosensor, the biosensor comprising the aforementioned phosphoenolpyruvate-responsive element.

[0013] A fourth objective of this invention is to provide recombinant Escherichia coli containing the aforementioned biosensor.

[0014] Furthermore, the recombinant Escherichia coli uses Escherichia coli Nissle 1917 as the chassis strain.

[0015] Furthermore, the recombinant E. coli knocks out the glucose-specific phosphotransferase encoding gene.

[0016] The fifth objective of this invention is to provide the application of the above-mentioned phosphoenolpyruvate responsive element, the above-mentioned biosensor, and the above-mentioned recombinant Escherichia coli in biological dynamic monitoring.

[0017] The sixth objective of this invention is to provide a method for real-time monitoring of changes in intracellular phosphoenolpyruvate metabolic flux, wherein the above-mentioned biosensor is introduced into the test strain, and after cultivation, the intensity of fluorescent protein is detected, wherein the change in the intensity of the fluorescent protein reflects the change in phosphoenolpyruvate metabolic flux.

[0018] The beneficial effects of this invention are:

[0019] This invention utilizes the natural response mechanism of phosphoenolpyruvate (PEP) and the transcription factor CbbR to construct a PEP-responsive biosensor. This avoids interference from other intracellular metabolites and overcomes the shortcomings of traditional PEP concentration monitoring methods, such as complex operation, time consumption, and inability to reflect real-time dynamic changes in intracellular metabolites. It can non-invasively track instantaneous changes in intracellular PEP concentration in real time. Through optimization of components such as the promoter and RBS sequence in this biosensor, the biosensor obtained by this invention exhibits high sensitivity and excellent linear response, with a dynamic range of approximately 6-fold, and can accurately distinguish changes in PEP concentration at low, medium, and high levels. Validation in engineered strains with different genetic backgrounds confirms that its output signal can accurately and reliably reflect the expected changes in PEP metabolic flux caused by genetic modification, showing promising application prospects in metabolic flux monitoring, strain screening, and metabolic regulation research. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0021] Figure 1 This is an AlphaFold3 modeling diagram of the interaction between CbbR protein and DNA in Example 1 of this invention;

[0022] Figure 2 This is a schematic diagram illustrating the optimization of the second RBS sequence in the PEP biosensor of Embodiment 2 of the present invention;

[0023] Figure 3 This is a performance characterization of the PEP biosensor with the second RBS sequence optimized in Embodiment 2 of the present invention;

[0024] Figure 4 This is a schematic diagram illustrating the optimization of the sigma factor binding region of the PEP biosensor in Embodiment 3 of the present invention;

[0025] Figure 5 This is a characterization of the performance of the PEP biosensor after optimizing the promoter sigma factor binding region in Embodiment 3 of the present invention.

[0026] Figure 6This is a schematic diagram illustrating the optimization of the transcription factor promoter in the PEP biosensor of Embodiment 4 of the present invention;

[0027] Figure 7 This is a characterization of the performance of the PEP biosensor with optimized transcription factor promoters in Example 4 of the present invention.

[0028] Figure 8 This is an evaluation of the performance of the PEP biosensor in different engineered bacterial strains in Example 5 of the present invention. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0030] The materials and testing methods involved in the following embodiments are as follows:

[0031] Glucose, succinic acid, glycerol, sodium chloride, agarose, etc. were purchased from Shanghai Sinopharm Reagent Group; kanamycin and plasmid DNA extraction kits were purchased from Sangon Biotech (Shanghai) Co., Ltd.; yeast extract, peptone, column DNA purification kits, etc. were purchased from Thermo Fisher Scientific, USA; Gibson assembly and cloning kits were purchased from New England Biolabs, UK.

[0032] The bacterial seed culture was performed using LB medium containing: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The final concentration of kanamycin in the medium was 50 μg / mL.

[0033] SOC-1 medium contains: 20 g / L tryptone, 5 g / L yeast extract, 0.5 g / L sodium chloride, 5 g / L magnesium sulfate heptahydrate, and 3.6 g / L glucose.

[0034] SOC-2 medium contains: 20 g / L tryptone, 5 g / L yeast extract, 0.5 g / L sodium chloride, 5 g / L magnesium sulfate heptahydrate, and 3.6 g / L succinic acid.

[0035] Strains and vectors: Plasmid construction was performed in *Escherichia coli* Top10. The constructed plasmids were then transformed into *Escherichia coli* Nissle 1917 for fermentation testing. Growth curves and fluorescence intensity (sfGFP / OD) of each strain were measured using a microplate reader. 600 To evaluate the expression of different PEP biosensors.

[0036] Fluorescence assay: Fluorescence data of promoter characterization are expressed as fluorescence value / OD. 600 Based on this, the excitation wavelength of GFP is 488 nm, the emission wavelength is 523 nm, the gain is 60, and the OD... 600 Detected using an ELISA reader.

[0037] Example 1: CbbR protein structure prediction based on Alphafold3

[0038] Using *Escherichia coli*, a strain with a clear genetic background, mature gene manipulation system, rapid growth rate, and low culture cost, as the chassis strain, this study aims to construct a phosphoenolpyruvate (PEP) biosensor based on the CbbR protein through systematic molecular design and regulatory element optimization strategies. CbbR is a LysR-type transcriptional regulator (LTTR) that primarily participates in regulating the expression of genes related to the carbon dioxide fixation metabolic pathway (Calvin-Bassham-Benson cycle, or CBB cycle) in various microorganisms.

[0039] Ralstonia eutropha H16 is derived from CbbR proteins such as Figure 1 As shown, CbbRs, as a typical type of LTTR, contain two main functional domains: an N-terminal DNA-binding domain, typically with a helix-turn-helix (HTH) motif, responsible for binding to specific promoter regions; and a C-terminal regulatory domain, containing effector molecule binding sites and mediating protein oligomerization. Taking Ralstonia eutropha H16 as an example, CbbRs typically bind as tetramers to the activator and regulator binding sites upstream of the cbb operon, inducing DNA bending and thus regulating the transcriptional activity of both cbb operons.

[0040] Notably, PEP has been identified as a key signaling molecule for CbbR. Studies have shown that elevated intracellular PEP concentrations can significantly inhibit the transcriptional activity of two cbb operons associated with CbbR regulation. This regulatory mechanism is considered to reflect cellular feedback control of carbon metabolism. As a core metabolic intermediate, PEP plays a crucial role in cellular carbon metabolism; therefore, a CbbR-based PEP sensing mechanism has significant potential for biosensor applications.

[0041] Example 2: Optimizing PEP biosensor expression by replacing different RBS

[0042] The structure of the PEP biosensor includes a CbbR protein-coding gene, a first RBS sequence, a first promoter (for expressing transcription factors), a second promoter (a CbbR-specific promoter for expressing fluorescently labeled proteins), a second RBS sequence, and a superfolded green fluorescent protein reporter gene sfGFP.

[0043] To improve the translation initiation efficiency of the reporter gene sfGFP, the second RBS sequence was optimized through systematic screening and evaluation of ribosome binding site (RBS) sequences. Specifically, a series of RBS sequence variants were constructed, and the effects of different RBSs on sfGFP expression levels were evaluated. By comparing the differences in expression intensity among the variants, RBS sequences with higher translation efficiency were screened. An RBS sequence variant library was constructed to evaluate the impact of different RBSs on sfGFP expression levels, thereby optimizing translation efficiency. Three different ribosome binding site (RBS) sequences were screened, and three PEP biosensors were constructed, named PEP-Biosensor-1 (nucleotide sequence as shown in SEQ ID NO.1), PEP-Biosensor-2 (nucleotide sequence as shown in SEQ ID NO.2), and PEP-Biosensor-3 (nucleotide sequence as shown in SEQ ID NO.3). The sensor structures are shown below. Figure 2 As shown.

[0044] Previous studies have shown that the synthesis level of PEP differs significantly among *E. coli* cultured with different carbon sources: *E. coli* producing more PEP when succinic acid is used as the carbon source compared to glucose-based culture conditions. Based on this, *E. coli* strains carrying three biosensors were inoculated into cultures using glucose (SOC-1) or succinic acid (SOC-2) as the primary carbon source for fermentation to compare the effect of PEP levels on the fluorescence intensity of the biosensors under different carbon source conditions. The fluorescence intensity results are shown below. Figure 3 As shown, the fluorescence signal of PEP-Biosensor-3 differed significantly under the two culture conditions: its fluorescence intensity in SOC-1 medium was approximately 1.9 times that in SOC-2 medium, indicating that the construct can effectively respond to changes in intracellular PEP concentration and has good sensitivity and discrimination ability. This provides fundamental support for the subsequent construction of a sensitive and stable PEP biosensor. The second RBS sequence (RBS-2) corresponding to PEP-Biosensor-3 is shown in SEQ ID NO.4.

[0045] Example 3: Optimization of PEP biosensor expression by the σ-factor binding region of the CbbR-specific promoter

[0046] To improve transcription initiation efficiency, the core elements of the CbbR protein-specific promoter were optimized. Specifically, the sequences of the σ factor recognition sites (-35 and -10 regions) that guide RNA polymerase binding were modified to enhance transcriptional activity, and corresponding biosensors PEP-Biosensor-4 (nucleotide sequence as shown in SEQ ID NO.5), PEP-Biosensor-5 (nucleotide sequence as shown in SEQ ID NO.6), and PEP-Biosensor-6 (nucleotide sequence as shown in SEQ ID NO.7) were constructed. The sensor structures are shown below. Figure 4 As shown.

[0047] Fermentation experiments were conducted using the same SOC-1 and SOC-2 culture conditions as in Example 2 to verify its responsiveness to changes in intracellular PEP concentration. Through comparative analysis, a construct with significant fluorescence response, PEP-Biosensor-4, was selected. Its fluorescence intensity results are shown below. Figure 5 As shown, although the highest fluorescence signal intensity of PEP-Biosensor-4 decreased slightly compared to the previously screened PEP-Biosensor-3, its dynamic range was significantly improved, reaching 2.5 times. Furthermore, its baseline fluorescence signal intensity decreased significantly from 3939 to 1923, indicating a lower background signal, which is beneficial for improving the system's sensitivity and signal-to-noise ratio. The CbbR protein-specific promoter nucleotide sequence corresponding to PEP-Biosensor-4 is shown in SEQ ID NO. 8.

[0048] Future optimization efforts will focus on further improving the dynamic response range of the biosensor while maintaining a low baseline fluorescence intensity, in order to achieve more accurate and sensitive monitoring of intracellular PEP concentration.

[0049] Example 4: Optimization of PEP biosensor expression by transcription regulator promoters

[0050] To finely regulate and enhance the transcriptional activation mediated by the LysR-type transcription factor CbbR, a library of natural / artificial promoter variants regulated by the CbbR protein was constructed based on PEP-Biosensor-4 using promoter engineering strategies. Five different promoters were screened as the first promoter in the PEP biosensor. An RBS sequence RBS-1 (consistent with the RBS-1 sequence in Example 2) was added upstream of the CbbR coding gene, resulting in PEP-Biosensor-7 (nucleotide sequence as shown in SEQ ID NO. 9), PEP-Biosensor-8 (nucleotide sequence as shown in SEQ ID NO. 10), PEP-Biosensor-9 (nucleotide sequence as shown in SEQ ID NO. 11), PEP-Biosensor-10 (nucleotide sequence as shown in SEQ ID NO. 12), and PEP-Biosensor-11 (nucleotide sequence as shown in SEQ ID NO. 13). The sensor structures are as follows: Figure 6 As shown.

[0051] Fermentation experiments were conducted using the same SOC-1 and SOC-2 culture conditions as in Example 2 to verify its responsiveness to changes in intracellular PEP concentration. Comparative analysis revealed four molecules exhibiting significant fluorescence response (PEP-Biosensor-7, PEP-Biosensor-8, PEP-Biosensor-10, and PEP-Biosensor-11), with fluorescence intensities as shown below. Figure 7 As shown, among the four constructed PEP biosensors with fluorescence response capabilities, three sensors (PEP-Biosensor-7, PEP-Biosensor-8, and PEP-Biosensor-11) exhibited a reduced dynamic response range, indicating a decrease in their sensitivity to changes in intracellular PEP concentration. In contrast, PEP-Biosensor-10 demonstrated superior fluorescence response performance compared to the other constructs, with its dynamic range further improved by 2.7 times compared to PEP-Biosensor-4, exhibiting better response characteristics. It is currently the best-performing sensor construct in the screening. The first promoter sequence corresponding to PEP-Biosensor-10 is shown in SEQ ID NO. 14.

[0052] Example 5: Performance evaluation of PEP biosensors in engineered strains

[0053] like Figure 8As shown, strain N1 is the original control strain of *Escherichia coli* Nissle 1917. In strain N2, constructed based on N1, the ptsG gene (CP171242.1) encoding a glucose-specific phosphotransferase was knocked out, leading to inactivation of the PTS transport system and thus blocking glucose uptake via the PTS system. Since the PTS system consumes PEP (phosphoenolpyruvate) as a phosphate donor during transport, strain N2 exhibited a higher intracellular PEP accumulation level than N1 after system inactivation. Further, strain N3, constructed by simultaneously knocking out ptsG and in situ integrating the exogenous genes glf-glk (glf gene number WP296508388.1, glk gene number CP097883.1) on top of N1, not only blocked the PTS system but also constructed a novel active glucose transport and phosphorylation pathway. This pathway involves glucose transmembrane transport mediated by a glucose transporter protein encoded by glf, followed by phosphorylation to glucose-6-phosphate by glk-encoded glucokinase. This transport mechanism does not depend on PEP, but it consumes ATP in the phosphorylation step.

[0054] The PEP-responsive fluorescent biosensor PEP-Biosensor-10 was transformed into strains N1, N2, and N3, respectively, and fermentation experiments were conducted in SOC-1 medium. The results showed that, as expected, strain N2 exhibited a stronger fluorescence signal due to a significant increase in intracellular PEP concentration caused by the inactivation of the PTS system, leading to reduced PEP consumption. While strain N3 also blocked the PTS system, the newly introduced pathway may have resulted in an energy metabolic load due to ATP consumption, limiting the downstream PEP synthesis capacity, and its fluorescence intensity was lower than that of strain N2.

[0055] This demonstrates that the sensor possesses excellent sensitivity, good linear response, and a wide dynamic range (approximately 6-fold), effectively distinguishing between low, medium, and high PEP levels. The sensor exhibits excellent temporal resolution, capable of tracking dynamic changes in PEP levels in real time and capturing subtle differences between strains at different growth stages. Its specificity was validated by the expected response to gene manipulations known to affect PEP metabolism. As a non-invasive, real-time dynamic monitoring tool, this sensor shows broad application prospects in metabolic flux monitoring, strain screening, and metabolic regulation research.

[0056] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A CbbR protein-specific promoter, characterized in that: The nucleotide sequence of the CbbR protein-specific promoter is shown in SEQ ID NO.

8.

2. A phosphoenol pyruvate responsive element, characterized in that: The phosphoenolpyruvate responsive element expresses CbbR protein via a first promoter and a fluorescent reporter gene via a second promoter, as described in claim 1.

3. The phosphoenol pyruvate responsive element according to claim 2, characterized in that: The fluorescent protein is expressed under the regulation of an RBS sequence, which is shown in SEQ ID NO.

4.

4. The phosphoenol pyruvate responsive element according to claim 2, characterized in that: The first promoter is shown in SEQ ID NO.

14.

5. A polynucleotide encoding a phosphoenolpyruvate responsive element according to any one of claims 2-4.

6. A phosphoenol pyruvate-responsive biosensor, characterized in that: The biosensor comprises the phosphoenolpyruvate responsive element as described in any one of claims 2-4.

7. Recombinant Escherichia coli comprising the biosensor of claim 7.

8. The recombinant Escherichia coli according to claim 8, characterized in that: The recombinant E. coli gene encoding glucose-specific phosphotransferase was knocked out.

9. The application of the phosphoenol pyruvate responsive element according to any one of claims 2-4, the biosensor according to claim 6, and the recombinant Escherichia coli according to claim 7 or 8 in biological dynamic monitoring.

10. A method for real-time monitoring of changes in intracellular phosphoenolpyruvate metabolic flux, characterized in that: The biosensor described in claim 6 is introduced into the bacterial strain to be tested, and the intensity of the fluorescent protein is detected after cultivation. The change in the intensity of the fluorescent protein reflects the change in the metabolic flux of phosphoenolpyruvate.

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