Metabolic coupling light-controlled mutant protein p4c and application thereof

Abstract

The application provides a kind of metabolism coupling light control mutant protein P4C and its application, belong to synthetic biology and engineered active material technical field.The mutant protein P4C is with the template of photosensitive protein PSP2 containing non-natural amino acid p-benzoyl-L-phenylalanine, is obtained by the synergistic mutation of four sites V16C, E95C, Y151C and L221C.Captures the endogenous reducing agent NADH in cell as electron donor under 365nm ultraviolet irradiation, occurs photoreduction reaction and produces red color development output, its color development efficiency is increased by more than 4 times compared with parent protein, and the process strictly depends on the existence of ultraviolet light and NADH regeneration driven by metabolic substrate, constitutes "and gate" logic.The mutant protein P4C of the application and the "and gate" logic system constructed can be applied to high security information encryption, dynamic biosensing and intelligent anti-counterfeiting label and the like fields.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology and engineered living materials technology, specifically relating to a metabolically coupled light-controlled mutant protein P4C and its applications. Background Technology

[0002] With the deep integration of synthetic biology and materials science, engineered living materials have become a research hotspot and cutting-edge direction in the field of biomaterials. Unlike traditional static synthetic materials, engineered living materials integrate engineered living cells with specific functions (such as Escherichia coli and Bacillus subtilis) in situ into artificial synthetic matrices (such as hydrogels, cellulose, and elastomers), giving the materials not only physical support functions but also the dynamic characteristics of biological systems.

[0003] Currently, this field is at a critical stage of transitioning from basic research to advanced functionalization. In terms of functional integration, researchers have successfully achieved self-growth and self-repair of living materials, endowing them with sensitive sensitivity to heavy metals and specific chemical molecules in the environment. To achieve precise control of these functions, programmable control technology, which uses gene circuits to modify cells, has been widely applied, enabling materials to execute specific biochemical reactions based on external commands such as light, heat, and magnetism. Simultaneously, the integration of advanced manufacturing technologies has further propelled the evolution of this field. By combining high-throughput processing methods such as 3D bioprinting, engineered living materials are transitioning from simple bulk materials to intelligent devices with complex geometric topologies and spatial functional heterogeneity.

[0004] However, despite significant progress in engineered living materials' response to environmental signals, existing technologies still have limitations. Currently, most photocontrolled living material systems utilize photosensitive proteins based on non-natural amino acid modifications, with PSP2 protein being a representative prototype. Under ultraviolet light irradiation, the chromophores within these proteins enter an excited state, capturing electrons from the environment to transform into a free radical state and produce a color change. However, in practical applications, especially in the complex reducing microenvironment of living cells, the excited-state energy of these prototype proteins is easily dissipated through fluorescence decay, resulting in low electron capture efficiency, slow color change response, and insufficient signal strength. At the material integration and logic control level, most systems rely solely on a single external physical signal or chemical inducer to trigger the functional output; that is, the material produces a preset response as long as an external physical stimulus is present. This design cannot distinguish between random physical noise in the environment and specific biochemical inputs, leading to poor specificity in information extraction. Furthermore, in the field of information storage and encryption materials, existing solutions often lack deep coupling with the physiological state of the organism itself, and cannot use endogenous metabolites of cells as logic fuel to construct dual-locking logic gates. As a result, they are unable to provide sufficient security and logical accuracy when facing high-level bio-anti-counterfeiting, dynamic data storage, and autonomous decision-making tasks in complex environments.

[0005] Therefore, there is an urgent need to develop a living material system that can deeply couple with the endogenous metabolic state of cells and has high-dimensional logical operation capabilities. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a metabolically coupled light-controlled mutant protein P4C, its engineered cells, in vivo materials, construction methods and applications, so as to achieve highly sensitive dual-lock logic color development and in vivo information encryption.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a mutant protein P4C, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] The present invention also provides a nucleic acid molecule encoding the mutant protein P4C, the sequence of which is shown in SEQ ID NO: 2.

[0009] The present invention also provides a recombinant expression vector comprising the nucleic acid molecule.

[0010] The present invention also provides an engineered cell that expresses the mutant protein P4C, or contains the nucleic acid molecule, or contains the recombinant expression vector.

[0011] The present invention also provides a living material comprising the engineered cells and a matrix for embedding the engineered cells.

[0012] Preferably, the matrix is ​​a hydrogel; the hydrogel is sodium alginate and / or carrageenan hydrogel.

[0013] The present invention also provides a biological product prepared from the aforementioned living material by 3D printing or extrusion molding.

[0014] This invention also provides a method for constructing a metabolically coupled light-controlled AND-gate logic system, comprising the following steps: introducing the mutant protein P4C into cells to express the mutant protein P4C within the cells; providing the cells with a metabolic substrate capable of driving the regeneration of endogenous NADH; irradiating the cells with 365nm ultraviolet light to cause the mutant protein P4C to capture electrons from endogenous NADH and undergo a photoreduction reaction, producing a colorimetric output.

[0015] Preferably, the metabolic substrate is glucose.

[0016] The present invention also provides a method for encrypting or dynamically displaying information, comprising the following steps: Provide the living material or the biological product; place the living material or biological product in a metabolically silent starvation state, thus concealing the information; when the information needs to be read, simultaneously provide the metabolic substrate and 365nm ultraviolet light irradiation, and the information becomes visible; remove the metabolic substrate or expose to oxygen, and the information becomes concealed again.

[0017] Compared with the prior art, the present invention has the following advantages: (1) This invention uses the photosensitive protein PSP2, which contains the non-natural amino acid p-benzoyl-L-phenylalanine (BpA), as a template. Using a protein rational design method, four key cysteine ​​(C) mutations are introduced around the chromophore microenvironment. The specific mutation sites are: V16C, E95C, Y151C, and L221C. Through the synergistic mutation of these four sites, the electrostatic potential distribution inside the protein is reshaped, resulting in the mutant protein P4C of this invention. Ultraviolet-visible absorption spectroscopy shows that under 365nm ultraviolet light irradiation, its free radical generation rate and equilibrium concentration are far greater than those of the parent protein PSP2. Under 30s illumination, the absorbance of P4C at 500nm (0.311) is more than four times that of PSP2 (0.071), achieving a rapid, high-contrast color change that is visible to the naked eye. Fluorescence spectroscopy and lifetime decay curves confirmed that the four-point co-mutation (V16C, E95C, Y151C, L221C) efficiently captures electron donors by reshaping the electrostatic potential of the chromophore microenvironment and forcing the excited-state energy from the fluorescence radiation path to a non-radiative electron transfer path. In 10 photo-oxidation cycles, the absorbance of P4C remained highly stable, demonstrating excellent reversibility and fatigue resistance.

[0018] (2) This invention further constructs a rigorous AND-gate logic system. Intracellular NADH / NAD + Ratio dynamic monitoring revealed that the photoreduction process of P4C directly consumes endogenous NADH, and its colorimetric output is strictly dependent on substrate-driven NADH regeneration. After depleting intracellular reducing power through starvation treatment, the engineered bacteria remained completely colorless even under UV light; only when glucose (activating glycolysis and TCA cycle to regenerate NADH) and UV light irradiation were simultaneously provided did the system produce a significant red signal. Gray-scale quantitative analysis further confirmed this "double-lock" mechanism, completely eliminating interference from single physical noise or endogenous metabolic fluctuations, ensuring extremely high information security.

[0019] (3) This invention successfully prepared a living gel scaffold with complex geometric topology by co-extruding embedded 3D printing of engineered bacteria expressing P4C and sodium alginate / carrageenan composite bio-ink. Under starvation, the special pattern encoded in the scaffold is completely hidden; when glucose is introduced and ultraviolet light is applied, the hidden pattern is precisely lit up within minutes, realizing reversible information encryption of metabolic "silence-activation". In addition, the growth kinetics of the engineered bacteria were not affected after 5 complete light cycles, which proved the excellent biocompatibility and robustness of the system, laying a solid foundation for the development of a new generation of smart sensors, bio-anti-counterfeiting labels and dynamic data storage devices. Attached Figure Description

[0020] Figure 1The figures show the structure and electrostatic potential distribution of PSP2 and its mutant P4C. In the figure, (a) is the crystal structure of the genetically encoded light-sensitive protein PSP2 (PDBID: 5YR3), and the magnified view shows the chromophores. (b) is the structure of the P4C mutant predicted by AlphaFold2, with the four cysteine ​​mutation sites (V16C, E95C, Y151C and L221C) highlighted in orange. (c) is the electrostatic potential diagram calculated at pH 7.0, ranging from -5.0 to 5.0 kT / e, showing the effect of mutation on the protein's electrical environment. The E95C site shows the most significant electrostatic remodeling, with the induced positive potential signal (blue area) significantly enhanced compared to the wild type.

[0021] Figure 2 The figures show a comparison of the photophysical properties of PSP2 and P4C. In the figures, (a) and (b) show the evolution of the UV-Vis absorption spectra of PSP2 and P4C under 365 nm UV irradiation in 10 mM sodium dithionite solution, respectively. (c) shows the fluorescence emission spectra of 50 μM PSP2 and P4C under the same excitation conditions. (d) shows the fluorescence lifetime decay curves of PSP2 and P4C. (e) shows the fluorescence imaging of E. coli cells expressing PSP2 or P4C under UV irradiation. (f) shows a schematic diagram of the energy redistribution of the excited state of PSP2 and P4C. (g) shows that in PSP2, a large part of the absorbed photon energy is dissipated through radiative decay (fluorescence, FL). (h) shows that in P4C, four cysteine ​​mutations redirect the energy flow to non-radiative electron transfer pathways, thereby promoting the generation of red radical states.

[0022] Figure 3 Figure 1 shows the photoreduction characteristics and reversibility of the NADH-driven logic of P4C. Figure 2 shows the schematic diagram and truth table of the P4C logic gate. Input A is 365nm ultraviolet light and input B is NADH. The output is 1 (red) only when both inputs are simultaneously in the open state (1,1). Figure 3 shows the control group (lacking NADH, ultraviolet light or P4C protein) which remains colorless, while the complete system appears red. Figure 4 shows the ultraviolet-visible absorption spectrum. Characteristic peaks at 500nm and 550nm are observed only in the complete system. Figure 5 shows the photo-switching mechanism and cycle stability test results of P4C, showing that the absorbance at 500nm remains stable during 10 "photo-oxidation" cycles.

[0023] Figure 4Figure 1 shows the photochromic properties of P4C engineered bacteria and their application in in vivo information storage. Figure 2 shows: (a) a schematic diagram of the photoresponse cycle of P4C engineered bacteria, including the initial state, the photoreduction-driven coding state (accompanied by intracellular NADH consumption), and the oxygen-driven reset / erase state; (b) a real-time photograph of the photochromic dynamics of live bacteria, showing significant reddening after 60 seconds of UV irradiation, followed by gradual fading under oxygen exposure for 8 minutes; (c) the quantitative signal intensity analysis curve corresponding to (b); (d) the signal intensity stability test results after 5 complete cycles; and (e) the effect of multiple light cycles on the bacterial growth curve, OD... 600 Monitoring showed no significant toxicity; (f) Intracellular NADH / NAD ratio during repeated photoreduction cycles. + (g) A photograph of 3D-printed biostructures containing P4C engineered bacteria, including cacti, Christmas trees and dinosaur models (scale bar: 5 mm).

[0024] Figure 5 Figure 1 shows the application results of P4C engineered bacteria in in vivo optical information storage and dynamic display. Figure 2 shows the precise encoding of the "2026" pattern on the P4C engineered bacteria array using 365 nm ultraviolet light, showing the different states before encoding, after activation and during information retention. Figure 3 shows the dynamic demonstration of the digital signal "139". After photo-induced activation, the signal gradually fades over time and eventually spontaneously recovers to the initial colorless state.

[0025] Figure 6 The diagram shows the results of the "AND gate" logic for glucose metabolism in live bacteria used for dual-input information display. In the figure, (a) is a schematic diagram of the dual-input "AND gate" logic system. Glucose produces NADH through glycolysis and the tricarboxylic acid cycle as metabolic input A, which, together with ultraviolet light input B, drives P4C photoreduction to produce a red signal output. (b) shows the quantitative grayscale analysis results of bacterial spots under different conditions. Fresh bacteria show significant color development under ultraviolet irradiation, while the starvation treatment group remains colorless. (c) is a photograph showing glucose-dependent "AND gate" behavior. The "HZNU" pattern is lit only when glucose and ultraviolet light are present simultaneously. (d) is a macroscopic encrypted photograph achieved using a 3D printed living scaffold. The upper figure is a schematic diagram of the co-extrusion embedded printing process, and the lower figure is a photograph showing the information of the printed scaffold completely hidden in the starvation state, and the "HZNU" pattern precisely activated and displayed after glucose is provided and combined with ultraviolet irradiation (scale bar: 10mm).

[0026] Figure 7The figures show a comparison of the photochromic properties of the mutant protein and PSP2 in the comparative example. In the figure, (a) PSP2 exhibits obvious photochromic behavior, while P4C1 does not change color under the same conditions; (b) P4C1 does not show the generation of free radicals after light exposure when exposed to the electron donor ascorbic acid; (c) P4C1 does not show the generation of free radicals after light exposure when exposed to the electron donor NADH; and (d) P4C1 does not show the generation of free radicals and does not change color when exposed to light exposure when exposed to the electron donor sodium dithionite. Detailed Implementation

[0027] This invention provides a mutant protein P4C, the amino acid sequence of which is: MSKGEELFTGVVPILCELDGDVNGHKFSVRGEGEGDATIGKLTLKFISTTGKLPVPWPTLVTTLGXGLQCFARYPDHMKQHDFFKSAMPEGYVQCRTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSENVCITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSDQTVLSKDPNEKRDHMVLCEFVTAAGITLGMDELYKLE (SEQ ID NO: 1). The mutant protein P4C provided by this invention uses the photosensitive protein PSP2, which contains the non-natural amino acid p-benzoyl-L-phenylalanine, as a template. Through rational protein design, four key cysteine ​​mutation sites, namely V16C, E95C, Y151C, and L221C, are introduced around the chromophore microenvironment, and obtained after four-point co-mutation. The mutation sites V16C, E95C, Y151C, and L221C respectively represent: valine (V) at position 16 is mutated to cysteine ​​(C), glutamic acid (E) at position 95 is mutated to cysteine ​​(C), tyrosine (Y) at position 151 is mutated to cysteine ​​(C), and leucine (L) at position 221 is mutated to cysteine ​​(C). The amino acid sequence of the photosensitive protein PSP2 is as follows: MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFISTTGKLPVPWPTLVTTLGXGLQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSENVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSDQTVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLE (SEQ ID NO: 3). It should be noted that the amino acid sequence shown in SEQ ID NO: 1 is the core functional sequence of the mutant protein P4C of this invention. Its N-terminus or C-terminus can be further linked with purification tags, including but not limited to 6×His tags, GST tags, MBP tags, and FLAG tags. These tags do not affect the photochromic function and NADH capture function of the mutant protein P4C and can be removed by conventional enzymatic digestion or chemical methods. To obtain the mutant protein, the nucleotide sequence encoding P4C is cloned between the NdeI and XhoI restriction sites of the pET-22b(+) vector, which contains a T7 promoter and a C-terminal 6×His tag. The recombinant plasmid and the orthogonal aminoacyl-tRNA synthetase plasmid pEVOL-pBpF (Addgene, Plasmid #31190) were co-transformed into E. coli BL21(DE3) competent cells. After induction of expression with IPTG, L-arabinose, and BpA, the P4C protein with photochromic function was obtained by Ni-NTA affinity chromatography and gel filtration chromatography. This mutant protein can efficiently capture the endogenous reducing agent NADH under 365nm ultraviolet light irradiation, undergo a photoreduction reaction, and produce a red color output. Its free radical generation rate and equilibrium concentration are significantly better than those of the parent protein PSP2.

[0028] The present invention also provides a nucleic acid molecule encoding the mutant protein P4C, the sequence of which is shown in SEQ ID NO: 2. The nucleic acid molecule was obtained by site-directed mutagenesis of the gene encoding the photosensitive protein PSP2. Specifically, overlap extension PCR was used to introduce nucleotide substitutions corresponding to the four amino acid mutations V16C, E95C, Y151C, and L221C. The complete P4C coding sequence was obtained after sequencing verification.

[0029] This invention also provides a recombinant expression vector containing the aforementioned nucleic acid molecule. As a preferred embodiment, the nucleic acid molecule encoding the P4C mutant protein shown in SEQ ID NO: 2 is cloned into the pET-22b(+) expression vector via NdeI and XhoI restriction endonuclease sites. This vector contains a potent T7 promoter, a lac operon, a ribosome binding site, and an ampicillin resistance marker gene. Furthermore, a sequence encoding a 6×His tag is fused downstream of the multiple cloning site, resulting in a His tag at the C-terminus of the expressed recombinant protein for subsequent affinity purification. The ligation product is transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Single clones are then picked for colony PCR and double enzyme digestion verification. Positive clones are sequenced to confirm the inserted fragment sequence is completely correct, thus obtaining the recombinant expression vector, named pET-22b-P4C. In a specific embodiment of this invention, the construction of the expression vector was completed by Sangon Biotech (Shanghai) Co., Ltd. To achieve site-specific incorporation of the non-natural amino acid benzoyl-L-phenylalanine into host cells, this recombinant expression vector needs to be co-transformed with the orthocyanin-tRNA synthetase plasmid pEVOL-pBpF into the *E. coli* BL21(DE3) expression strain. pEVOL-pBpF provides an orthocyanin-tRNA synthetase and repressor tRNA that specifically recognizes BpA, while pET-22b-P4C provides the P4C coding gene with an amber stop codon. Those skilled in the art can obtain a recombinant expression vector for expressing the P4C mutant protein using the above construction method. Different expression vector backbones (such as pBAD, other pET series vectors) or tag sequences (such as GST, MBP, etc.) can be replaced as needed, as long as the soluble expression of the P4C protein and the correct incorporation of BpA are guaranteed.

[0030] This invention also provides an engineered cell that expresses the mutant protein P4C, or contains the nucleic acid molecule described above, or contains the recombinant expression vector described above. As a preferred embodiment, the recombinant expression vector pET-22b-P4C constructed above and the orthogonalyl-tRNA synthetase plasmid pEVOL-pBpF are co-transformed into *E. coli* BL21(DE3) competent cells. After transformation, the cells are plated on LB agar plates containing 100 μg / mL ampicillin and 27 μg / mL chloramphenicol, and cultured overnight at 37°C. Single colonies are picked to obtain engineered cells that respond to the non-natural amino acid BpA and express the P4C mutant protein. These engineered cells, after IPTG induction in a BpA-containing medium, can efficiently express the P4C protein and utilize the endogenous metabolite NADH as an electron donor, undergoing a photoreduction reaction under 365 nm ultraviolet light to produce a red color output. In addition to Escherichia coli, those skilled in the art can introduce the nucleic acid molecules or recombinant expression vectors described above into other suitable host cells as needed, including but not limited to Bacillus subtilis, yeast cells (such as Pichia pastoris and Saccharomyces cerevisiae), insect cells or mammalian cells (such as HEK293 and CHO cells), as long as a non-natural amino acid incorporation system (such as orthocyanin-tRNA synthetase / tRNA pair) is established in the corresponding host.

[0031] This invention also provides a living material comprising the engineered cells described in any of the preceding claims, and a matrix embedding the engineered cells. As a preferred embodiment, engineered *E. coli* expressing the P4C mutant protein are cultured to a suitable density according to the method described in the examples. After centrifugation to collect the bacterial cells, they are resuspended in sterile PBS buffer, and the bacterial concentration is adjusted to 120 mg / mL (wet weight). Using 4% (w / v) sodium alginate and 2% (w / v) carrageenan as matrix materials, equal volumes of the two are mixed uniformly, and then the above-mentioned concentrated engineered bacterial solution is added. The mixture is stirred thoroughly to form a homogeneous composite bio-ink. During this process, sodium alginate provides ionic cross-linking ability, and carrageenan enhances the thermal reversibility and mechanical strength of the gel; the two work synergistically to form a three-dimensional network structure suitable for 3D printing. The mixed bio-ink is loaded into the barrel of an extrusion 3D printer and printed through a nozzle with an inner diameter of 0.23 mm at a printing speed of 10 mm / s and an extrusion pressure of 50-80 kPa. The printing path is pre-programmed using Rhino 7 software. After printing, immediately spray or soak the product in a 2% (w / v) CaCl2 solution for 1-2 minutes. 2+Ionic crosslinking with guluronic acid fragments in sodium alginate rapidly solidifies the scaffold surface, forming a stable three-dimensional structure. After starvation treatment (i.e., incubation at 37°C for 1 hour in carbon-free Tris buffer), the prepared living material exhibits complete depletion of intracellular NADH, resulting in a colorless and transparent material. When transferred to a solution containing 50 mM glucose and simultaneously irradiated with 365 nm UV light, the engineered bacteria within the material utilize glucose metabolism to regenerate NADH, driving a photoreduction reaction in the P4C protein and producing a red color output, enabling precise information display. Besides sodium alginate / carrageenan composite hydrogels, other biocompatible hydrogel materials capable of encapsulating living cells can also be used to construct the living material described in this invention, including but not limited to gelatin methacryloyl ester, polyethylene glycol diacrylate, hyaluronic acid and its derivatives, fibrinogen, collagen, and thermosensitive hydrogels such as Pluronic F127. Those skilled in the art can select appropriate matrix materials based on specific application scenarios and printing process requirements.

[0032] This invention also provides a biological product prepared from the aforementioned living material via 3D printing or extrusion molding. As a preferred embodiment, a composite bio-ink containing engineered bacteria expressing the P4C mutant protein (e.g., a mixture of 4% sodium alginate and 2% carrageenan, with a bacterial concentration of 120 mg / mL) is loaded into the barrel of an extrusion 3D printer. A co-extrusion embedded printing strategy is employed: a background ink prepared with the same formula using a blank strain not expressing P4C is used as the base material, while the P4C engineered bacterial ink serves as the information carrier. Specific patterns or structures are precisely extruded according to a pre-designed printing path. Using Rhino 7 software programming as an example, models with complex geometric shapes such as cacti, Christmas trees, and dinosaurs can be designed, or text and number patterns such as "HZNU", "2026", and "139" can be designed. Printing parameters are set as follows: nozzle inner diameter 0.23 mm, printing speed 10 mm / s, extrusion pressure 50-80 kPa, and layer height adjusted according to the complexity of the model. Immediately after printing, the scaffold is sprayed or soaked in a 2% CaCl2 solution for 1-2 minutes to induce ionic cross-linking of sodium alginate, thereby solidifying the scaffold surface and maintaining the stability of the three-dimensional structure. Taking the "HZNU" pattern as an example, the P4C engineered bacterial ink is precisely extruded to form the "HZNU" lettering, with a blank bacterial ink background. After the printed in vivo gel scaffold is starved (carbon-free Tris buffer, incubated at 37°C for 1 hour), the intracellular NADH is depleted, and the pattern completely disappears. When the bioproduct is transferred to a solution containing 50 mM glucose and simultaneously irradiated with 365 nm ultraviolet light for 5 minutes, the pattern is precisely activated to red, while the control group that did not receive glucose or sucrose treatment remains colorless.

[0033] This invention also provides a method for constructing a metabolically coupled light-controlled AND-gate logic system. The core of this method lies in introducing the mutant protein P4C into cells for expression and utilizing the endogenous cellular metabolite NADH as an electron donor. This achieves an AND gate logic where colorimetric output is only generated when a physical signal (ultraviolet light) and a biochemical signal (substrate-driven NADH regeneration) are simultaneously present. As a preferred embodiment, firstly, the nucleic acid molecule encoding the P4C mutant protein (e.g., SEQ ID NO: 2) is cloned into a suitable expression vector and co-transformed with the orthocyanin-tRNA synthetase plasmid pEVOL-pBpF into *E. coli* BL21(DE3). Expression is induced by IPTG in a medium containing the non-natural amino acid BpA, allowing the P4C protein to be efficiently expressed and correctly folded within the cells. Subsequently, the engineered cells are provided with a metabolic substrate capable of driving the regeneration of endogenous NADH. In practice, the basal level of intracellular NADH can be depleted first through starvation treatment: P4C-expressing engineered bacteria are collected by centrifugation and resuspended in carbon-free Tris buffer, then incubated at 37°C with shaking for 1 hour. At this point, the intracellular NADH / NAD ratio is... + The ratio drops to an extremely low level, and the system is in a "logic 0" silent state, producing no color output even under ultraviolet light irradiation. Based on this, a metabolic substrate, preferably glucose, is added to the starved cells. For example, the starved bacteria are resuspended in a solution containing 50 mM glucose and incubated at 37°C for 30 min to 2 h, allowing glucose to regenerate NADH through glycolysis and the tricarboxylic acid cycle, restoring the intracellular reducing power level. Finally, the cells are illuminated with 365 nm ultraviolet light (50 mW / cm²). 2 The cells were irradiated for 30 to 60 seconds. Under ultraviolet light excitation, the chromophore of the P4C protein entered the excited state, efficiently capturing electrons from endogenous NADH regenerated from glucose metabolism, undergoing a photoreduction reaction. The chromophore changed from a torsional state to a flat state, forming a highly delocalized π-conjugated system, thus producing a visible red color output. Experimental results showed that the system only appeared red when glucose and ultraviolet light were present simultaneously; when glucose was applied alone (without ultraviolet light), or when ultraviolet light was applied alone (without glucose), or when neither was present, the system remained colorless, conforming to the AND gate truth table. The metabolic substrate in the method of this invention is not limited to glucose; any carbon source that can regenerate NADH through cellular metabolic pathways can be used, such as glycerol and lactose, but glucose is preferred due to its high efficiency and wide applicability.

[0034] The present invention also provides a method for information encryption or dynamic display, the method preferably comprising the following steps: providing the living material or the biological product; placing the living material or biological product in a metabolically silent starvation state to conceal the information; when the information needs to be read, simultaneously providing a metabolic substrate and 365nm ultraviolet light irradiation to reveal the information; removing the metabolic substrate or exposing to oxygen to conceal the information again.

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0037] Example 1: Gene Construction and Expression of P4C Mutant Protein Using the photosensitizing protein PSP2, which contains the non-natural amino acid p-benzoyl-L-phenylalanine (BpA), as a template, four key cysteine ​​mutations were introduced around the chromophore microenvironment using a protein rational design approach. The specific mutation sites were: V16C, E95C, Y151C, and L221C. Figure 1 a and Figure 1 (b) Through coordinated mutation at the above four sites, the electrostatic potential distribution inside the protein is reshaped to obtain the mutant protein P4C ( Figure 1 (c) The target gene sequence was cloned between the NdeI and XhoI restriction sites of the pET-22b(+) vector. This vector contains a potent T7 promoter and a C-terminal 6×His tag to facilitate downstream affinity purification. To achieve site-specific incorporation of the non-classical amino acid BpA, the orthocyanin-tRNA synthetase plasmid (pEVOL-pBpF) was co-transformed with pET-22b-PSP2 or pET-22b-P4C into BL21(DE3) chemocompetent cells. The same host cells transformed with the empty pET-22b plasmid served as a negative control. Detailed protein sequences are shown in Table 1. The C-terminal 6×His tag used in this embodiment was only for purification purposes.

[0038] Table 1 Protein Sequences

[0039] The transformed E. coli BL21(DE3) glycerol bacteria were thawed and streaked onto LB agar plates supplemented with 100 μg / mL ampicillin and 27 μg / mL chloramphenicol. After incubation overnight at 37°C, single colonies were picked and inoculated into 5 mL of LB liquid medium containing the same antibiotics, and incubated at 37°C and 200 rpm for 12 h. The primary culture was then diluted 1:100 to 500 mL of fresh LB medium and cultured until OD (October Expected). 600 The target concentration was 0.8. Before induction, the culture was pre-cooled at 30°C for 30 min. Protein expression was initiated by adding 1 mM IPTG, 0.02% (w / v) L-arabinose, and 1 mM BpA (the latter added under low light conditions). Induction conditions were 30°C, 200 rpm, for 12 h. Cells were collected by centrifugation (5000 rpm, 10 min, 4°C) and stored at -80°C.

[0040] Example 2 Purification of P4C mutant protein The bacterial pellet collected in Example 1 was resuspended in 50 mM phosphate buffer (PB, pH 7.4) containing 100 mM NaCl and 20 mM imidazole. The pellet was sonicated on ice for 15 min and centrifuged at 15,000 rpm for 30 min at 4°C. The supernatant was filtered through a 0.22 μm filter and loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with equilibration buffer (50 mM PB, 100 mM NaCl, 20 mM imidazole, pH 7.4). Non-specifically binding proteins were washed away with washing buffer (50 mM PB, 100 mM NaCl, 50 mM imidazole, pH 7.4), and the target protein P4C was eluted with elution buffer (50 mM PB, 100 mM NaCl, 250 mM imidazole, pH 7.4). The protein was concentrated and desalted using a centrifugal filter with a 10 kDa molecular weight cutoff, and finally purified using a Superdex™ 75 Increase gel filtration chromatography column (AKTA Pure 25 system) to obtain the purified P4C protein. The parent protein PSP2 was expressed and purified using the same method as a control.

[0041] Example 3: Photochromic performance test of P4C protein The purified P4C or PSP2 protein from Example 2 was dissolved in 100 mM Tris-HCl buffer (pH 7.0) to a final concentration of 50 μM. Sodium dithionite, an exogenous reducing agent, was added to a final concentration of 10 mM, mixed thoroughly, and transferred to a 1 cm path length cuvette. The mixture was then incubated under 365 nm UV light (50 mW / cm²). 2 Irradiation was performed, and the ultraviolet-visible absorption spectrum (300-700 nm) was recorded at regular intervals. For example... Figure 2As shown, after only 30 seconds of illumination, the absorbance of P4C at 500 nm reached 0.311, while that of PSP2 under the same conditions was only 0.071. More importantly, when the reaction reached equilibrium after 90 seconds, the steady-state absorbance of P4C was still significantly better than that of PSP2. Figure 2 a and Figure 2 (b) This increase in equilibrium absorbance indicates that the design of P4C not only significantly lowers the energy barrier for the chromophore's transition from the "torsed state" to the "flat state," but also substantially increases the upper limit of the chromophore's transition to the free ground state, thereby maintaining a higher concentration of active product under the same light intensity. To further explore the physical mechanism of P4C's performance improvement, its spectral characteristics were analyzed in this embodiment. Compared with PSP2, P4C exhibits significant fluorescence quenching (…). Figure 2 c) and shortened fluorescence lifetime ( Figure 2 This phenomenon has also been verified in in vivo E. coli imaging (d). Figure 2 (e). This reveals the logic of the redistribution of excited-state energy ( Figure 2 f, Figure 2 Zhongg and Figure 2 (h): In P4C, the energy of the chromophore after absorbing photons is reduced and consumed in the radiative transition (fluorescence generation) path, and is instead more efficiently invested in driving conformational rotation and charge transfer processes.

[0042] Example 4: Logic Gate Testing of P4C and NADH After confirming the superior photochromic properties of P4C, this embodiment further explores its logical behavior in a simulated biological environment. The following control groups were set up: lack of NADH, lack of ultraviolet light, lack of P4C protein, and a complete system (P4C protein + NADH + ultraviolet light). Specifically: purified P4C protein (50 μM) was prepared in 100 mM Tris-HCl buffer (pH 7.0), and 5 mM NADH was added as an electron donor. The system was then exposed to 365 nm ultraviolet light (50 mW / cm²). 2 Observe the color change under stimulation. For example... Figure 3 As shown, the system only outputs a red signal when both ultraviolet light (input A) and NADH (input B) are present simultaneously. Figure 3 a, Figure 3 (b) Spectroscopic analysis and 10 "light-oxidation" cycles demonstrated that this protein logic switch not only exhibits rigorous logical characteristics but also possesses excellent reversibility and cycle tolerance, laying the foundation for subsequent biological logic reconstruction in living cells. Figure 3 c in the middle Figure 3 (d).

[0043] Example 5: Photochromism and Intracellular Metabolic Coupling of Engineered Bacteria Expressing P4C Having confirmed the feasibility of the P4C in vitro logic system, this embodiment extends its functional evaluation to the complex intracellular environment. Given the highly complex internal environment of living cells and the significantly lower concentrations of endogenous electron donors (such as NADH) compared to commonly used chemical reducing agents in the laboratory, it is hypothesized that the parent protein PSP2 may lack an efficient charge transfer pathway, making intracellular photoreduction difficult. To verify this hypothesis, this embodiment constructs a non-destructive photocycling system based on live engineered bacteria (…). Figure 4 (a). The system comprises three key phases: an initial state (phase 1), a coding state driven by 365 nm ultraviolet light and coupled with intracellular NADH consumption (phase 2), and a signal erasure / reset state driven by oxygen (phase 3).

[0044] like Figure 4 As shown, the engineered bacterial culture expressing P4C rapidly changed from colorless to deep red after 60 seconds of UV irradiation. Figure 4 (b); Subsequently, the dark red bacteria were exposed to the air, and the system could automatically reset within 8 minutes. Figure 4 (c) This cyclic process exhibits extremely high robustness: the signal strength remains highly consistent across 5 consecutive cyclic tests. Figure 4 (d), and multiple light treatments had no significant effect on the normal growth kinetics of the bacteria ( Figure 4 (e) demonstrates the good biocompatibility of the P4C logic system. Simultaneously, intracellular NADH and NAD+ levels were quantified using a commercial coenzyme I NAD(H) content assay kit. + Levels. Results showed that during each photoreduction cycle, intracellular NADH / NAD levels... + The ratios all decreased ( Figure 4 (f) confirms that the P4C logic system selectively utilizes intracellular NADH as the main electron source, and its photochromic phenomenon is deeply coupled with the cellular metabolic state.

[0045] Example 6: 3D Printing Living Materials Based on P4C Engineered Bacteria In this embodiment, 4% (w / v) sodium alginate and 2% (w / v) carrageenan were mixed, and engineered bacteria were added to a final concentration of 120 mg / mL to prepare a composite bio-ink. The mixture was loaded into an extrusion system for 3D printing. The cell-loaded composite was extruded through a nozzle (0.23 mm inner diameter) at a printing speed of 10 mm / s and an extrusion pressure of 50-80 kPa. The printing path was programmed using Rhino 7 software. After printing, the scaffold surface was cured using CaCl2 spray to achieve structural shape stability. Bio-constructs with geometric shapes such as cacti, Christmas trees, and dinosaurs were successfully prepared. Figure 4g). These bio-components exhibit vibrant color contrasts and precise patterning capabilities under ultraviolet light excitation. This application demonstrates that P4C can serve not only as a molecular-level logic device but also as a macroscopic-scale functional component, offering possibilities for the development of novel photoresponsive biomaterials.

[0046] Example 7: Optical Information Storage and Dynamic Display Based on P4C Engineered Bacteria Engineered bacteria expressing P4C (prepared according to the method in Example 1) were arranged in an array on the surface of a solid culture medium. The bacterial array was locally excited using 365nm ultraviolet light as a "writing beam" through a mask or focused spot. Figure 5 As shown, in the ultraviolet light irradiation area, the bacteria changed from colorless to red, successfully achieving high-resolution encoding of the letters "2026". At room temperature, the written optical information exhibited good retention. Figure 5 (a)

[0047] To further simulate the working principle of an e-ink screen, the real-time processing of the digital signal "139" was demonstrated. The engineered bacterial array was patterned under 365nm ultraviolet light, and the system quickly activated, displaying a clear red digital signal "139." Subsequently, the light exposure was stopped, and the sample was placed in air. The red signal gradually faded naturally over time, eventually returning to its initial colorless state. This process requires no external physical or chemical intervention. Figure 5 (b) The above results indicate that P4C engineered bacteria can not only serve as static "biological hard drives," but also possess the characteristics of dynamic information carriers.

[0048] Example 8: Double-lock information encryption based on glucose metabolism regulation To achieve higher-level controlled processing of biological information, this embodiment couples the P4C system with the bacterial substrate metabolic pathway, constructing a logic operation system for living bacteria regulated by sugar metabolism. Figure 6 (a) In this model, the reducing equivalent (NADH) of glucose produced through glycolysis and the TCA cycle is defined as the metabolic input signal, which, together with the ultraviolet light physical signal, drives P4C color development.

[0049] To eliminate interference from endogenous metabolic background and confirm logical controllability, this embodiment first established an evaluation system based on metabolic "zeroing". The experiment introduced fresh bacterial strain (P4C), starved bacterial strain (Starved & UV), and blank control bacterial strain (Control) for comparison. The results showed that the Starved & UV group maintained its original color after light exposure, just like the Control group, and its grayscale quantification results ( Figure 6(b) The results were highly consistent with and significantly lower than those of the Fresh group. This demonstrates that starvation treatment can completely deplete the intracellular reducing background, providing a reliable "low-level (Logic 0)" benchmark for constructing rigorous logic systems.

[0050] Based on this, the logic "AND gate" was successfully reconstructed by reintroducing glucose. Figure 6 (c). Experiments showed that the colorimetric output of the "HZNU" pattern is strictly dependent on glucose supply: the system is in a quiescent state when glucose metabolism is absent, and the information is only illuminated under the dual action of "light + glucose". This characteristic has been further applied to the encryption of macroscopic materials ( Figure 6 (d). The living scaffold prepared by co-extrusion embedded printing technology exhibited excellent information concealment in a starved state (NADH-depleted).

[0051] Engineered bacteria expressing P4C were subjected to starvation treatment: bacteria were collected by centrifugation, resuspended in carbon-free Tris buffer, and incubated with shaking at 37°C for 1 hour to consume intracellular NADH. The starved bacteria were then spotted onto agar plates under different conditions: fresh bacteria (unstarved) plus UV light, starved bacteria plus UV light, and starved bacteria plus glucose plus UV light. The results showed that the starved bacteria remained colorless under UV light, while UV light irradiation restored color after glucose supplementation. Figure 6 b, Figure 6 (c) Using co-extrusion embedded printing technology, P4C engineered bacteria were precisely encoded as "HZNU" patterns and embedded in sodium alginate / carrageenan composite bio-ink to prepare a living gel scaffold. After starvation treatment, the information on the printed scaffold was completely hidden; when glucose was provided and combined with ultraviolet light irradiation, the hidden "HZNU" pattern was precisely activated and revealed. Figure 6 (d). The addition of sucrose failed to activate the color development, demonstrating the system's specificity for glucose metabolism. This system achieves a high-security information encryption function through a dual "light + sugar" locking mechanism.

[0052] Comparative Example 1 Using the photosensitive protein PSP2 as a template, four cysteine ​​mutations were introduced at different sites: E17C, E95C, F165C, and L221C. These mutation sites represent the following mutations in the PSP2 protein amino acid sequence (SEQ ID NO:3): glutamic acid (E) at position 17 is mutated to cysteine ​​(C), glutamic acid (E) at position 95 is mutated to cysteine ​​(C), phenylalanine (F) at position 165 is mutated to cysteine ​​(C), and leucine (L) at position 221 is mutated to cysteine ​​(C). Except for the different mutation sites, the P4C1 protein was obtained using the same gene construction, protein expression, and purification methods as in Example 1.

[0053] The photochromic properties of P4C1 were tested according to the methods described in Examples 3 and 4. The color development behavior and free radical generation of P4C1 under 365 nm ultraviolet light irradiation were evaluated under different electron donor conditions (ascorbic acid, NADH, and sodium dithionite).

[0054] like Figure 7 As shown, unlike the parent protein PSP2 which exhibits obvious photochromic behavior, P4C1 did not undergo any color change after ultraviolet light irradiation. Figure 7 (a) Under conditions containing the electron donor ascorbic acid, no free radicals were detected after light irradiation. Figure 7 (b) Under conditions containing the electron donor NADH, no free radicals were detected after illumination. Figure 7 (c) No free radicals were detected after irradiation under conditions containing the electron donor sodium dithionite. Figure 7 (d).

[0055] The above results demonstrate that not every combination of cysteine ​​mutation sites can endow photosensitive proteins with photochromic or electron-trapping functions. Compared to the P4C protein (V16C, E95C, Y151C, L221C) described in this invention, P4C1, although introducing four cysteine ​​mutations, has different mutation locations, resulting in the complete loss of photochromic ability and photoinduced free radical generation pathway. This further proves that the four specific mutation sites selected in this invention and their synergistic effects are unpredictable and cannot be easily obtained by those skilled in the art through conventional optimization.

[0056] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A mutant protein P4C, characterized in that, The amino acid sequence of the mutant protein P4C is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the mutant protein P4C of claim 1, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

3. A recombinant expression vector comprising the nucleic acid molecule of claim 2.

4. An engineered cell, characterized in that, The cell expresses the mutant protein P4C of claim 1, or contains the nucleic acid molecule of claim 2, or contains the recombinant expression vector of claim 3.

5. A living material, characterized in that, It includes the engineered cells as described in claim 4, and the matrix for embedding the engineered cells.

6. The living material according to claim 5, characterized in that, The matrix is ​​a hydrogel; the hydrogel is sodium alginate and / or carrageenan hydrogel.

7. A biological product, characterized in that, The living material described in claim 5 or 6 is prepared by 3D printing or extrusion molding.

8. A method for constructing a metabolically coupled, light-controlled AND-gate logic system, characterized in that, The method includes the following steps: introducing the mutant protein P4C of claim 1 into cells to express the mutant protein P4C in the cells; providing the cells with a metabolic substrate that can drive the regeneration of endogenous NADH; irradiating the cells with 365nm ultraviolet light to cause the mutant protein P4C to capture electrons from endogenous NADH and undergo a photoreduction reaction to produce a colorimetric output.

9. The method according to claim 8, characterized in that, The metabolic substrate is glucose.

10. A method for encrypting or dynamically displaying information, characterized in that, Includes the following steps: Provide the living material as described in claim 5 or 6, or the biological product as described in claim 7; The living material or biological product is placed in a state of metabolic quiescence and starvation, thus concealing its information. When information needs to be read, both metabolic substrates and 365nm ultraviolet light are provided for information to be displayed. Once the metabolic substrate is removed or the body is exposed to oxygen, the information is hidden again.

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