Protein metabolism method based on combination of complementary enzyme digestion

Abstract

The invention relates to the technical field of proteometabolism, and provides a proteometabolism method based on combination of complementary enzyme digestion, which comprises the following steps: firstly, selecting a key rate-limiting enzyme in a target metabolic pathway, modifying a coding sequence of the key rate-limiting enzyme, and inserting a specific protease recognition sequence; then, a pair of functional complementary protease fragments is constructed, expression strategies of the protease fragments in cells are designed respectively, one fragment achieves constant low-level expression, and the other fragment is expressed under the induction of a specific signal; when the two fragments coexist in cells, the two fragments can be combined to form an enzyme complex with cleavage activity, so that a target sequence on a rate-limiting enzyme is recognized and cleaved. And after the signal is removed, the enzyme complex is gradually decomposed, and the expression level of the target rate-limiting enzyme is recovered. In addition, according to the method, metabolic modeling and protein expression cost parameters are combined, and the expression proportion of protease to rate-limiting enzyme is synergistically optimized, so that the system stability and regulation efficiency are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein metabolism, and more particularly to a protein metabolism method based on the combination of complementary enzyme cleavage. Background Art

[0002] With the rapid development of synthetic biology, how to achieve precise regulation of metabolic pathways at the cellular level, especially the dynamic regulation of key enzymes that determine flux, has become a research hotspot in metabolic engineering. In the prior art, a representative control strategy is a switch-type expression system based on RNA regulatory elements. For example, document 1 (Gao X, et al., A programmable proteolysis-based post-translational controller for metabolic flux redirection in E. coli. Nature Communications, 2019) proposed an RNA control network with CRISPRi and transcription terminator as the core, which achieves product-directed metabolic flux guidance by applying inducible activation or inhibition signals to target metabolic genes. The advantages of this type of method are high modularity and fast regulation speed, which are suitable for rapid reconstruction of metabolic pathways. However, it relies on chemical inducers as input signals, and the response threshold is significantly affected by environmental concentrations, making it difficult to form steady-state adaptive regulation in complex dynamic environments. At the same time, this strategy still remains at the regulatory level of gene expression and cannot solve the core problems of "lag", "residual" and "imprecision" at the protein level during the regulation of protease activity.

[0003] In terms of protein level regulation, proteases are gradually being used in the construction of synthetic biology systems because of their ability to achieve specific cleavage of structural domains and thus regulate the function of target enzymes. Document 2 (Pogson M, et al., Directed evolution for improved protein engineering: understanding selection criteria and sequence diversity. Current Opinion in Structural Biology, 2009) systematically sorted out the research results in substrate adaptation, cleavage sequence optimization and protease structure modification, emphasizing that modular control of protein levels can be achieved through the careful design of enzyme recognition sequences. However, this type of research mostly stays on the performance improvement of proteases themselves, and has not yet formed a complete strategic path that can achieve dynamic feedback and reversible regulation at the cellular level. Especially in the activity control mechanism of proteases themselves, there is a general lack of endogenous regulatory methods for their activation and inactivation processes. Most still use the traditional method of exogenous expression and full-process activity, which results in limited performance of the system in terms of regulation accuracy and biological burden.

[0004] In reality, most existing protein-level regulation systems still rely on direct expression of full-length proteases, a model that presents several key challenges in its application. Most existing control strategies rely on direct expression of full-length proteases, which often suffer from background expression leading to residual activity, potentially triggering nonspecific premature cleavage of rate-limiting enzymes and reducing regulatory precision. Furthermore, these regulation processes are often irreversible and lack automatic response mechanisms for flux recovery, making them difficult to adapt to industrial applications requiring dynamic, reversible control. Summary of the Invention

[0005] To overcome the aforementioned shortcomings of the prior art, the present invention provides a protein metabolism method based on a combination of complementary enzymatic cleavages. This method utilizes a two-fragment complementary protease system that, under induction conditions, activates catalytic activity and specifically cleaves the recognition sequence embedded in the rate-limiting enzyme, thereby dynamically regulating metabolic flux. This method addresses the issues of background expression leading to miscleavage and irreversible flux loss in existing protease regulation, offering advantages such as high selectivity, reversibility, and low interference.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A protein metabolism method based on complementary enzyme cleavage, comprising the following steps:

[0008] Step S1, screening the rate-limiting enzyme in the target metabolic pathway, and embedding a protease recognition sequence that can be recognized and specifically cleaved by a two-fragment complementary protease system into the coding sequence of the rate-limiting enzyme through genetic engineering, wherein the recognition sequence is embedded in the non-catalytic region of the rate-limiting enzyme;

[0009] Step S2, constructing a two-fragment complementary protease system, wherein the target protease is divided into a first fragment and a second fragment, wherein the first fragment is constantly expressed at a low level in the cell, and the second fragment is expressed in a controlled manner. The first fragment and the second fragment are both catalytically inactive when present alone, and the two fragments are co-expressed in the cell and combine to form a protease complex with catalytic activity;

[0010] Step S3, introducing an expression regulatory element to control the expression level of the second fragment so that the expression is initiated only under the action of an induction signal and automatically returns to the background level after the induction signal disappears;

[0011] Step S4, when the second fragment is activated by the induction signal and binds to the first fragment, an active protease complex is formed, which recognizes and cleaves the recognition sequence in the rate-limiting enzyme, thereby reducing the activity of the rate-limiting enzyme and thus reducing the carbon flux of the target metabolic pathway;

[0012] In step S5, when the expression control condition is released, the expression level of the second fragment of the protease decreases, the protease activity weakens, and the cells replenish the rate-limiting enzyme that has not been cut by continuous expression, gradually restoring the metabolic pathway flux.

[0013] As a further embodiment of the present invention, screening the rate-limiting enzyme in step S1 includes analyzing the flux control coefficient of each candidate enzyme in the target metabolic pathway, and selecting the rate-limiting enzyme with the largest flux control coefficient as the regulatory target for inserting the protease recognition sequence;

[0014] The non-catalytic region is a predicted flexible loop region or connecting peptide segment in the three-dimensional structure of the rate-limiting enzyme. By identifying the three-dimensional structure of the rate-limiting enzyme, the flexible loop region located on the enzyme surface or the connecting region between the two functional domains is selected as the insertion site of the protease recognition sequence; the effect of the insertion site on the enzyme activity is predicted by molecular simulation, and the substrate binding free energy change value before and after insertion, the spatial displacement root mean square difference of the catalytic residues, and the local structure stability score are calculated based on the protein three-dimensional structure model. Finally, among the candidate regions, the substrate binding free energy change value, the spatial displacement root mean square difference of the catalytic residues, and the local structure stability score are all better than the median of the corresponding indicators, and at least two indicators are located in the top 25% percentile segment of all candidate sites.

[0015] As a further scheme of the present invention, based on the three-dimensional structure, the flexible loop area located on the enzyme surface or the connecting peptide segment between two functional domains is preliminarily screened as potential insertion areas; and the protease recognition short peptide sequence is respectively inserted into the candidate area, and the local structure is subjected to conformational optimization and energy minimization treatment; then the change in the binding free energy of the enzyme and the substrate, the spatial offset root mean square difference of the catalytic center residues and the thermodynamic stability change score of the local area before and after the insertion are respectively calculated, and the above indicators are used as comprehensive evaluation criteria, and the enzyme-substrate binding free energy change, catalytic center RMSD value and local structure stability score are all better than the median of the corresponding indicators, and at least two indicators are located in the top 25% percentile segment of all candidate sites, as the preferred embedding site of the recognition sequence to achieve minimal disturbance to the enzyme activity.

[0016] As a further embodiment of the present invention, the method for constructing a two-fragment complementary protease system in step S2 includes:

[0017] The target protease is divided into N-terminal fragment and C-terminal fragment according to the boundary of its functional domain;

[0018] A pair of complementary binding domains are fused to the N-terminal fragment and the C-terminal fragment, respectively. The complementary binding domain is a GCN4 leucine zipper, whose amino acid sequence is SEQ ID NO: 1: MKQLEDKVEELLSKNYHLENEVARLKKLV; the protease recovers catalytic activity only when the N-terminal fragment and the C-terminal fragment form a parallel α-helical dimer through the leucine zipper;

[0019] Designing gene expression elements that allow for constant low-level expression of the N-terminal fragment in cells, including using weak promoters and weak ribosome binding sites;

[0020] Design of gene expression elements for controlled expression of the C-terminal fragment includes the use of inducible promoters or promoter sequences that are feedback regulated.

[0021] As a further approach to the present invention, the construction of a two-fragment complementary protease system first requires segmenting the target protease into an N-terminal fragment and a C-terminal fragment according to the boundaries of its functional domains, which are determined based on the structural properties of the protease. The specific method includes: analyzing the domain composition of the protease to identify relatively independent functional structural units; determining suitable segmentation sites by referring to the resolved three-dimensional structure data of the protease; and ensuring that the segmented N-terminal and C-terminal fragments are catalytically inactive when present alone, but regain activity after co-expression and complexation in cells.

[0022] As a further embodiment of the present invention, the complementary binding domain utilizes the GCN4 leucine zipper (SEQ ID NO: 1, amino acid sequence MKQLEDKVEELLSKNYHLENEVARLKKLV). This zipper, through the leucine residues at positions a and d, primarily facilitates the "knobs-into-holes" binding of the two α-helices. The design of the complementary binding domain ensures that the two protease fragments can efficiently recognize, bind, and form a catalytically active complex within the cellular environment, thereby achieving specific cleavage of the rate-limiting enzyme.

[0023] As a further embodiment of the present invention, the expression control elements introduced in step S3 include one or more of the following:

[0024] Chemically inducible promoter elements, including the IPTG-responsive lac promoter;

[0025] Temperature-responsive elements, including the cI857 temperature-sensitive regulatory sequence;

[0026] Metabolite-responsive elements, including transcription factors and corresponding operator sequences that detect the concentration of target metabolites;

[0027] Light-induced expression elements, including a coupling system of light-sensitive proteins and promoters.

[0028] As a further embodiment of the present invention, the protease in step S4 is TEV protease, which recognizes and cleaves the sequence ENLYFQ / S or a homologous peptide thereof, wherein the cleavage occurs between Q and S.

[0029] As a further embodiment of the present invention, the mechanism by which the rate-limiting enzyme is inactivated after the protease cleaves it in step S4 includes one or more of the following:

[0030] Cleavage causes the catalytic domain of the rate-limiting enzyme to separate from the substrate binding domain, causing it to lose its catalytic function;

[0031] After cleavage, the unstable N-terminal amino acid is exposed, triggering the N-end rule-mediated intracellular degradation pathway;

[0032] Cleavage causes the three-dimensional conformation of the rate-limiting enzyme to disintegrate, leading to its aggregation or degradation.

[0033] As a further embodiment of the present invention, the method further comprises the step of determining the optimal expression ratio of the protease to the rate-limiting enzyme, which step utilizes an enzyme cost minimization optimization algorithm, specifically comprising:

[0034] Step A1, using a metabolic model to calculate the minimum catalytic amount of rate-limiting enzymes and proteases required to support metabolic stability under given target product flux conditions;

[0035] Step A2, evaluating the unit expression cost of proteases and rate-limiting enzymes, including the number of required amino acids, translation energy, and ribosome occupancy time;

[0036] Step A3, predicting the rate-limiting enzyme concentration range required for different product output levels based on the metabolic flux modeling results, and establishing an expression ratio optimization model based on the unit expression cost;

[0037] Step A4: Based on the above calculation results, matching gene expression regulatory elements are selected, including promoters and ribosome binding sites, to achieve an optimal expression ratio of the protease and the rate-limiting enzyme.

[0038] Compared with the prior art, the protein metabolism method based on complementary enzyme cleavage of the present invention has the following beneficial effects:

[0039] This invention utilizes a two-fragment complementary protease system. By splitting the target protease into two functional fragments, the fragments recombine to form a catalytically active protease complex only under induction conditions, thereby achieving specific recognition and cleavage of the rate-limiting enzyme. This overcomes the background activity issue common in traditional full-length protease expression models, improves regulatory accuracy, and reduces the risk of inadvertent cleavage of non-target proteins.

[0040] This method utilizes a combined mechanism of induced and basal expression to gradually attenuate protease activity after exogenous signals are removed. Pathway activity is then gradually restored through the continued synthesis of the uncleaved rate-limiting enzyme within the cell, achieving reversible regulation of metabolic states. Compared to existing control strategies that are irreversible or rely on complex feedback loops, this method offers a more flexible response, a clearer structure, and more stable regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a fluorescence image of the cell status of different strains during the xylonic acid production process in Reference 1.

[0042] Figure 2 This is a single-cell micrograph of the dynamic changes in cell fluorescence under the pbO system in Reference 1.

[0043] Figure 3 Schematic diagram of the leucine zipper from the gcn4 protein.

[0044] Figure 4 The figure is a schematic flow chart of a protein metabolism method based on the combination of complementary enzyme cleavage according to the present invention.

[0045] Figure 5 This is a logical framework diagram of the double-fragment complementary protease system of the present invention.

[0046] Figure 6 This is a recovery curve diagram of TrpE activity and tryptophan production according to the present invention as a function of induction release time.

[0047] Figure 7 This is a diagram showing the effect of expression ratio regulation on TrpE activity and cell growth according to the present invention.

[0048] In the figure, Brightfield: bright field; pHluorin: pHluorin fluorescent protein; PI: propidium iodide; Merge: merged image; XP: XP strain of experimental group; XO: XO strain of experimental group; XN: XN strain of control group. DETAILED DESCRIPTION

[0049] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] Example 1

[0051] This example describes a protein metabolism method based on complementary enzyme cleavage combined with the implementation of the regulation of the L-tryptophan biosynthesis pathway in Escherichia coli MG1655, comprising the following steps:

[0052] In this example, we first performed a flux control coefficient (FCC) analysis on key enzymes in the Escherichia coli tryptophan biosynthesis pathway. Using the COBRApy tool, we constructed a metabolic pathway model. By simulating perturbations to each enzyme, we calculated the extent to which these perturbations affected the overall pathway flux. The results showed that TrpE (the catalytic subunit of anthranilate synthase) had the highest control coefficient on the pathway flux, and therefore was selected as the target rate-limiting enzyme.

[0053] The three-dimensional structure of the TrpE protein was then obtained and analyzed using the Rosetta software package. Based on this structural analysis, peptides located in the flexible loop region and the connecting domain on the TrpE surface were initially selected as candidate insertion sites. These regions included the N-terminal region (amino acids 25-35), the linker between the catalytic and regulatory domains (amino acids 188-203), and the C-terminal flexible loop region. For each candidate region, the TEV protease recognition sequence ENLYFQ / S (where / represents the cleavage site) was embedded to construct a virtual mutant model.

[0054] FoldX molecular simulation software was used to calculate the evaluation indicators of each insertion region, including the change in substrate binding free energy, the root mean square difference of the spatial displacement of the catalytic residues, and the local structural stability score. Comparative analysis showed that the linker region between amino acids 190-196 performed best, with the smallest change in substrate binding free energy and the root mean square difference of the spatial displacement of the catalytic residues being less than 1. The local structural stability score was in the top 15% of all candidate sites. In particular, after the TEV recognition sequence was inserted into position R193, the overall protein structure remained stable with minimal effect on enzyme activity.

[0055] In this example, a trpE gene containing an embedded TEV recognition sequence (designated trpE-TEV) was designed and synthesized. The TrpE protein encoded by this gene contains an ENLYFQ / S sequence at position R193. This gene was cloned into the low-copy plasmid pSC101 and its expression was controlled by the moderately strong promoter J23108.

[0056] The present invention uses tobacco etch virus (TEV) protease as a cleavage tool. Based on the boundaries of its functional domains, the TEV protease is split into two fragments: an N-terminal fragment (amino acids 1-118) and a C-terminal fragment (amino acids 119-242). This segmentation ensures that the two fragments are catalytically inactive in isolation and only form an active complex when combined within the cell.

[0057] To promote efficient binding of the two fragments, a pair of complementary binding domains—GCN4 leucine zippers (sequence: MKQLEDKVEELLSKNYHLENEVARLKKLV)—were fused to the C-terminus of the N-terminal fragment and the N-terminus of the C-terminal fragment, respectively. These leucine zippers form stable parallel α-helical dimers in cells through a "knobs-into-holes" mechanism, thereby drawing the two TEV fragments together and promoting their recombination into an active form.

[0058] When constructing the expression vector, the N-terminal fragment gene is integrated into the host strain's chromosome, achieving constant low-level expression under the control of the weak promoter lacUV5. The C-terminal fragment gene is cloned into the pBAD plasmid and placed under the control of the araBAD promoter, enabling L-arabinose-inducible expression. This design ensures that the C-terminal fragment is barely expressed in the absence of induction, and even if a small amount of the N-terminal fragment is present, no active protease is formed, thus avoiding background cleavage activity.

[0059] In the present invention, an arabinose (L-arabinose) induction system is used to control the expression of the C-terminal fragment of TEV protease. On the pBAD plasmid, the expression of the C-terminal fragment is regulated by the araBAD promoter, which is almost inactive in the absence of arabinose but is rapidly activated upon addition of arabinose.

[0060] In the present examples, a ribosome binding site (RBS B0032) was used to ensure sufficient C-terminal fragment production under induction conditions while avoiding cellular burden caused by overexpression. Furthermore, to prevent metabolic degradation of arabinose, which could affect the induction effect, a host strain deficient in arabinose metabolism (ΔaraBA) was selected to ensure stable intracellular arabinose concentrations and achieve a sustained and controllable induction effect.

[0061] When the induction signal exists, the C-terminal fragment is expressed in large quantities and combines with the N-terminal fragment to form an active protease; when the induction signal disappears, the expression of the C-terminal fragment decreases rapidly, and the protease activity weakens accordingly, thus achieving the control of protease activity.

[0062] When expression is induced by the addition of 0.2% L-arabinose, the C-terminal fragment of TEV protease begins to accumulate in the cells and combines with the constant-expressed N-terminal fragment to form an active protease complex. This complex specifically recognizes and cleaves the ENLYFQ / S sequence embedded in the TrpE-TEV protein, resulting in cleavage of the TrpE protein at site R193.

[0063] The resulting TrpE fragments no longer maintain their correct three-dimensional structure, leading to a significant decrease in enzyme activity. Furthermore, cleavage may expose unstable N-terminal amino acids, triggering N-end rule-mediated protein degradation, further reducing the amount of active TrpE. Western blot and enzyme activity analysis confirmed that after 6 hours of induction, TrpE activity was reduced by approximately 75%, resulting in a corresponding decrease in L-tryptophan biosynthesis flux.

[0064] This decrease in activity is directly reflected in product formation. HPLC analysis showed that the L-tryptophan concentration in the culture medium was reduced by about 60% compared with the uninduced control group, demonstrating that the embodiments of the present invention can effectively regulate metabolic pathway flux.

[0065] When the induction signal is removed, the expression level of the C-terminal fragment of TEV protease in the cell begins to decline. Due to the natural degradation of the protein and the dilution effect of cell division, the amount of active protease complex gradually decreases, and the cleavage pressure of the rate-limiting enzyme TrpE-TEV decreases accordingly.

[0066] At the same time, the host cells continued to express the TrpE-TEV protein at low levels via the low-copy plasmid pSC101, and newly synthesized, intact TrpE-TEV protein began to accumulate within the cells. As the amount of uncleaved TrpE-TEV increased, the activity of the tryptophan biosynthesis pathway gradually recovered. Regular sampling and analysis revealed that TrpE activity had recovered to approximately 70% of its original level 24 hours after the removal of arabinose. After 48 hours, activity had recovered to over 90%, and tryptophan production also rebounded.

[0067] This reversible regulatory property provides flexibility for industrial applications, allowing operators to dynamically regulate metabolic flux by simply adding or removing inducers as needed without the need for genetic modification or other complex interventions.

[0068] Example 2

[0069] The embodiment of the present invention describes a specific calculation process for optimizing the expression ratio of each component in a complementary enzyme-cleaved protein metabolism regulation system using an enzyme cost minimization algorithm.

[0070] First, a kinetic model was constructed encompassing TrpE, TEV protease, and related metabolic reactions. This model included the rate-limiting enzyme containing the TEV recognition site (TrpE-TEV), the N-terminal fragment of TEV protease fused to a leucine zipper (TEV-N-Zip), the C-terminal fragment of TEV protease fused to a leucine zipper (Zip-TEV-C), the complete TEV protease complex assembled from the N- and C-terminal fragments, and the cleaved, inactivated TrpE fragment.

[0071] Minimize the total protein expression cost while meeting the target flux. The expression is:

[0072] minE total =h TrpE ·[TrpE-TEV]+h N ·[TEV-N-Zip]+h C ·[Zip-

[0073] TEV-C];

[0074] Where h TrpE 、h N and h C Represents the unit expression cost of TrpE-TEV, TEV-N-Zip and Zip-TEV-C, respectively, including:

[0075] Energy cost:

[0076] E energy =4·L protein ;

[0077] Where, L protein is the number of amino acids;

[0078] Material cost, which is the weighted sum of each amino acid:

[0079] E material =∑(w aa ·n aa );

[0080] Ribosome occupancy cost:

[0081]

[0082] Based on protein sequence analysis, the cost coefficients of TrpE-TEV, TEV-N-Zip, and Zip-TEV-C were calculated as follows: h TrpE =1.45,h N =0.55, h C =0.65.

[0083] Convert the above model to a convex optimization problem. The following is a Python code example. Please note that this example is only a starting point and may need to be adjusted according to the actual situation and device interface in actual application.

[0084]

[0085]

[0086]

[0087] This code is only an example and needs to be modified and adjusted appropriately according to specific circumstances in actual applications.

[0088] The optimal protein expression ratio was obtained, including the optimal TrpE-TEV concentration: 2.85 μM; the optimal TEV-N-Zip concentration: 0.14 μM (close to the set minimum value); and the optimal Zip-TEV-C concentration: 0.58 μM.

[0089] Calculations showed that the optimal expression molar ratio of the TEV protease C-terminal fragment to TrpE-TEV was approximately 1:5.

[0090] To verify the actual effect of the optimal ratio, three groups of strains with different expression ratios were constructed as shown in Table 1:

[0091] Table 1

[0092] Group A: TEV-C Group B: TEV-C Group C: TEV-C 1:2 1:5 1:10

[0093] like Figure 7 As shown, TrpE activity in group B was inhibited by 75% after 6 hours of induction, and cell growth was only reduced by 17%; although the TrpE inhibition rate in group A was as high as 85%, cell growth was reduced by more than 30%; TrpE inhibition in group C was only 45%, and the regulatory effect was insufficient.

[0094] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of this application. Therefore, the scope of protection of this application should be based on the scope of protection of the claims.

[0095] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are within the scope of protection of the present invention.

Claims

1. A protein metabolism method based on complementary enzyme cleavage, characterized in that: The following steps are involved: Step S1, screening the rate-limiting enzyme in the target metabolic pathway, and embedding a protease recognition sequence that can be recognized and specifically cleaved by a two-fragment complementary protease system into the coding sequence of the rate-limiting enzyme through genetic engineering, wherein the recognition sequence is embedded in the non-catalytic region of the rate-limiting enzyme; Step S2, constructing a two-fragment complementary protease system, splitting the target protease into two functionally complementary fragments, wherein the first fragment is constantly expressed at a low level in the cell, and the second fragment is expressed in a controlled manner; Step S3, introducing an expression regulatory element to control the expression level of the second fragment so that it is only expressed under the action of an induction signal; Step S4, when the second fragment is activated for expression by the induction signal and combines with the first fragment, an active protease complex is formed, which recognizes and cleaves the recognition sequence in the rate-limiting enzyme; In step S5, the expression regulation conditions are released, the expression level of the second fragment of the protease decreases, the protease activity is weakened, and the cells replenish the rate-limiting enzyme that is not cut by continuous expression to restore the metabolic pathway flux.

2. A protein metabolism method based on complementary enzyme cleavage according to claim 1, characterized in that: Screening the rate-limiting enzyme in step S1 includes analyzing the flux control coefficient of each candidate enzyme in the target metabolic pathway, and selecting the rate-limiting enzyme with the largest flux control coefficient as the regulatory target for inserting the protease recognition sequence; The non-catalytic region is a flexible loop region or connecting peptide segment predicted in the three-dimensional structure of the rate-limiting enzyme. By identifying the three-dimensional structure of the rate-limiting enzyme, the flexible loop region located on the enzyme surface or the connecting region between the two functional domains is selected as the insertion site of the protease recognition sequence; the effect of the insertion site on the enzyme activity is predicted by molecular simulation, and the change value of the substrate binding free energy before and after insertion, the root mean square difference of the spatial displacement of the catalytic residues, and the local structure stability score are calculated based on the three-dimensional structure model of the protein. Finally, among the candidate regions, the change value of the substrate binding free energy, the root mean square difference of the spatial displacement of the catalytic residues, and the local structure stability score are all better than the median of the corresponding indicators, and at least two indicators are located in the top 25% percentile segment of all candidate sites.

3. The protein metabolism method based on complementary enzyme cleavage according to claim 1, characterized in that: The method for constructing a two-fragment complementary protease system in step S2 includes: The target protease is divided into N-terminal fragment and C-terminal fragment according to the boundary of its functional domain; A pair of complementary binding domains are fused to the N-terminal fragment and the C-terminal fragment, respectively. The complementary binding domain is a GCN4 leucine zipper, whose amino acid sequence is SEQ ID NO: 1: MKQLEDKVEELLSKNYHLENEVARLKKLV; the protease recovers catalytic activity only when the N-terminal fragment and the C-terminal fragment form a parallel α-helical dimer through the leucine zipper; Designing gene expression elements that allow for constant low-level expression of the N-terminal fragment in cells, including the use of weak promoters and ribosome binding sites; Design of gene expression elements for controlled expression of the C-terminal fragment includes the use of inducible promoters or promoter sequences that are feedback regulated.

4. The protein metabolism method based on complementary enzyme cleavage according to claim 1, characterized in that: The expression control element introduced in step S3 is selected from one or more of the following: a chemically inducible promoter element, including an IPTG-responsive lac promoter; a temperature-responsive element, including a cI 857 temperature-sensitive regulatory sequence; Metabolite-responsive elements include transcription factors and corresponding operator sequences that detect the concentration of target metabolites; light-induced expression elements include a coupling system of light-sensitive proteins and promoters.

5. The protein metabolism method based on complementary enzyme cleavage according to claim 1, characterized in that: The protease described in step S4 is TEV protease, which recognizes and cleaves the sequence ENLYFQS or a homologous peptide thereof, wherein the cleavage occurs between Q and S.

6. The protein metabolism method based on complementary enzyme cleavage according to claim 1, characterized in that: The enzyme cost minimization optimization algorithm is used to determine the expression ratio of protease and rate-limiting enzyme, including the following steps: Step A1, using a metabolic model to calculate the minimum catalytic amount of rate-limiting enzymes and proteases required to support metabolic stability under given target product flux conditions; Step A2, evaluating the unit expression cost of proteases and rate-limiting enzymes, including the number of required amino acids, translation energy, and ribosome occupancy time; Step A3, predicting the rate-limiting enzyme concentration range required for different product output levels based on the metabolic flux modeling results, and establishing an expression ratio optimization model based on the unit expression cost; Step A4: Based on the above calculation results, matching gene expression regulatory elements are selected, including promoters and ribosome binding sites, to achieve an optimal expression ratio of the protease and the rate-limiting enzyme.

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