IntroductionIn synthetic biology, precise manipulation of cellular activities and functions typically requires the regulation of intracellular protein abundance at the DNA, RNA, and protein levels. Compared with DNA/RNA level regulation, directly regulating the protein level by degradation technology does not require genome modification and provides benefits such as rapid response1, high tunability2,3, and broad applicability4. Additionally, it can also be used to study the functions of lethal or essential genes, eliminate long-lived proteins, and regulate protein levels in specific organelles. In recent years, protein degradation approaches have achieved rapid advancements in eukaryotic systems. For instance, chemically induced strategies such as PROTACs5, molecular glues6, and LYTACs7, as well as degron-based fusion systems like degronLOCKR8 and SD409, have emerged as powerful tools. These methods have significantly advanced both therapeutic development and fundamental research, enabling precise control over disease-related proteins10 and cellular processes11. However, the protein degradation methodologies in bacterial systems remain limited and underdeveloped. This scarcity of versatile tools has hindered the application and advancement of protein-level regulation in bacterial synthetic biology and related fields.Bacterial protein degradation methods can be categorized into two types: degrader addition and degron fusion. BacPROTAC (Bacterial-based PROTAC), currently the only reported degrader in bacteria, is a typical PROTAC-like chemical molecule composed of a phosphorylated arginine analog (pArg) linked to a target protein ligand (biotin) via a chemical linker. In Bacillus subtilis, the pArg is responsible for binding the protease ClpC, while biotin recruits the target protein, enabling the ClpCP protease complex to proximity-induce degradation12. According to this design, recent works successfully achieved specific degradation of multiple target proteins in B. subtilis and Mycobacteria, and highlight the potential of BacPROTAC for treating bacterial infections and antibiotic tolerance12,13,14. However, its high cost and reliance on membrane permeability limit its scalability for microbial manufacturing and industrial applications. In contrast, degron fusion—characterized by simple design, editability, and low cost—has been widely adopted in bacterial systems, particularly in synthetic biology and industrial biotechnology. Representative bacterial degron fusion strategies fall into two categories: (i) Orthogonal degron degradation systems and (ii) Degron masking/exposure systems. The first systems fuse a degron sequence to the terminal of target proteins, requiring co-expression of a cognate heterologous protease15,16,17. For example, GFP fused to the Mesoplasma florum degron tag (mf-ssrA) is stable in E. coli but rapidly degraded upon mf-Lon protease induction, forming an orthogonal degradation system15. For degron masking/exposure systems, a masking sequence (e.g., a TEV protease cleavage site) is fused alongside the degron to shield it during protein folding, ensuring stable target expression. Inducing TEV protease expression removes the masking sequence, exposing the degron and triggering degradation18. This approach has been implemented in genetic circuit construction19, cellular function regulation20, and metabolic flux control21. Despite these advances, degron fusion methods require the pre-fusion of degron sequences to the target protein, which is time-consuming and may disrupt the protein’s normal function. To address these challenges, there is an urgent need to develop degradation strategies that: (i) operate without exogenous degraders; (ii) eliminate the need for pre-fused degrons; (iii) directly recognize and degrade target proteins.In this study, we develop a protein-targeted degradation technology termed the Guided Protein Labeling and Degradation (GPlad) system. The GPlad system comprises three key components: the marking protein (MP), the guide protein (GP), and the protease. Its mechanism operates as follows: first, a de novo-designed guide protein positions the marking protein close to the target protein; next, the marking protein introduces labels to the target protein; finally, the labeled target protein is degraded by a specific protease (Fig. 1a). With this design, directly target protein degradation can be achieved without the need for additional degraders or prior degron fusion. Only a plasmid expressing the GPlad system needs to be constructed. This method has the advantages of simple operation, low cost, and plug-and-play, making it particularly suitable for application in synthetic biology and related fields. As proof of concept, various types of proteins, including fluorescent proteins, metabolic enzymes, and human proteins, are precisely degraded by the GPlad system. Additionally, we develop regulatory tools such as antiGPlad, OptoGPlad, and GPTAC to enable reversible inhibition, optogenetic regulation, and biological chimerization, respectively. Furthermore, these tools are applied to design programmable protein switches, accelerate adaptive laboratory evolution, and rewire metabolic flux in microbial cell factories.Fig. 1: Design and construction of the GPlad system.a Schematic representation of GPlad. First, a guide protein (GP) is fused with a marking protein (MP) via a linker, forming a GP-MP complex. GP binds the target protein and brings MP close to the target protein. The MP labels the target protein, and then the labeled target protein is subsequently recognized and degraded by the corresponding proteases. b Left: Western blot (WB) analysis showing McsBE121A and McsB-mediated arginine phosphorylation of mKate2 guided by 1/1′ heterodimer. Right: WB analysis showing PafAH123A and PafA-mediated ubiquitin-like modification of mKate2 guided by 1/1′ heterodimer. c Flow cytometry analysis of intracellular mKate2 fluorescence intensity at 0, 3, and 6 h after induction of McsB or PafA expression. P-value was determined by a two-tailed unpaired Student’s t-test. ****p