Engineering an SspB-mediated degron for novel controllable protein degradation |
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| Institution: | 1. CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China;2. Beijing Key Laboratory of Plant Resources Research and Development, Beijing Technology and Business University, Beijing, 100048, China;3. State Key Laboratory of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China;4. University of Chinese Academy of Sciences, Beijing, 100049, China;1. Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, 266101, China;2. Shandong Energy Institute, Qingdao, Shandong, 266101, China;3. Qingdao New Energy Shandong Laboratory, Qingdao, Shandong, 266101, China;4. University of Chinese Academy of Sciences, Beijing, 100049, China;5. State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China;6. Shandong Lukang Pharmaceutical Co. Ltd., No. 88, Deyuan Road, Jining, Shandong, 272021, China;7. Marine Biology and Biotechnology Laboratory, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, 266237, China;1. Institute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China;2. College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China;3. School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China;4. Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology, Nanjing, 210014, China;1. Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, Saint Louis, MO, 63130, USA;2. The Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine in St. Louis, Saint Louis, MO, 63110, USA;3. Department of Pathology and Immunology, Washington University School of Medicine in St. Louis, Saint Louis, MO, 63110, USA;4. Mass Spectrometry Resource, Division of Endocrinology, Metabolism, and Lipid Research, Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA;5. Department of Molecular Microbiology, Washington University School of Medicine in St. Louis, Saint Louis, MO, 63110, USA;6. Department of Biomedical Engineering, Washington University in St. Louis, Saint Louis, MO, 63130, USA;7. Institute of Materials Science & Engineering, Washington University in St. Louis, Saint Louis, MO, 63130, USA;1. Institute of Quantitative and Theoretical Biology, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany;2. Cluster of Excellence on Plant Sciences, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany;3. Department for AI in Society, Science, and Technology, Zuse Institute Berlin, Takustraße 7, 14195, Berlin, Germany;4. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Campus Belval, L-4367, Belvaux, Luxembourg;1. Key Laboratory of Industrial Biocatalysis (Ministry of Education), Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China;2. Tsinghua Innovation Center in Dongguan, Dongguan, 523808, China;3. Center for Synthetic and Systems Biology, Tsinghua University, Beijing, 100084, China;4. School of Physiology, Pharmacology & Neuroscience, Faculty of Life Sciences, University of Bristol, Bristol, BS8 1TD, United Kingdom |
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| Abstract: | Elegant controllable protein degradation tools have great applications in metabolic engineering and synthetic biology designs. SspB-mediated ClpXP proteolysis system is well characterized, and SspB acts as an adaptor tethering ssrA-tagged substrates to the ClpXP protease. This degron was applied in metabolism optimization, but the efficiency was barely satisfactory. Limited high-quality tools are available for controllable protein degradation. By coupling structure-guided modeling and directed evolution, we establish state-of-the-art high-throughput screening strategies for engineering both degradation efficiency and SspB-ssrA binding specificity of this degron. The reliability of our approach is confirmed by functional validation of both SspB and ssrA mutants using fluorescence assays and metabolic engineering of itaconic acid or ferulic acid biosynthesis. Isothermal titration calorimetry analysis and molecular modeling revealed that an appropriate instead of excessively strong interaction between SspB and ssrA benefited degradation efficiency. Mutated SspB-ssrA pairs with 7–22-fold higher binding KD than the wild-type pair led to higher degradation efficiency, revealing the advantage of directed evolution over rational design in degradation efficiency optimization. Furthermore, an artificial SspB-ssrA pair exhibiting low crosstalk of interactions with the wild-type SspB-ssrA pair was also developed. Efforts in this study have demonstrated the plasticity of SspB-ssrA binding pocket for designing high-quality controllable protein degradation tools. The obtained mutated degrons enriched the tool box of metabolic engineering designs. |
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