將目標蛋白質表達在膜上不僅可以防止受質受到外膜的阻礙，還有助於評估酵素的可重複使用性，這對於酵素的回收至關重要。在這項研究中，我們利用不同的跨膜系統 (包括IgA、Ag43和eCPX) 優化大腸桿菌 (E. coli) 膜上目標蛋白質的表面展示。比較分析顯示，Ag43是最佳的表達系統，具有優越的蛋白質表達能力，並且在蛋白質表達在膜外時仍能保持酵素活性。為了驗證這一概念，我們以紅色螢光蛋白 (RFP) 和4,5多巴雙加氧酶為例，展示Ag43系統在表面展示中的效率。這些發現對未來的酵素表達實驗尤其在生物降解應用中具有潛力，本研究篩選用不同系統表達的氟乙酸脫鹵酶 (FAcD)，結果顯示IgA系統具有較高的活性。並且通過MALDI-TOF MS驗證FAcD降解受質後的產物，結果與預期一致。此外，證明IgA-FAcD系統具有良好的重複使用性，以及FAcD的高度穩定性。最後，探討表達於膜外的FAcD在生物降解應用中的潛力，為未來的研究提供重要的參考價值。

Expressing the target protein on the membrane prevents substrates from encountering barriers posed by the outer membrane. This approach not only broadens the range of reacting substrates but also facilitates the assessment of enzyme reusability, essential for enzyme recovery. In this study, we optimized the surface presentation of target proteins on the Escherichia coli (E. coli) membrane, employing various transmembrane systems, including IgA, Ag43, and eCPX. Comparative analysis revealed Ag43 as the optimal expression system, showcasing its superior protein expression capability and the ability to maintain enzyme activity when proteins are expressed outside the membrane. To validate the concept, we utilized Red Fluorescent Protein (RFP) and DOPA-dioxygenase as examples, demonstrating the efficiency of the Ag43 system in surface presentation. The findings hold promise for future enzyme expression experiments, particularly in biodegradation applications. We screened for fluoroacetate dehalogenase expressed using different systems, with results indicating higher activity in the IgA system. Additionally, we confirmed the products of FAcD substrate degradation using MALDI-TOF MS, which matched expectations. Furthermore, we demonstrated the excellent reusability of the IgA-FAcD system and the high stability of FAcD. Finally, we discussed the potential of extracellularly expressed FAcD in biodegradation applications, providing valuable insights for future research.

1. Liu, Q.; Yuan, F.; Liang, Y.; Li, Z., Cadmium adsorption by E. coli with surface displayed CadR. RSC Advances 2015, 5 (21), 16089-16092.
2. Lee, J. S.; Shin, K. S.; Pan, J. G.; Kim, C. J., Surface-displayed viral antigens on Salmonella carrier vaccine. Nat Biotechnol 2000, 18 (6), 645-8.
3. Zhang, H.; Chu, W.; Sun, J.; Liu, Z.; Huang, W. C.; Xue, C.; Mao, X., Combining Cell Surface Display and DNA-Shuffling Technology for Directed Evolution of Streptomyces Phospholipase D and Synthesis of Phosphatidylserine. J Agric Food Chem 2019, 67 (47), 13119-13126.
4. Cribari, M. A.; Unger, M. J.; Unarta, I. C.; Ogorek, A. N.; Huang, X.; Martell, J. D., Ultrahigh-Throughput Directed Evolution of Polymer-Degrading Enzymes Using Yeast Display. J Am Chem Soc 2023, 145 (50), 27380-27389.
5. Tsai, D.-Y.; Tsai, Y.-J.; Yen, C.-H.; Ouyang, C.-Y.; Yeh, Y.-C., Bacterial surface display of metal binding peptides as whole-cell biocatalysts for 4-nitroaniline reduction. RSC Advances 2015, 5 (107), 87998-88001.
6. Martineau, P.; Charbit, A.; Leclerc, C.; Werts, C.; O'Callaghan, D.; Hofnung, M., A genetic system to elicit and monitor antipeptide antibodies without peptide synthesis. Biotechnology (N Y) 1991, 9 (2), 170-2.
7. Lee, S. Y.; Choi, J. H.; Xu, Z., Microbial cell-surface display. Trends Biotechnol 2003, 21 (1), 45-52.
8. Jose, J.; Meyer, T. F., The autodisplay story, from discovery to biotechnical and biomedical applications. Microbiol Mol Biol Rev 2007, 71 (4), 600-19.
9. Rice, J. J.; Schohn, A.; Bessette, P. H.; Boulware, K. T.; Daugherty, P. S., Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands. Protein Sci 2006, 15 (4), 825-36.
10. Park, M.; Yoo, G.; Bong, J. H.; Jose, J.; Kang, M. J.; Pyun, J. C., Isolation and characterization of the outer membrane of Escherichia coli with autodisplayed Z-domains. Biochim Biophys Acta 2015, 1848 (3), 842-7.
11. Freudl, R.; MacIntyre, S.; Degen, M.; Henning, U., Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12. J Mol Biol 1986, 188 (3), 491-4.
12. Isticato, R.; Cangiano, G.; Tran, H. T.; Ciabattini, A.; Medaglini, D.; Oggioni, M. R.; De Felice, M.; Pozzi, G.; Ricca, E., Surface display of recombinant proteins on Bacillus subtilis spores. J Bacteriol 2001, 183 (21), 6294-301.
13. Shibasaki, S.; Tanaka, A.; Ueda, M., Development of combinatorial bioengineering using yeast cell surface display--order-made design of cell and protein for bio-monitoring. Biosens Bioelectron 2003, 19 (2), 123-30.
14. Tanaka, T.; Yamada, R.; Ogino, C.; Kondo, A., Recent developments in yeast cell surface display toward extended applications in biotechnology. Appl Microbiol Biotechnol 2012, 95 (3), 577-91.
15. Chen, X., Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance. Bioengineered 2017, 8 (2), 115-119.
16. Stahl, S.; Robert, A.; Gunneriusson, E.; Wernerus, H.; Cano, F.; Liljeqvist, S.; Hansson, M.; Nguyen, T. N.; Samuelson, P., Staphylococcal surface display and its applications. Int J Med Microbiol 2000, 290 (7), 571-7.
17. Hansson, M.; Samuelson, P.; Gunneriusson, E.; Stahl, S., Surface display on gram positive bacteria. Comb Chem High Throughput Screen 2001, 4 (2), 171-84.
18. Jeong, H.; Kim, H. J.; Lee, S. J. J. G. a., Complete genome sequence of Escherichia coli strain BL21. 2015, 3 (2).
19. Yeh, Y. C.; Muller, J.; Bi, C.; Hillson, N. J.; Beller, H. R.; Chhabra, S. R.; Singer, S. W., Functionalizing bacterial cell surfaces with a phage protein. Chem Commun (Camb) 2013, 49 (9), 910-2.
20. Ahan, R. E.; Kirpat, B. M.; Saltepe, B.; Seker, U. O. S., A Self-Actuated Cellular Protein Delivery Machine. ACS Synth Biol 2019, 8 (4), 686-696.
21. Veiga, E.; Sugawara, E.; Nikaido, H.; de Lorenzo, V.; Fernández, L. A., Export of autotransported proteins proceeds through an oligomeric ring shaped by C‐terminal domains. The EMBO Journal 2002, 21 (9), 2122-2131-2131.
22. Biondo, R.; da Silva, F. A.; Vicente, E. J.; Souza Sarkis, J. E.; Schenberg, A. C. G., Synthetic Phytochelatin Surface Display in Cupriavidus metallidurans CH34 for Enhanced Metals Bioremediation. Environmental Science & Technology 2012, 46 (15), 8325-8332.
23. Veiga, E.; de Lorenzo, V.; Fernandez, L. A., Probing secretion and translocation of a beta-autotransporter using a reporter single-chain Fv as a cognate passenger domain. Mol Microbiol 1999, 33 (6), 1232-43.
24. Export of autotransported proteins proceeds through an oligomeric ring shaped by C‐terminal domains.
25. Kjaergaard, K.; Hasman, H.; Schembri, M. A.; Klemm, P., Antigen 43-mediated autotransporter display, a versatile bacterial cell surface presentation system. J Bacteriol 2002, 184 (15), 4197-204.
26. Dvorak, P.; Bayer, E. A.; de Lorenzo, V., Surface Display of Designer Protein Scaffolds on Genome-Reduced Strains of Pseudomonas putida. ACS Synth Biol 2020, 9 (10), 2749-2764.
27. Rice, J. J.; Daugherty, P. S., Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides. Protein Eng Des Sel 2008, 21 (7), 435-42.
28. Dong, H.; Sarkes, D. A.; Rice, J. J.; Hurley, M. M.; Fu, A. J.; Stratis-Cullum, D. N., Living Bacteria-Nanoparticle Hybrids Mediated through Surface-Displayed Peptides. Langmuir 2018, 34 (20), 5837-5848.
29. Henderson, I. R.; Owen, P.; Nataro, J. P., Molecular switches--the ON and OFF of bacterial phase variation. Mol Microbiol 1999, 33 (5), 919-32.
30. Dautin, N.; Bernstein, H. D., Protein secretion in gram-negative bacteria via the autotransporter pathway. Annu Rev Microbiol 2007, 61, 89-112.
31. Henderson, I. R.; Navarro-Garcia, F.; Nataro, J. P., The great escape: structure and function of the autotransporter proteins. Trends Microbiol 1998, 6 (9), 370-8.
32. Leyton, D. L.; Rossiter, A. E.; Henderson, I. R., From self sufficiency to dependence: mechanisms and factors important for autotransporter biogenesis. Nat Rev Microbiol 2012, 10 (3), 213-25.
33. van der Woude, M. W.; Henderson, I. R., Regulation and function of Ag43 (flu). Annu Rev Microbiol 2008, 62, 153-69.
34. Sasaki, N.; Abe, Y.; Goda, Y.; Adachi, T.; Kasahara, K.; Ozeki, Y., Detection of DOPA 4,5-Dioxygenase (DOD) Activity Using Recombinant Protein Prepared from Escherichia coli Cells Harboring cDNA Encoding DOD from Mirabilis jalapa. Plant and Cell Physiology 2009, 50 (5), 1012-1016.
35. Cimmino, L.; Duarte, A. G.; Ni, D.; Ekundayo, B. E.; Pereira, I. A. C.; Stahlberg, H.; Holliger, C.; Maillard, J., Structure of a membrane-bound menaquinol:organohalide oxidoreductase. Nat Commun 2023, 14 (1), 7038.
36. Payne, K. A.; Quezada, C. P.; Fisher, K.; Dunstan, M. S.; Collins, F. A.; Sjuts, H.; Levy, C.; Hay, S.; Rigby, S. E.; Leys, D., Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation. Nature 2015, 517 (7535), 513-516.
37. Wang, Y.; Feng, Y.; Cao, X.; Liu, Y.; Xue, S., Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures. Scientific Reports 2018, 8 (1), 1454.
38. Tian, C.; Yang, J.; Liu, C.; Chen, P.; Zhang, T.; Men, Y.; Ma, H.; Sun, Y.; Ma, Y., Engineering substrate specificity of HAD phosphatases and multienzyme systems development for the thermodynamic-driven manufacturing sugars. Nat Commun 2022, 13 (1), 3582.
39. Zhang, H.; Tian, S.; Yue, Y.; Li, M.; Tong, W.; Xu, G.; Chen, B.; Ma, M.; Li, Y.; Wang, J.-b., Semirational Design of Fluoroacetate Dehalogenase RPA1163 for Kinetic Resolution of α-Fluorocarboxylic Acids on a Gram Scale. ACS Catalysis 2020, 10 (5), 3143-3151.
40. Chan, P. W.; Yakunin, A. F.; Edwards, E. A.; Pai, E. F., Mapping the reaction coordinates of enzymatic defluorination. J Am Chem Soc 2011, 133 (19), 7461-8.
41. Yue, Y.; Fan, J.; Xin, G.; Huang, Q.; Wang, J. B.; Li, Y.; Zhang, Q.; Wang, W., Comprehensive Understanding of Fluoroacetate Dehalogenase-Catalyzed Degradation of Fluorocarboxylic Acids: A QM/MM Approach. Environ Sci Technol 2021, 55 (14), 9817-9825.
42. Wang, J.-b.; Ilie, A.; Yuan, S.; Reetz, M. T., Investigating Substrate Scope and Enantioselectivity of a Defluorinase by a Stereochemical Probe. J Am Chem Soc 2017, 139 (32), 11241-11247.
43. Holloway, P.; Trevors, J. T.; Lee*, H., A colorimetric assay for detecting haloalkane dehalogenase activity. Journal of Microbiological Methods 1998, 32 (1), 31-36.
44. Hui, J. O.; Flick, T.; Loo, J. A.; Campuzano, I. D. G., Unequivocal Identification of Aspartic Acid and isoAspartic Acid by MALDI-TOF/TOF: From Peptide Standards to a Therapeutic Antibody. Journal of the American Society for Mass Spectrometry 2021, 32 (8), 1901-1909.
45. Karbassi, I. D.; Nyalwidhe, J. O.; Wilkins, C. E.; Cazares, L. H.; Lance, R. S.; Semmes, O. J.; Drake, R. R., Proteomic Expression Profiling and Identification of Serum Proteins Using Immobilized Trypsin Beads with MALDI-TOF/TOF. Journal of Proteome Research 2009, 8 (9), 4182-4192.
46. Li, D.; Yi, J.; Han, G.; Qiao, L., MALDI-TOF Mass Spectrometry in Clinical Analysis and Research. ACS Measurement Science Au 2022, 2 (5), 385-404.
47. Chen, W.; Xu, D.; Liu, L.; Peng, C.; Zhu, Y.; Ma, W.; Bian, A.; Li, Z.; Yuanyuan; Jin, Z.; Zhu, S.; Xu, C.; Wang, L., Ultrasensitive Detection of Trace Protein by Western Blot Based on POLY-Quantum Dot Probes. Analytical Chemistry 2009, 81 (21), 9194-9198.
48. Bakalova, R.; Zhelev, Z.; Ohba, H.; Baba, Y., Quantum Dot-Based Western Blot Technology for Ultrasensitive Detection of Tracer Proteins. J Am Chem Soc 2005, 127 (26), 9328-9329.
49. Maqbool, M.; Bhat, A.; Sumi, M. P.; Baba, M. A., Laboratory Protocols in Medical Biotechnology I. In Fundamentals and Advances in Medical Biotechnology, Anwar, M.; Ahmad Rather, R.; Farooq, Z., Eds. Springer International Publishing: Cham, 2022; pp 363-389.
50. Liu, T.; Zhang, W.; Zhang, Z.; Chen, M.; Wang, J.; Qian, X.; Qin, W., Sensitive Western-Blot Analysis of Azide-Tagged Protein Post Translational Modifications Using Thermoresponsive Polymer Self-Assembly. Analytical Chemistry 2018, 90 (3), 2186-2192.
51. van den Berg, B.; Prathyusha Bhamidimarri, S.; Dahyabhai Prajapati, J.; Kleinekathofer, U.; Winterhalter, M., Outer-membrane translocation of bulky small molecules by passive diffusion. Proc Natl Acad Sci U S A 2015, 112 (23), E2991-9.
52. Nikaido, H.; Vaara, M., Molecular basis of bacterial outer membrane permeability. Microbiol Rev 1985, 49 (1), 1-32.
53. Jap, B. K.; Walian, P. J., Structure and functional mechanism of porins. Physiol Rev 1996, 76 (4), 1073-88.
54. Ageorges, V.; Schiavone, M.; Jubelin, G.; Caccia, N.; Ruiz, P.; Chafsey, I.; Bailly, X.; Dague, E.; Leroy, S.; Paxman, J.; Heras, B.; Chaucheyras-Durand, F.; Rossiter, A. E.; Henderson, I. R.; Desvaux, M., Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation. Sci Rep 2019, 9 (1), 11100.