The bacterium Pseudomonas putida KT2440 is gaining considerable interest as a microbial platform for biotechnological valorization of polymeric organic materials, such as lignocellulosic residues or plastics. However, P. putida on its own cannot make much use of such complex substrates, mainly because it lacks an efficient extracellular depolymerizing apparatus. We seek to address this limitation by adopting a recombinant cellulosome strategy for this host. In this work, we report an essential step in this endeavor—a display of designer enzyme-anchoring protein “scaffoldins”, encompassing cohesin binding domains from divergent cellulolytic bacterial species on the P. putida surface. Two P. putida chassis strains, EM42 and EM371, with streamlined genomes and differences in the composition of the outer membrane were employed in this study. Scaffoldin variants were optimally delivered to their surface with one of four tested autotransporter systems (Ag43 from Escherichia coli), and the efficient display was confirmed by extracellular attachment of chimeric β-glucosidase and fluorescent proteins. Our results not only highlight the value of cell surface engineering for presentation of recombinant proteins on the envelope of Gram-negative bacteria but also pave the way toward designer cellulosome strategies tailored for P. putida.

中文翻译：

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设计蛋白支架在恶臭假单胞菌基因组减少菌株上的表面展示。

恶臭假单胞菌KT2440细菌作为聚合有机材料（如木质纤维素残留物或塑料）的生物技术价值化的微生物平台，引起了极大的兴趣。然而，恶臭假单胞菌本身并不能充分利用这种复杂的底物，主要是因为它缺乏有效的细胞外解聚装置。我们试图通过为该宿主采用重组纤维素体策略来解决这一限制。在这项工作中，我们报告了这项工作的一个重要步骤——展示设计酶锚定蛋白“支架素”，包括来自恶臭假单胞菌表面不同纤维素分解细菌物种的黏连蛋白结合域。两个恶臭假单胞菌本研究采用了具有流线型基因组和外膜组成差异的底盘菌株 EM42 和 EM371。支架素变体通过四种经过测试的自转运系统之一（来自大肠杆菌的Ag43 ）最佳地递送到其表面，并且通过嵌合 β-葡萄糖苷酶和荧光蛋白的细胞外附着证实了有效展示。我们的结果不仅突出了细胞表面工程在革兰氏阴性菌包膜上呈现重组蛋白的价值，而且还为设计为恶臭假单胞菌量身定制的纤维素体策略铺平了道路。