The bacterium Pseudomonas putida KT2440 is gaining considerable interest as a microbial platform for biotechnological valorization of polymeric organic materials, such as waste lignocellulose 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 meet this challenge by adopting a recombinant cellulosome strategy for this attractive host. Here, 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 substantial differences in the composition of the outer membrane were employed in this study. Scaffoldin variants were 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 chimaeric beta-glucosidase and fluorescent proteins. Our results highlight the importance of cell surface engineering for display of recombinant proteins in Gram-negative bacteria and pave the way towards designer cellulosome strategies, tailored for P. putida.

中文翻译：

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设计者蛋白质支架在基因组减少的恶臭假单胞菌菌株上的表面展示

恶臭假单胞菌（Pseudomonas putida）KT2440作为一种微生物平台，正对诸如废弃的木质纤维素或塑料之类的聚合有机材料进行生物技术价值评估，引起了广泛的关注。但是，恶臭假单胞菌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设备。我们寻求通过针对这种有吸引力的宿主采取重组纤维素体策略来应对这一挑战。在这里，我们报告了这项工作中必不可少的一步，展示了一种设计酶固定蛋白支架蛋白，包括来自恶臭假单胞菌表面上不同纤维素分解细菌物种的粘着蛋白结合域。在这项研究中，使用了两个恶臭假单胞菌底盘菌株EM42和EM371，它们具有简化的基因组和外膜组成的显着差异。用四个经过测试的自动转运蛋白系统（来自大肠杆菌的Ag43）之一将支架蛋白变体递送至其表面，并且通过嵌合的β-葡萄糖苷酶和荧光蛋白的细胞外附着确认了有效的展示。我们的结果凸显了细胞表面工程对于展示革兰氏阴性细菌中重组蛋白的重要性，并为专为恶臭假单胞菌量身定制的设计纤维素体策略铺平了道路。