Surface display of designer protein scaffolds on genome-reduced strains of Pseudomonas putida

Abstract

AbstractThe 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 chimeric β-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.