Cell density-dependent auto-inducible promoters for expression of recombinant proteins in Pseudomonas putida.

Inducible promoters such as P are of limited usability for industrial protein production with Pseudomonas putida. We therefore utilized cell density-dependent auto-inducible promoters for recombinant gene expression in P. putida KT2440 based on the RoxS/RoxR Quorum Sensing (QS) system of the bacterium.

Improving the autotransporter-based surface display of enzymes in Pseudomonas putida KT2440.

Pseudomonas putida can be used as a host for the autotransporter-mediated surface display of enzymes (autodisplay), resulting in whole-cell biocatalysts with recombinant functionalities on their cell envelope. The efficiency of autotransporter-mediated secretion depends on the N-terminal signal peptide as well as on the C-terminal translocator domain of autotransporter fusion proteins. We set out to optimize autodisplay for P.

Proof of concept for the simplified breakdown of cellulose by combining Pseudomonas putida strains with surface displayed thermophilic endocellulase, exocellulase and β-glucosidase.

Background: The production and employment of cellulases still represents an economic bottleneck in the conversion of lignocellulosic biomass to biofuels and other biocommodities. This process could be simplified by displaying the necessary enzymes on a microbial cell surface. Such an approach, however, requires an appropriate host organism which on the one hand can withstand the rough environment coming along with lignocellulose hydrolysis, and on the other hand does not consume the generated glucose so that it remains available for subsequent fermentation steps.

Maximized Autotransporter-Mediated Expression (MATE) for Surface Display and Secretion of Recombinant Proteins in .

A new optimized system for the surface display and secretion of recombinant proteins is described, termed MATE (maximized autotransporter-mediated expression). It is based on an artificial gene consisting of the coding region for the signal peptide of CtxB, a multiple cloning site for passenger gene insertion, flanked by coding sequences for linear epitopes for monoclonal antibodies and OmpT, and factor Xa protease cleavage sites followed by a codon-optimized DNA sequence of the linker and the β-barrel of the type V autotransporter EhaA from under control of an IPTG-inducible T5 promoter. The MATE system enabled the continuous secretion of recombinant passenger mCherry OmpT-mediated cleavage, using native OmpT protease activity in when grown at 37 °C.

Going beyond E. coli: autotransporter based surface display on alternative host organisms.

Autotransporters represent one of the most popular anchoring motifs used to display peptides, proteins or enzymes on the cell surface of a Gram-negative bacterium. Applications range from vaccine delivery to library screenings to biocatalysis and bioremediation. Although the underlying secretion mechanism is supposed to be available in most, if not all, Gram-negative bacteria, autotransporters have to date almost exclusively been used for surface display on Escherichia coli.

Autotransporter mediated esterase display on Zymomonas mobilis and Zymobacter palmae.

Autodisplay, i.e. surface expression of recombinant proteins by virtue of the autotransporter secretion pathway, has been used predominantly with Escherichia coli as host organism, which often limits the applicability of this technique to laboratory purposes and scales.

Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood-brain barrier after CITCO activation.

ATP-driven efflux transporters are considered to be the major hurdle in the treatment of central nervous system (CNS) diseases. Abcb1 (P-glycoprotein) and Abcg2 (breast cancer resistance protein/brain multidrug resistance protein) belong to the best known ABC-transporters. These ABC-transporters limit the permeability of the blood-brain barrier and protect the brain against toxic compounds in the blood but on the other hand they also reduce the efficacy of CNS pharmacotherapy.

Pregnane X receptor upregulates ABC-transporter Abcg2 and Abcb1 at the blood-brain barrier.

ATP-driven efflux transporters are important, selective elements of the blood-brain barrier. Abcg2 (also brain multidrug resistance protein) and Abcb1 (P-glycoprotein) belong to the best known ABC-transporters which limit the access of therapeutic drugs to the brain and impair pharmacotherapy of central nervous system (CNS) disorders. To investigate the question how ATP-binding cassette (ABC)-transporters are regulated, we analyzed the influence of the nuclear receptor, pregnane X receptor (PXR) on transporter expression.