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. putida as the host bacterium by comparing different signal peptides and translocator domains for the surface display of an esterase. The translocator domain did not have a considerable effect on the activity of the whole-cell catalysts. In contrast, by using the signal peptide of the P. putida outer membrane protein OprF, the activity was more than 12-fold enhanced to 638 mU ml-1  OD-1 compared with the signal peptide of V. cholerae CtxB (52 mU ml-1  OD-1 ). This positive effect was confirmed with a ß-glucosidase as a second example enzyme. Here, cells expressing the protein with N-terminal OprF signal peptide showed more than fourfold higher ß-glucosidase activity (181 mU ml-1  OD-1 ) than with the CtxB signal peptide (42 mU ml-1  OD-1 ). SDS-PAGE and flow cytometry analyses indicated that the increased activities correlated with an increased amount of recombinant protein in the outer membrane and a higher number of enzymes detectable on the cell surface.

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

Inducible promoters such as Plac 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. To this end, genetic regions upstream of the RoxS/RoxR-regulated genes ddcA (PR ox132 ) and PP_3332 (PR ox306 ) were inserted into plasmids that mediated the expression of superfolder green fluorescent protein (sfGFP) and surface displayed mCherry, confirming their promoter functionalities. Mutation of the Pribnow box of PR ox306 to the σ70 consensus sequence (PR ox3061 ) resulted in a more than threefold increase of sfGFP production. All three promoters caused cell density-dependent expression, starting transcription at optical densities (OD578 ) of approximately 1.0 (PR ox132 , PR ox306 ) or 0.7 (PR ox3061 ) as determined by RT-qPCR. The QS dependency of PR ox306 was further shown by cultivating P. putida in media that had already been used for cultivation and thus contained bacterial signal molecules. The longer P. putida had grown in these media before, the earlier protein expression in freshly inoculated P. putida appeared with PR ox306 . This confirmed previous findings that a bacterial compound accumulates within the culture and induces protein expression.

Proof of concept for the simplified breakdown of cellulose by combining Pseudomonas putida strains with surface displayed thermophilic endocellulase, exocellulase and ß-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. RESULTS: The robust soil bacterium Pseudomonas putida showed a strongly reduced uptake of glucose above a temperature of 50 °C, while remaining structurally intact hence recyclable, which makes it suitable for cellulose hydrolysis at elevated temperatures. Consequently, three complementary, thermophilic cellulases from Ruminiclostridium thermocellum were displayed on the surface of the bacterium. All three enzymes retained their activity on the cell surface. A mixture of three strains displaying each one of these enzymes was able to synergistically hydrolyze filter paper at 55 °C, producing 20 µg glucose per mL cell suspension in 24 h. CONCLUSION: We could establish Pseudomonas putida as host for the surface display of cellulases, and provided proof-of-concept for a fast and simple cellulose breakdown process at elevated temperatures. This study opens up new perspectives for the application of P. putida in the production of biofuels and other biotechnological products.

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. However, for their utilisation beyond a laboratory scale, in particular for biocatalysis, host bacteria with specific features and industrial applicability are required. A few groups have addressed this issue and demonstrated that bacteria other than E. coli can also be used for autotransporter based surface display. We summarise these studies and discuss opportunities and challenges that arise from surface display of recombinant proteins using the autotransporter pathway in alternative hosts.

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

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 ß-barrel of the type V autotransporter EhaA from Escherichia coli under control of an IPTG-inducible T5 promoter. The MATE system enabled the continuous secretion of recombinant passenger mCherry via OmpT-mediated cleavage, using native OmpT protease activity in E. coli when grown at 37 °C. It is the first example to show that native OmpT activity is sufficient to facilitate the secretion of a correctly folded target protein in preparative amounts obtaining 240 µg of purified mCherry from 800 mL of crude culture supernatant. Because the release of mCherry was achieved by a simple transfer of the encoding plasmid from an OmpT-negative to an OmpT-positive strain, it bears the option to use surface display for screening purposes and secretion for production of the selected variant. A single plasmid could therefore be used for continuous secretion in OmpT-positive strains or surface display in OmpT-negative strains. In conclusion, the MATE system appears to be a versatile tool for the surface display and for the secretion of target proteins in E. 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. The aim of this study was to investigate if the fermentative bacteria Zymomonas mobilis and Zymobacter palmae, representing attractive candidates for industrial applications, can serve as host organisms for autodisplay. We therefore used the carboxylesterase EstA from Burkholderia gladioli as an autotransporter passenger to display it on the surfaces of Z. palmae and Z. mobilis. Expression and outer membrane localization of the EstA-autotransporter fusion protein were verified by SDS-PAGE, and surface display of the enzyme was demonstrated by ELISA and flow cytometer analysis. Whole-cell activity assays revealed that EstA retained its activity on the cell surface. Recombinant Z. palmae cells exhibited significant higher esterase activity (294mU/mL/OD 1) in comparison to Z. mobilis (88mU/mL/OD 1) and the control E. coli (113mU/mL/OD 1). This appears even more noteworthy, as about 30% of EstA was released from the cell surface of Z. palmae. Nevertheless, our results indicate that both species are suitable autodisplay hosts, in particular Z. palmae for displaying esterase, opening up new horizons for biocatalytic applications.

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. Even after 40 years of extensive research, the regulatory mechanisms of these efflux transporters are still not completely understood. To unravel the efflux transporter regulation, we analyzed the effect of the nuclear receptor CAR (constitutive androstane receptor) on the expression of Abcb1 and Abcg2 in primary cultures of porcine brain capillary endothelial cells (PBCEC). CAR is a xenobiotic-activated transcription factor, which is, like the other important nuclear receptor pregnane X receptor (PXR), highly expressed in barrier tissue and known to be a positive regulator of ABC-transporters. We demonstrate that activation of porcine CAR by the human CAR (hCAR) ligand CITCO (6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde) leads to an up-regulation of both transporters, whereas the mouse-specific CAR ligand TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene) had no effect on transporter expression. The stimulation of PBCEC with CITCO caused a significant up-regulation of both efflux-transporters on RNA-level, protein level and transport level. Furthermore the additional application of a CAR inhibitor significantly decreased the transporter expression to control niveau. In conclusion our data prove CAR activation only by the human ligand CITCO leading to an increased ABC-transporter expression and transport activity.

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. PXR is a xenobiotic-activated transcription factor that is highly expressed in barrier tissue. Xenobiotics like rifampicin and hyperforin activate PXR and induce Abcb1 expression. ABC-transporter up-regulation could have potential effects on pharmacokinetics of different drugs. To study the influence of PXR on the two most prominent efflux transporters we used a primary culture of porcine brain capillary endothelial cells (PBCEC) due to higher homologies to the human form of PXR. For activation of the pregnane X receptor the ligands hyperforin and rifampicin were used. We investigated the effects on the transporters on RNA level (quantitative real time PCR), protein level (Western blotting) and transport level (uptake assay, active transport). The stimulation of the PBCEC with rifampicin or hyperforin showed a significant up-regulation of both ABC-transporters on RNA level after 6h, whereas an increased protein expression was strongest after 12h. Also the transport activity intensified after a period of 12h for Abcg2 and Abcb1. In conclusion our data prove PXR activation by rifampicin and hyperforin lead to an increased ABC-transporter expression and transport activity.