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. 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.<br

Sustav za izlaganje na površini i sekreciju rekombinantnih proteina u bakteriji Escherichia coli, posredovan ekspresijom autotransportera

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 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.U radu je opisan novi, optimirani sustav za izlaganje na površini stanice i sekreciju rekombinantnih proteina, nazvan MATE (engl. maximized autotransporter-mediated expression). Zasnovan je na sintetiziranoj sekvenciji DNA, koja se sastoji od dijela što kodira za signalni peptid CtxB, višestrukog mjesta za kloniranje u kojeg se ugrađuje gen za željeni protein, omeđenog sekvencijama za linearne epitope monoklonskih antitijela i proteaze OmpT, te mjestom cijepanja za proteazu Xa. Nakon toga slijedi optimirana sekvencija za vezni dio i β-bačvasti dio autotransportera EhaA tipa V iz bakterije Escherichia coli pod kontrolom promotora T5 induciranog pomoću spoja IPTG. Zahvaljujući cijepanju pomoću proteaze OmpT nativno prisutne u E. coli tijekom uzgoja pri 37 °C omogućena je kontinuirana sekrecija rekombinantnog proteina mCherry u sustavu MATE. Ovo je prvi dokaz da je nativna aktivnost OmpT dovoljna za sekreciju proteina ispravne konformacije, pri čemu je iz 800 mL sirovog supernatanta bakterijske kulture dobiveno 240 μg pročišćenog proteina mCherry. Budući da je za oslobađanje proteina odgovorna aktivnost proteaze OmpT, isti se plazmid može koristiti za kontinuiranu sekreciju u OmpT-pozitivnom soju ili za izlaganje proteina na površini stanice u OmpT-negativnom soju. Stoga je zaključeno da se sustav MATE može koristiti za izlaganje proteina na površini stanice i sekreciju odabranih proteina u bakteriji E. coli

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 (PRox132) and PP_3332 (PRox306) 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 PRox306 to the σ70 consensus sequence (PRox3061) 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 (PRox132, PRox306) or 0.7 (PRox3061) as determined by RT‐qPCR. The QS dependency of PRox306 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 PRox306. This confirmed previous findings that a bacterial compound accumulates within the culture and induces protein expression

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 β‐glucosidase as a second example enzyme. Here, cells expressing the protein with N‐terminal OprF signal peptide showed more than fourfold higher β‐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