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.


CONCLUSIONS
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.
Institute of Pharmaceutical and Medicinal Chemistry, Westfälische Wilhelms-Universität Münster, PharmaCampus, Corrensstraße 48, 48149, Münster, Germany.|NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, PharmaCampus, Corrensstraße 48, 48149, Münster, Germany.
Iasson E P
IE
Tozakidis
Autodisplay Biotech GmbH, Merowingerplatz 1a, 40225, Düsseldorf, Germany.
Tatjana
T
Brossette
Institute of Pharmaceutical and Medicinal Chemistry, Westfälische Wilhelms-Universität Münster, PharmaCampus, Corrensstraße 48, 48149, Münster, Germany.
Florian
F
Lenz
Autodisplay Biotech GmbH, Merowingerplatz 1a, 40225, Düsseldorf, Germany.
Ruth M
RM
Maas
Institute of Pharmaceutical and Medicinal Chemistry, Westfälische Wilhelms-Universität Münster, PharmaCampus, Corrensstraße 48, 48149, Münster, Germany. joachim.jose@uni-muenster.de.|NRW Graduate School of Chemistry, Westfälische Wilhelms-Universität Münster, PharmaCampus, Corrensstraße 48, 48149, Münster, Germany. joachim.jose@uni-muenster.de.
Joachim
J
Jose
D055162:Biocatalysis; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006868:Hydrolysis; D016958:Pseudomonas putida / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*; D013499:Surface Properties; D013696:Temperature; D000069320:Type V Secretion Systems; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D000069320:Type V Secretion Systems; D002482:Cellulose; D001617:beta-Glucosidase; D002480:Cellulase; D005947:Glucose
Autotransporter; Cellulases; Cellulose breakdown; Glucose production; Maximized autotransporter mediated expression (MATE); Pseudomonas putida; Surface display; Whole cell biocatalysis