Abstract
BackgroundThe 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.ResultsThe 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0505-8) contains supplementary material, which is available to authorized users.
Highlights
The production and employment of cellulases still represents an economic bottleneck in the conversion of lignocellulosic biomass to biofuels and other biocommodities
We have recently developed a surface display system named mediated expression (MATE), which is based on the autotransporter secretion mechanism using EhaA from E. coli, and which was designed for its application in a broad range of Gram-negative bacteria [32]
Glucose uptake and recyclability of P. putida at different temperatures For the economic success of a cellulose hydrolysis process it is crucial that the generated glucose can be recovered
Summary
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, 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. Several studies showed that the degree of synergism strongly depends on the ratio of the applied cellulases, which implies that precise control over the composition of the enzyme mixture is important to maximize hydrolysis efficiency [9,10,11] Such a control is difficult to obtain when a single organism produces multiple enzymes. The availability of a set of bacterial strains that can be cultivated and in large amounts, each displaying a single enzyme, would bring a great deal of flexibility and simplicity into the lignocellulose degradation process
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