Novel cellulases from hyperthermophilic organisms

Enzymatic hydrolysis of (ligno)cellulose by cellulases is key for the production of second generation biofuels which represent a long-standing leading theme in the field of sustainable energy. Cellulases which work under extreme conditions such as extreme pH values, high temperatures and pressure, organic solvents or ionic liquids are highly desired for industrial processes exploiting enzymatic breakdown of cellulose. Such enzymes could work under conditions similar to those often used in pretreatment of lignocellulosic biomass. Currently, there is a paucity of enzymes which are able to degrade (ligno)cellulose efficiently and at elevated temperatures. Cellulases with exquisite hyperthermostability were discovered in archaeal microorganisms dwelling in extreme environments, such as hot springs or ocean vents. Unfortunately, industrial exploitation of archaeal extremozymes is generally hampered by difficulties in laboratory cultivation and a lack of genetic tools. Deciphering the structural basis of (hyper)thermostability in archaeal enzymes, however, will provide a toolkit for rational engineering of traditional enzymes towards increased thermostability. Here we propose to express and characterize two cellulases (EBI-244 and EBI-108) from a hyperthermophilic consortium. EBI-244 was already successfully expressed in Escherichia coli and tobacco. The recently isolated consortium consists of three hyperthermophilic Archaea and is capable of growing on crystalline cellulose at 90C. Several glycosyl hydrolases including putative cellulases - were identified by metagenomic analysis, among them a multidomain cellulase (EBI-244) which proved to be remarkably resistant towards harsh conditions and has a temperature optimum at 109C. While the optimum temperature of these enzymes might actually be too high for some processes, this and other cellulases from hyperthermophilic organisms can teach us an important lesson of how proteins achieve stability and sustain activity in extreme environments. We chose the two enzymes among the several putative cellulases identified in the consortium because EBI-244, a member of glycosyl hydrolase family 5 (GH5), was shown to be the most active among them; and because the sequence of EBI-108 (GH12) exhibits high homology with an archaeal cellulase with a known crystal structure, which provides a good basis for studying and understanding structure/function relationships. We furthermore want to systematically analyze putative glycosylation sites in these enzymes through mutational analysis. Generally, little is known about the role of glycosylation in Archaea; and although it has been hypothesized to have a possible important role in protein activity and thermostability especially in cellulases it is not well studied to date. We anticipate that understanding the factors conferring (hyper)thermostability in archaeal cellulases will make novel cellulases with unique properties available. Detailed comprehension of the mechanism, molecular architecture, influence of glycosylation and substrate specificities will be available. Through gained knowledge on archaeal cellulases with unique properties, this project will thus open up new strategies to make biofuel production from lignocellulosic feedstock economic.

In order to use plant-generated (ligno)cellulosic biomass as a feedstock for second generation biofuels, efficient enzymatic degradation of the substrate by biocatalysts such as cellulases is pivotal. Due to the resilient and complex nature of (ligno)cellulase, pretreatment is required to facilitate access of the enzymes. Pretreatment needs harsh conditions such as extreme pH values, high temperatures and pressure, organic solvents or ionic liquids which are harmful to most enzymes. This prevents more efficient one-pot pretreatment processes. Therefore, biocatalysts with superior stability in these conditions are required for industrial processes exploiting enzymatic breakdown of cellulose. A hyperthermostable cellulase EBI-244 - was discovered in an archaeal microorganism in a hot spring in Nevada. This enzyme was successfully expressed in tobacco plants (Nicotiana benthamiana) in this project. Expression of archaeal extremozymes in their native host is not feasible in laboratory settings. The hyperthermophilic archaeal cellulase EBI-244 has an optimal temperature of 109C, a melting temperature of 113C, and is resilient towards salt, detergents, and ionic liquids. This makes it a promising candidate for an industrial glycosidase. EBI-244 had been previously expressed in the bacterial model organism Escherichia coli, however, at disappointingly low yields. The plant expressed enzyme showed the same temperature and pH stability as the previously bacterial expressed version at increased expression levels (0.3 mg protein per g leaves). After successful production of high amounts (2 g) of EBI-244 in plants, I characterized the enzyme with regard of its temperature and pH stability, activity on soluble and insoluble substrates, and posttranslational modifications. It turned out that EBI-244 is most active on Lichenan and Mannan. Compared to the activity of EBI-244 on these substrates, activity on carboxymethylcellulose and Barley ß-glucan was reduced approximately two-fold and five-fold, respectively. Low activity on crystalline cellulose and marginal activity on corn stover and miscanthus was also detected. Interestingly, when studying posttranslational modifications, besides determining several N-glycosylation sites, I also identified some O- glycosylated peptides and identified the respective O-glycosylation sites. This is especially intriguing, since O-glycosylation on serine or threonine residues has been rarely reported in plants. Complementary to these experiments, I also tested whether EBI-244 might be suited in high temperature pretreatment of biomass. This would be of particular interest for the current process, since EBI-244 is resilient towards high temperatures, salt concentrations, and solvents. However, comparative testing of EBI-244 pretreated and untreated biomass with a standard complete cellulase system did not show any differences in soluble sugar release. Therefore, its use as an industrial cellulase might be limited.