Abstract
BackgroundMetagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals.ResultsTwenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C.ConclusionsSeven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.
Highlights
Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes
The characterized enzymes pH optima and thermal stability profiles are quite consistent with the culture conditions of the compost community [8], further highlighting the importance of altering culture conditions in metagenomics experiments to enrich for enzymes with desired properties
Twenty-two putative CAZy ORFs were cloned into expression vectors and screened for soluble expression and activity using various glycoside hydrolase assays
Summary
Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Recent advances in DNA sequencing technology has made it possible to interrogate the sequence space of an organism or community to recover large numbers of putative coding regions in a high throughput manner without the need for laboratory culturing and propagation [5]. This approach allows for the detection of functional genes encoding biocatalysts of interest and has recently been successfully used for GH gene discovery using samples from the cow rumen microbial community [6] and in the microbial community associated with earthworm casts [7]
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