Discovering novel hydrolases from hot environments

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this recordNovel hydrolases from hot and other extreme environments showing appropriate performance and/or novel functionalities and new approaches for their systematic screening are of great interest for developing new processes, for improving safety, health and environment issues. Existing processes could benefit as well from their properties. The workflow, based on the HotZyme project, describes a multitude of technologies and their integration from discovery to application, providing new tools for discovering, identifying and characterizing more novel thermostable hydrolases with desired functions from hot terrestrial and marine environments. To this end, hot springs worldwide were mined, resulting in hundreds of environmental samples and thousands of enrichment cultures growing on polymeric substrates of industrial interest. Using high-throughput sequencing and bioinformatics, 15 hot spring metagenomes, as well as several sequenced isolate genomes and transcriptomes were obtained. To facilitate the discovery of novel hydrolases, the annotation platform Anastasia and a whole-cell bioreporter-based functional screening method were developed. Sequence-based screening and functional screening together resulted in about 100 potentially new hydrolases of which more than a dozen have been characterized comprehensively from a biochemical and structural perspective. The characterized hydrolases include thermostable carboxylesterases, enol lactonases, quorum sensing lactonases, gluconolactonases, epoxide hydrolases, and cellulases. Apart from these novel thermostable hydrolases, the project generated an enormous amount of samples and data, thereby allowing the future discovery of even more novel enzymes.European CommissionEuropean Union FP

The Phylum dictyoglomi

The phylum Dictyoglomi consists a single genus, Dictyoglomus, with two type strains and several related pure culture isolates. All isolates are thermophilic anaerobic Gram-type negative rods. A major distinguishing phenotypic feature is the formation of spherical bodies in late stationary phase of growth, the function of which is not understood.Most isolates are fermentative using a range of simple carbohydrates, but some isolates are able to grow on crystalline cellulose and chemolithotrophy using carbon monoxide as energy source has been reported for one pure culture. There have been relatively few applications for Dictyoglomus enzymes as a result of a number of factors. Although many of their kinetic properties are exceptional, they have had to be cloned and expressed in standard fermentation strains as hosts, and their low G:C content has required significant genetic manipulation to provide expression. Some of the main applications have required inexpensive enzymes in bulk (e.g., pulp bleaching in paper manufacture), and they have had to be regarded as a replacement for well-established enzymes currently used in the industry. The major applications have involved glycosyl hydrolases, but new uses in value-added products involving drug precursor transformations have been reported recently.10 page(s