Abstract

The Crenarchaeotes constitute a kindom of the Archaea that contains a very diverse range of microorganisms many of which are extreme- or hyperthermophiles. They differ extensively genetically and biochemically from Bacteria and Eukaryotes. Sulfolobus is the best studied of these organisms and most of groundbreaking work on this organism has been done in Europe. The purpose of this project is to exploit this European advantage and to screen for novel enzyme activities both at the genomic level and in an extensive and unique European collection of thermophilic Crenarchaeotes. Moreover, we will develop and refine vector systems for expressing these proteins in a range of Bacterial and Eukaryotic hosts by modifying the Archaeal genetic signals. In particular, for the first time we will develop a vector-host system for expressing Crenarchaeal enzymes in a Sulfolobus host on a large scale.

Objectives

• To identity new or improved enzymes from extremely thermophilic and hypethermophilic crenarchaeotes using an integrated genomics, microarray and screening approach.
• To develop and refine Archaeal shuttle vectors for high expression of crenarchaeal enzymes in bacterial and lower eukaryotic hosts.
• To develop the first vector-host system for high expression of crenarchaeal enzymes in the crenarchaeote Sulfolobus.
• Perform large-scale fermentation to express new crenarchaeal enzymes and to thermoadapt mesophilic enzymes in Sulfolobus.

Description of the work

Sulfolobus solfataricus is the only Crenarchaeote that has been widely studied by biologists, almost exclusively in Europe where a wealth of expertise lies. Given that the Archaea differ considerably in their biology from Bacteria and Eukaryotes - at least 30% of their genes have no detectable homologues in the other two domains - they are a rich source of novel enzymes. Moreover, since many of the archaeal homologs of eukaryotic multi-subunit enzymes are monomeric, this greatly extends their applicability to biotechnological processes. The project involves a genomic approach where we exploit the finished genome of S. solfataricus and perform low pass sequencing of the genomes of two other diverse and divergent Crenarchaeotes Sulfolobus acidocaldarius and Hyperthermus butylicus. Parallel microarray analyses will be made on S. solfataricus grown under widely different conditions in order to monitor gene expression accurately. The genome and microarray data will then be analysed using state of the art bioinformatics techniques to identify expressible novel enzymes. Screening for enzyme activities will also be performed on a large and unique collection of Crenarchaeotes assembled from all over the world. New vectors will be developed and modified to express Archaeal genes in bacterial, lower eukaryotic and archaeal hosts. Given the difficulty in expressing many archaeal proteins efficiently in non-archaeal hosts, we will develop a vector-host system for expression in Sulfolobus for the first time. Large-scale fermentation of the Sulfolobus for enzyme production will also be developed.

Deliverables

• Expertly annotated low pass sequences from S. acidocaldarius and H. butylicus.
• Microarray analysis of gene expression from the S. solfataricus genome.
• Enzyme screening of the three genomes and of a large and unique collection of Crenarchaeotes.
• Vector development for expression of extremely thermophilic and hyperthermophilic Crenarchaeote genes.
• Development of a host-vector system for Sulfolobus.
• Large-scale fermentation of Sulfolobus carrying recombinant enzymes.

Contacts

Coordinator

• Roger GARRETT / University of Copenhagen Institute of Molecular Biology / DENMARK

Participant

• Dr Neil RAVEN / Centre for Applied Microbiology & Research / UNITED KINGDOM
• Simonetta BARTOLUCCI / Dipartimento di Chimica organica e Biologica ITALY
• Hans-Peter KLENK / Epidauros Biotechnologie AG GERMANY
• Julio SALINAS / Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) / SPAIN
• Groth CLAUSEN / Novo Nordisk A/S DENMARK
• Technische Universität Darmstadt Institute of Microbiology and Genetics / GERMANY
• Rolf BERNANDER / Uppsala University Department of Cell and Molecular Biology / SWEDEN
• Wageningen University Laboratory of Microbiology / NETHERLANDS