Abstract

The project aims to exploit the very high reducing power of hyperthermophilic microorganisms for the on-line production of fine chemicals for a range of industrial applications. The metabolic and genetic diversity of Thermococcales, including Pyrococcus and Thermococcus spp. will be exploited by screening available strain collections for specific bio-reducing capacities, as well as the three established Pyrococcus genomes for enzymes catalyzing the reductive conversions via genome and proteome based approaches. Further development of genetic tools will enable the in vivo bioreductions at 50-100 ºC by metabolic engineering to prevent side reactions to take place. The project will develop fermentation conditions and reactor design, and on-line product distillation will be established. Finally, a new, stable co-factor regenerating system will be applied to the in vitro bio-reduction system.

Objectives

The limited range of substrates that can be efficiently converted restricts the production of fine chemicals, by chemical methods or by biological systems such as yeast. The PYRED project will develop new novel bioreduction processes for natural and on-line generation of a broad spectrum of specific products at elevated temperatures. For this purpose, we will exploit the extraordinary high reductive power of anaerobic, hyperthermophilic microbes that belong to the archaebacterial order Thermococcales. Strains of Pyrococcus and Thermococcus spp. have established biotechnological potential, have favourable physiological properties (fast growth on cheap substrates), and are metabolically quite diverse. By using natural and engineered hyperthermophiles and their extremely stable enzymes, we will develop integrated in vivo and in vitro bio-reduction reactor systems, respectively, for the production of valuable fine chemicals.

Description of the work

A multidisciplinary approach will be used for the development of novel bio-reduction processes. In addition to three recently-completed pyrococcal genome sequences (in which the current partners played a role), we will have access to a large Thermococcales strain collection. The metabolic diversity of Thermococcales will be screened for the bio-reducing capacity of strains, converting specific precursor substrates for the production of valuble aldehydes, (chiral) alcohols, as well as for the selective reduction of double bonds. The available hyperthermophile fermentation expertise will be used for cultivation of selected strains, aiming at optimal conversion efficiency in high cell-density, pilot-scale dialysis reactors. A qualitative and quantitative analysis of new strains will be performed during studies on the type strain P. furiosus. Development of the recently established genetic tools enables the metabolic engineering to take place, based on insight gained from genome and proteome analysis. This is aimed at improving the in vivo production process. Apart from such an in vivo production system, an in vitro system will be developed with isolated enzymes. In addition to the more common aldehyde-/alcohol-producing enzymes, novel catalysts will be identified by a combined genome/proteome screening approach, and produced in (heterologous) expression systems. We will develop a robust co-factor regeneration system required for the in vitro reactor, by making use of a pyrococcal hydrogenase. In addition, with further development of genetic tools the homologous production of complex redox enzymes can take place, and as such will be instrumental in the developing the biocatalyst as well as the co-factor regenerating components of the in vitro bioreduction process. Another innovative aspect of these bioreduction systems at elevated temperatures is the possibility of on-line distillation of the volatile products.

Deliverables

• Selection of Thermococcales with novel bioreductive capacities from an unexploited strain collection.
• Genome/proteome-based identification of novel reductive biocatalysts from Pyrococcus spp.
• In vivo production of fine chemicals by natural and engineered methods in dialysis fermentors.
• In vitro production of fine chemicals by novel reductive biocatalysts from Thermococcales in a bio-reduction reactor based on a novel H₂-driven in vitro NAD(P)H-regeneration system.
• Natural, environmental-friendly, cheap bioconversion of raw materials (renewables) to specific fine chemicals by an integrated process with on-line product distillation.

Contacts

Coordinator

• Wageningen University Laboratory of Microbiology / NETHERLANDS

Participant

• Kornel KOVACS / Hungarian Academy of Sciences Biological Research Centre / HUNGARY
• Chris WINKEL / Quest International B.V. Food Science and Technology Center / NETHERLANDS
• Wilfred R. HAGEN / Technical University Delft Kluyver Department of Biotechnology / NETHERLANDS
• Herbert MÄRKL / Technologie GmbH Technische Universität Hamburg-Harburg / GERMANY
• Daniel PRIEUR / Universite de Bretagne Occidentale Laboratoire des Sciences de l'Environnement Marin / FRANCE
• Lubbert DIJKHUIZEN / University of Groningen Microbial Physiology / NETHERLANDS
• Martijn HUYNEN / University of Nijmegen Centre for Molecular and Biomolecular Informatics / NETHERLANDS