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5th RTD Framework Programme

1. Objectives and expected achievements

The ultimate goal of the PYRED project is the development of novel, natural bio-reduction processes by exploiting the metabolic and genetic diversity of a recently discovered group of hyperthermophilic microorganisms, i.e. the order of Thermococcales, including Pyrococcus and Thermococcus subspecies. These microorganisms have established biotechnological potential, since they are a source of a variety of novel enzymes with a high reducing power. This project will focus on the identification, characterisation and exploitation of enzymes involved in (1) reduction of acid to aldehyde, (2) reduction of acid to alcohol; (3) reduction of keton to chiral alcohol; (4) selective reduction of double bonds. The resulting aldehydes, (chiral) alcohols and selectively saturated compounds, some of which can not be produced by currently applied industrial production organisms, will have applications in food, pharmaceutical and chemical industries.

The Thermococcales have been selected for several reasons. Apart from their high reducing power that has been demonstrated to drive both in vivo and in vitro at least some of the aforementioned conversions, the low risk of biological contamination is an additional advantage of operating the bio-reduction processes at high temperatures. Moreover, Thermococcales have the potential of a very efficient down-stream processing, i.e. by on-line destillation of the volatile product. A practical advantage of at least some Thermococcales strains is the fast growth (40 min generation time on carbohydrates) on cheap, renewable substrates such as starch. Hence, the described process is an environmental-friendly alternative for the chemical conversions that are currently used in industry. The reductive enzymes from Thermococcales have great potential for food applications since these hosts are archaea, among which no pathogens exist and their genes can be expressed in food-grade hosts.

The last decade many new strains have been isolated from a variety of extreme environments, and have been characterized in considerable detail: physiologically, biochemically and genetically. Moreover, three pyrococcal genome sequences have been completed. Recently, a breakthrough in the development of a hyperthermophile genetic system has been accomplished: a highly efficient plating method has been established, an E. coli-Pyrococcus shuttle vector has been developed, and transformation studies have been performed aiming at complementation of pyrococcal uracil auxotrophs. Using these techniques, metabolic engineering of pyrococcal strains aiming to increase the efficiency of the in vivo bioreductive conversion will become feasible for the first time.

Objectives of PYRED project

Screening the metabolic and the genetic diversity in order to identify enzymes that are capable of driving specific bioreduction processes

Hyperthermophilic strains from collections of Thermococcales will be screened for enzymes (gene-products) that are capable of driving specific bioreduction processes. In this respect, strains will be screened on natural variants with a different specificity than the currently studied type strain, Pyrococcus furiosus. Specific precursors of products with industrial interest will be used as substrate. The specific bio-reduction of such substrates will be assayed by standard chromatographic means (HPLC);

The three available pyrococcal genome sequences (P.furiosus, P.horikoshii, P.abyssi) will be screened for biocatalysts that are potentially involved in bioreductions, by using sophisticated bio-information tools to predict enzyme function and to predict relevant metabolic pathways. A subsequent analysis of the expression of the obtained group of enzymes with a proteome approach under different conditions is expected to reveal insight in their metabolic role.

Optimization of the bioreduction process, in vivo and in vitro

Improvement of the in vivo production process of selected Thermococcales strains by a factorial analysis of physical parameters (temperature, pH, etc.), as well as by metabolic engineering of a Pyrococcus host, by introducing or deleting certain key enzymes at some stage of the bioreductive pathway. For this purpose, the recently developed pyrococcal genetic system (see above) will be applied, if necessary after optimization, to allow for directed overexpression or gene disruption;

Optimization of the in vitro process, by using a variety of substrates, biocatalysts, and by a recently developed NAD(P)H-regeneration system, based on hyperthermophilic, extremely stable components; although technically difficult, this system may give rise to enhanced bioconversion specificity;

(3) Establishment of a highly efficient production system of the desired, valuable fine chemicals, both in vivo and in vitro. This should result in improved hyperthermophile fermentation, aiming at high cell densities at pilot scale, using a recently established dialysis-based fed-batch system for Pyrococcus, in which growth-inhibiting components are continuously removed from the reactor. Currently, cell densities of 3 gram biomass (wet weight) per liter are obtained, but higher yields are certainly envisaged. In the course of this project limiting factors will be identified, aiming at further optimization of the cell density. In combination with a novel, on-line method for product recovery, this will result in efficient production of selected fine chemicals.

Expected achievements of PYRED project

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 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.