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FP6

DATAGENOM Informe resumido

Project ID: 503017
Financiado con arreglo a: FP6-LIFESCIHEALTH
País: Iceland

Final Report Summary - DATAGENOM (Post-genomic datamining of enzymes for the synthesis of chiral pharmaceutical intermediates)

The key focus of the DATAGENOM project was on exploiting the recent explosive growth of genomic data that has been made available from the massive Deoxyribonucleic acid (DNA) sequencing programmes. As large-scale DNA sequencing of various life forms has become easier and less expensive, the available data has since long surpassed the 'wet chemistry' capabilities to clone the found genes and to study the properties of the expressed proteins. The key targets in DATAGENOM were three classes of enzymes, useful in the industry for making the correct versions (chiral enantiomers) of intermediate chemical compounds, which can then serve as building blocks for drug making.

Chirality is a key factor in the efficacy of many drugs and the production of single enantiomers of chiral intermediates has therefore become increasingly important. Biocatalysis offers high enantioselectivity and regioselectivity in chiral synthesis through enzyme-catalysed reactions and thus has an important advantage over chemical synthesis.

The DATAGENOM project covered the fields of bioinformatic genome analysis, cloning, expression, enzyme production, and screening and protein engineering, to the production of chiralbiomolecules. The design of the project used the broad funnel approach, starting with innovative data-mining and processing of a large number of genes to ensure a high flow-through in the process and rational selection of the best enzyme candidates.

A highly regulated L-rhamnose-inducible E. coli system for expression was improved and the optimised vector was used with success. Temperature-regulated expression vectors were constructed, giving great improvements and better scale-up. A Thermus thermophilus thermophilic system was improved and another thermophilic Rhodothermus marinus shuttle vector expression system was established with stable markers, promoters and reporter genes.

Synthesis of (S)-Dapoxetine was developed with a good yield. Kinetic resolution of chiral building blocks that are possible intermediates of interesting drugs was successful. Statistical optimised reactions using oxidase biocatalysts yielded specific benefits applied to oxidase biotransformations to prepare two unnatural amino acids. Some AAO enzymes moved to 10 L scale and stabilised whole cells of D-AAO to pilot industrial manufacturing. Formulation of improved oxidases lowered the overall biomass by 8- to 20-fold, greatly improving productivity. This manufacturing process for L-2-aminobutyric acid and two further unnatural L-amino acids was evaluated at 100-1000 L scale, en route to full commercial manufacturing.

Novel and improved cofactor regeneration enzymes were formulated as whole cell biocatalysts. Three new ADHs are already sold commercially for the bioproduction of speciality compounds on 5-10 L scale.

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Contacto

Jakob K. KRISTJANSSON
Tel.: +354-570-7900
Fax: +354-570-7901
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