Enzymatic Reduction of Ketones

Sammanfattning: Methods have been studied for the production of chiral alcohols, which are of importance in organic synthesis of pharmaceuticals, agro-chemicals and fragrances, etc., through the enzymatic or microbial reduction of ketones. Classical reaction engineering was combined with genetic engineering to improve the production of chiral alcohols and kinetic resolution of racemic ketones with high enantiomeric and diastereomeric purity, productivity, product yield and co-substrate yield. Reductases originating from baker’s yeast, Saccharomyces cerevisiae, were over-expressed in either S. cerevisiae or in the Gram-negative bacterium Escherichia coli. This increased reaction rates and selectivity towards the substrates for whole-cell reduction. Also, several mutants of S. cerevisiae and E. coli with altered metabolic carbon fluxes were constructed and compared regarding their ability to supply the aldo-keto reductase YPR1 with the essential coenzyme NADPH at a non-limiting rate. In order not to limit the reductase, S. cerevisiae required a higher NADPH regeneration rate, which was achieved by directing the carbon flux through the main NADPH-generating pathway, the pentose–phosphate pathway. However, this created a disturbance in the redox balance, which led to the need for fine-tuning of the glucose concentration during biomass production. The same modifications of the central carbon metabolism in E. coli did not increase the reaction rate. In fact, the highest initial reaction rate, productivity and co-substrate yield were obtained with the strain only over-expressing YPR1. This was even higher than for the best S. cerevisiae strain. However, the highest degree of conversion was obtained with S. cerevisiae, because of its significantly greater robustness. In fact, S. cerevisiae has potential to be recycled for sequential bioreductions. The second part of this work involved the study of the inhibition of human reductases, with the aim of reducing the side-effects of anthracycline antibiotics, a group of drugs commonly used to treat a wide variety of cancer diseases. Anthracyclines are reduced in the body to less potent and cardiotoxic alcohol metabolites. To reduce the side-effects of anthracyclines, reductase inhibitors could be administered concomitantly to prevent metabolite formation. Important functionalities for binding of flavonoid compounds to human carbonyl reductase 1 (CBR1) were identified by determining IC50 values for 11 different flavonoids and performing computational docking experiments with four of the best inhibitors. The knowledge acquired on the binding of different flavonoids to the catalytic site of CBR1 is of importance for the design of an optimal inhibitor.