Bioreduction of Carbonyl Compounds to Chiral Alcohols by Whole Yeast Cells: Process Optimisation, Strain Design and Non-Conventional Yeast Screening

Abstract: Chiral building blocks are needed for the production of drugs and fine chemicals, which requires the use of several synthetic routes to produce a specific enantiomer of interest. One promising approach to introduce chirality into molecules is the stereo-selective whole cell bioreduction of carbonyl compounds or ketones to the corresponding chiral alcohols. The aim of this thesis was to develop efficient whole cell bioreduction processes with yeast as a biocatalyst. Three parallel and complementary ways were investigated: (i) the optimisation of the process such as medium and reactor engineering, (ii) the optimisation of the Saccharomyces cerevisiae biocatalyst via genetic engineering, and (iii) the screening of non-conventional yeasts with novel properties stemming from natural diversity. The reduction of the bicyclic diketone, bicyclo[2.2.2]octane-2,6-dione or BCO2,6D, was used as model reaction since the reduced product is a starting material of interest in organic synthesis. Saccharomyces cerevisiae cells convert BCO2,6D to the corresponding ketoalcohol, (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octane-2-one or endo-alcohol, at high optical purity using NADPH as co-factor. Process parameters, such as the presence of a co-substrate (glucose or ethanol), initial bicyclic diketone concentration, ratio of yeast to glucose, medium composition and pH were shown to affect the whole cell bioreduction. The co-substrate yield (formed chiral ketoalcohol per consumed glucose co-substrate) was further enhanced by genetically engineered S. cerevisiae strains with a reduced phosphoglucose isomerase activity or with the alcohol dehydrogenase gene deleted. To identify the reductases involved in the reduction of BCO2,6D a spectrophotometric screening method was developed. This method quickly identified cytosolic reductases active against specific carbonyl compounds (diacetyl, ethyl acetoacetate and BCO2,6D) by comparing the cytosolic activities in a control strain to the activity in strains having a single reductase gene deleted or overexpressed. Five reductases encoded by YOR120w, YDR368w, YMR226c, YGL157w and YGL039w accepted BCO2,6D as substrate and produced (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octane-2-one. The reductases encoded by YOR120w, YDR368w and YMR226c were purified and characterised. The overexpression of BCO2,6D-reductases in S. cerevisiae under a strong constitutive promoter generated strains with increased reduction rates and enabled a process with lowered co-substrate yield. Further decrease in co-substrate yield was achieved by combining high reductase activity with low phosphoglucose isomerase activity. Non-conventional yeasts (non S. cerevisiae yeasts) were also screened for BCO2,6D reduction. It was shown that Candida species generated another diastereomer ketoalcohol, (1S,4R,6S)-6-hydroxybicyclo[2.2.2]octane-2-one or exo-alcohol, as major product from BCO2,6D. Candida tropicalis was identified as the best producer. The reductase responsible for exo-alcohol formation, that was found to be located in the membrane fraction of C. tropicalis, should enable the development of yeast catalysts for the production of a different diastereomer at high yield and optical purity.