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

Michael Katz

Research output: ThesisDoctoral Thesis (compilation)


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.
Original languageEnglish
Awarding Institution
  • Applied Microbiology
  • [unknown], [unknown], Supervisor, External person
Award date2004 Jan 12
Publication statusPublished - 2003

Bibliographical note

Defence details

Date: 2004-01-12
Time: 10:30
Place: Lecture Hall B, Chemical Center, Sölvegatan 39, Lund Institute of Technology

External reviewer(s)

Name: Kula, Maria-Regina
Title: Professor
Affiliation: Munchen, Germany


Article: Katz M, Sarvary I, Frejd T, Hahn-Hägerdal B, Gorwa-Grauslund MF (2002)An improved stereoselective reduction of a bicyclic diketone by Saccharomyces cerevisiae combining process optimization and strain engineeringApplied Microbiology and Biotechnology 2002; 59: 641-648

Article: Katz M, Hahn-Hägerdal B, Gorwa-Grauslund MF (2003)Screening of two complementary collections of Saccharomyces cerevisiae to identify enzymes involved in stereo-selective reduction of specific carbonyl compounds: an alternative to protein purification.Enzyme and Microbial Technology 2003; 33: 163-172

Article: Katz M, Frejd T, Hahn-Hägerdal B , Gorwa-Grauslund MF (2003) Efficient anaerobic whole cell stereo-selective bioreduction with recombinant Saccharomyces cerevisiae.Biotechnology and Bioengineering 2003; 84: 573-582

Article: Botes AL, Harvig D, van Dyk MS, Sarvary I, Frejd T, Katz M, Hahn-Hägerdal B, Gorwa-Grauslund MF (2002) Screening of yeast species for the stereo-selective reduction of bicyclo[2.2.2]octane-2,6-dione. Journal of the Chemical Society, Perkin Transactions 1 2002; 1111-1114

Article: Katz M, Johanson T, Gorwa-Grauslund MFMild detergent treatment of Candida tropicalis reveals a NADPH dependent reductase in the crude membrane fraction, which enables the production of pure bicyclic exo alcohol.Submitted

Subject classification (UKÄ)

  • Industrial Biotechnology


  • bicyclic diketone
  • carbonyl
  • yeast
  • candida
  • cerevisiae
  • reductase
  • ketone
  • reduction
  • whole cell
  • YMR226c
  • YDR368w
  • Microbiology
  • bacteriology
  • virology
  • mycology
  • Biokemisk teknik
  • Biochemical technology
  • mykologi
  • virologi
  • Mikrobiologi
  • bakteriologi


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