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

Katz, Michael LU (2003)
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... (More)
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. (Less)
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author
supervisor
opponent
  • Professor Kula, Maria-Regina, Munchen, Germany
organization
publishing date
type
Thesis
publication status
published
subject
keywords
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
pages
168 pages
publisher
Secretary, Applied Microbiology Lund University
defense location
Lecture Hall B, Chemical Center, Sölvegatan 39, Lund Institute of Technology
defense date
2004-01-12 10:30:00
language
English
LU publication?
yes
additional info
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
id
0cd70227-7d9f-44d9-9c5d-c02db739c533 (old id 466569)
date added to LUP
2016-04-04 11:45:01
date last changed
2018-11-21 21:06:57
@phdthesis{0cd70227-7d9f-44d9-9c5d-c02db739c533,
  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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.},
  author       = {Katz, Michael},
  language     = {eng},
  publisher    = {Secretary, Applied Microbiology Lund University},
  school       = {Lund University},
  title        = {Bioreduction of Carbonyl Compounds to Chiral Alcohols by Whole Yeast Cells: Process Optimisation, Strain Design and Non-Conventional Yeast Screening.},
  year         = {2003},
}