Bioreduction of Carbonyl Compounds to Chiral Alcohols by Whole Yeast Cells: Process Optimisation, Strain Design and Non-Conventional Yeast Screening.
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/466569
- author
- Katz, Michael LU
- supervisor
- opponent
-
- Professor Kula, Maria-Regina, Munchen, Germany
- organization
- publishing date
- 2003
- 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}},
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}},
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}},
}