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Engineered Yeast as Biocatalyst for Stereoselective reductions of Dicarbonyl Compounds

Johanson, Ted LU (2007)
Abstract
Chiral building blocks are needed in the chemical and pharmaceutical industries for the production of fine chemicals and pharmaceuticals. The utilisation of microorganisms and enzymes to perform stereoselective reductions of carbonyl compounds is an efficient and widely applied method for the generation of chiral molecules.



The aim of this thesis was to develop and improve yeast-catalysed bioreduction processes by yeast strain engineering and process engineering, the main aim being the reduction of xenobiotic bicyclic diketones. The hydroxy ketone products obtained constitute interesting building blocks for the synthesis of chiral chemical catalysts and Taxol analogues.



An unusual... (More)
Chiral building blocks are needed in the chemical and pharmaceutical industries for the production of fine chemicals and pharmaceuticals. The utilisation of microorganisms and enzymes to perform stereoselective reductions of carbonyl compounds is an efficient and widely applied method for the generation of chiral molecules.



The aim of this thesis was to develop and improve yeast-catalysed bioreduction processes by yeast strain engineering and process engineering, the main aim being the reduction of xenobiotic bicyclic diketones. The hydroxy ketone products obtained constitute interesting building blocks for the synthesis of chiral chemical catalysts and Taxol analogues.



An unusual bicyclo[2.2.2]octane-2,6-dione (BCO2,6D) activity generating (1S ,4R, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one (exo-alcohol) was investigated in Candida tropicalis and C. albicans. These yeasts produce a mixture of the exo-alcohol and its (1R , 4S, 6S)-diastereomer, the endo-alcohol. The exo-alcohol was observed to be predominantly produced in the membrane fraction of detergent-treated cells and the activity was therefore suspected to be membrane-associated. Purification of the exoalcohol-generating enzyme from C. tropicalis was unsuccessful, as the activity was weak and diminished rapidly.



An in vitro screening identified seven putative exo-reductases in C. albicans. A C. albicans expression plasmid was used for heterologous expression of the candidate genes in C. tropicalis. Five of the seven genes were expressed in C. tropicalis, one of which (AYR1) produced an increased exo-to-endo ratio in both whole cells and crude membranes. The S. cerevisiae homologue of the putative exo-generating gene was cloned in C. tropicalis, but no increase in exo-to-endo ratio was detected. Both genes were subsequently cloned and expressed in S. cerevisiae and a small increase in exo-to-endo ratio was detected in strains expressing either gene. However, exo-to-endo ratio and substrate conversion rate were several folds lower compared to C. tropicalis.



Improved reduction of the prochiral BCO2,6D to endo-alcohol was achieved using a recombinant S. cerevisiae strain and process engineering. Substrate inhibition was modelled using a Han-Levenspiel kinetic model and an efficient concentration window was found in which the activity was maintained above 95%. Growth stage of the yeast, substrate concentration and a stable pH were shown to be important parameters for efficient conversion. By exchanging the reductase gene YMR226c for YPR1, the diastereomeric excess was significantly improved. Complete conversion of 40 g/l substrate was achived with high stereoselectivity, allowing facile isolation of the optically pure hydroxy ketone.



A reductase for the reduction of the racemic bicyclic diketones, bicyclo[2.2.2]octane-2,5-one and bicyclo[2.2.2]oct-7-ene-2,5-dione, was identified by screening a small collection of recombinant S. cerevisiae strains, baker's yeast and non-conventional yeasts. S. cerevisiae expressing YMR226c was found to convert both substrates with high efficiency and stereoselectivity. Production and isolation of the corresponding hydroxy ketones was achieved on a preparative scale using a semi-fed-batch approach.



A concentration-dependent diastereoselectivity in the reduction of BCO2,6D was discovered for the S. cerevisiae reductase encoded by YMR226c. The protein was purified and the selectivity investigated at different substrate concentrations. The concentration dependence was explained by the existence of two substrate-binding configurations at the active site, with separate affinity (KM) and maximum activity (Vmax), each configuration yielding a distinct hydroxy ketone diastereomer. Furthermore, the concentration dependence observed could be modelled with Michaelis-Menten reaction kinetics and the apparent Km and Vmax values for the generation of each diastereomer obtained. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Dr. Bringer-Meyer, Stephanie, Institute of Biotechnology 1, Forschungzentrum Jülich GmbH, Jülich, Germany
organization
publishing date
type
Thesis
publication status
published
subject
keywords
chiral, screening, process optimisation, metabolic engineering, dicarbonyl, biocatalysis, yeast, bioreduction, Biotechnology, Bioteknik
pages
123 pages
publisher
Applied Microbiology (LTH)
defense location
Hall B, Center of Chemistry and Chemical Engineering, Getingevägen 60, Lund
defense date
2007-10-25 10:30
ISBN
978-91-6287297-7
language
English
LU publication?
yes
id
600062dd-3ceb-49dc-b582-6d80447ce05f (old id 599049)
date added to LUP
2007-11-13 08:05:41
date last changed
2016-09-19 08:45:13
@misc{600062dd-3ceb-49dc-b582-6d80447ce05f,
  abstract     = {Chiral building blocks are needed in the chemical and pharmaceutical industries for the production of fine chemicals and pharmaceuticals. The utilisation of microorganisms and enzymes to perform stereoselective reductions of carbonyl compounds is an efficient and widely applied method for the generation of chiral molecules.<br/><br>
<br/><br>
The aim of this thesis was to develop and improve yeast-catalysed bioreduction processes by yeast strain engineering and process engineering, the main aim being the reduction of xenobiotic bicyclic diketones. The hydroxy ketone products obtained constitute interesting building blocks for the synthesis of chiral chemical catalysts and Taxol analogues.<br/><br>
<br/><br>
An unusual bicyclo[2.2.2]octane-2,6-dione (BCO2,6D) activity generating (1S ,4R, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one (exo-alcohol) was investigated in Candida tropicalis and C. albicans. These yeasts produce a mixture of the exo-alcohol and its (1R , 4S, 6S)-diastereomer, the endo-alcohol. The exo-alcohol was observed to be predominantly produced in the membrane fraction of detergent-treated cells and the activity was therefore suspected to be membrane-associated. Purification of the exoalcohol-generating enzyme from C. tropicalis was unsuccessful, as the activity was weak and diminished rapidly.<br/><br>
<br/><br>
An in vitro screening identified seven putative exo-reductases in C. albicans. A C. albicans expression plasmid was used for heterologous expression of the candidate genes in C. tropicalis. Five of the seven genes were expressed in C. tropicalis, one of which (AYR1) produced an increased exo-to-endo ratio in both whole cells and crude membranes. The S. cerevisiae homologue of the putative exo-generating gene was cloned in C. tropicalis, but no increase in exo-to-endo ratio was detected. Both genes were subsequently cloned and expressed in S. cerevisiae and a small increase in exo-to-endo ratio was detected in strains expressing either gene. However, exo-to-endo ratio and substrate conversion rate were several folds lower compared to C. tropicalis.<br/><br>
<br/><br>
Improved reduction of the prochiral BCO2,6D to endo-alcohol was achieved using a recombinant S. cerevisiae strain and process engineering. Substrate inhibition was modelled using a Han-Levenspiel kinetic model and an efficient concentration window was found in which the activity was maintained above 95%. Growth stage of the yeast, substrate concentration and a stable pH were shown to be important parameters for efficient conversion. By exchanging the reductase gene YMR226c for YPR1, the diastereomeric excess was significantly improved. Complete conversion of 40 g/l substrate was achived with high stereoselectivity, allowing facile isolation of the optically pure hydroxy ketone.<br/><br>
<br/><br>
A reductase for the reduction of the racemic bicyclic diketones, bicyclo[2.2.2]octane-2,5-one and bicyclo[2.2.2]oct-7-ene-2,5-dione, was identified by screening a small collection of recombinant S. cerevisiae strains, baker's yeast and non-conventional yeasts. S. cerevisiae expressing YMR226c was found to convert both substrates with high efficiency and stereoselectivity. Production and isolation of the corresponding hydroxy ketones was achieved on a preparative scale using a semi-fed-batch approach.<br/><br>
<br/><br>
A concentration-dependent diastereoselectivity in the reduction of BCO2,6D was discovered for the S. cerevisiae reductase encoded by YMR226c. The protein was purified and the selectivity investigated at different substrate concentrations. The concentration dependence was explained by the existence of two substrate-binding configurations at the active site, with separate affinity (KM) and maximum activity (Vmax), each configuration yielding a distinct hydroxy ketone diastereomer. Furthermore, the concentration dependence observed could be modelled with Michaelis-Menten reaction kinetics and the apparent Km and Vmax values for the generation of each diastereomer obtained.},
  author       = {Johanson, Ted},
  isbn         = {978-91-6287297-7},
  keyword      = {chiral,screening,process optimisation,metabolic engineering,dicarbonyl,biocatalysis,yeast,bioreduction,Biotechnology,Bioteknik},
  language     = {eng},
  pages        = {123},
  publisher    = {ARRAY(0x932b158)},
  title        = {Engineered Yeast as Biocatalyst for Stereoselective reductions of Dicarbonyl Compounds},
  year         = {2007},
}