| 1. |
- Carlquist, Magnus, et al.
(författare)
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Rationalisation of the substrate concentration dependent diastereoselectivity of a Saccharomyces cerevisiae short-chain dehydrogenase
- 2007
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Ingår i: Tetrahedron: Asymmetry. - : Elsevier BV. - 0957-4166. ; 18:21, s. 2554-2556
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Tidskriftsartikel (refereegranskat)abstract
- The diastereoselectivity of the carbonyl reduction of bicyclo[2.2.2]octane-2,6-dione, catalysed by the purified yeast cytosolic short-chain dehydrogenase Ymr226cp, was shown to be substrate concentration dependent. The changing selectivity was attributed to two distinct binding configurations of the substrate in the active site, each yielding a distinct hydroxy ketone diastereomer. By applying individual KM and Vmax values for each binding configuration, the concentration dependence could be modelled with Michaelis–Menten kinetics and the apparent KM and Vmax values for the generation of each diastereomer determined. This is to the best of our knowledge the first rationalisation of a substrate dependent stereoselectivity for a pro-chiral substrate with an isolated enzyme.
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| 2. |
- Friberg, Annika, et al.
(författare)
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Efficient bioreduction of bicyclo[2.2.2]octane-2,5-dione and bicyclo[2.2.2]oct-7-ene-2,5-dione by genetically engineered Saccharomyces cerevisiae
- 2006
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 4:11, s. 2304-2312
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Tidskriftsartikel (refereegranskat)abstract
- A screening of non-conventional yeast species and several Saccharomyces cerevisiae ( baker's yeast) strains overexpressing known carbonyl reductases revealed the S. cerevisiae reductase encoded by YMR226c as highly efficient for the reduction of the diketones 1 and 2 to their corresponding hydroxyketones 3 - 6 ( Scheme 1) in excellent enantiomeric excesses. Bioreduction of 1 using the genetically engineered yeast TMB4100, overexpressing YMR226c, resulted in > 99% ee for hydroxyketone (+)- 4 and 84 - 98% ee for (-)- 3, depending on the degree of conversion. Baker's yeast reduction of diketone 2 resulted in > 98% ee for the hydroxyketones (+)- 5 and (+)- 6. However, TMB4100 led to significantly higher conversion rates ( over 40 fold faster) and also a minor improvement of the enantiomeric excesses (> 99%).
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| 3. |
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| 4. |
- Johanson, Ted
(författare)
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Engineered Yeast as Biocatalyst for Stereoselective reductions of Dicarbonyl Compounds
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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 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.
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| 5. |
- Johanson, Ted, et al.
(författare)
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Identification of a Candida sp reductase behind bicyclic exo-alcohol production
- 2009
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 59:4, s. 286-291
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Tidskriftsartikel (refereegranskat)abstract
- Stereoselective baker's yeast-catalysed bioreduction of bicyclo [2.2.2]octane-2.6-dione generates (1R, 4S, 6S)-6-hydroxy-bicyclo [2.2.2]octane-2-one (endo-alcohol) with high enantiomeric and diastereomeric excess. In contrast, whole cells and crude membrane fractions of Candida sp. have been reported to produce the unusual (I R, 4S, 6S)-diastereomer (exo-alcohol) as a major product. Previous in silica screening has identified seven membrane or membrane-bound reductases in C albicans as candidates for the exoactivity. In this work, purification of the corresponding exo-reductase(s) as well as the heterologous cloning of the seven candidate genes was attempted in C tropicalis. The overexpression of IPF4033 (AYR1) gene generated an increased exo-to-endo ratio and exo-alcohol production in whole cells and membranes of C tropicalis. In addition, a slight increased exo-to-endo ratio was observed when overexpressing IPF4033 in S. cerevisiae, although the reduction rate and exo-to-endo ratio were several fold lower compared to those obtained with C. tropicalis. (C) 2008 Elsevier B.V. All rights reserved.
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| 6. |
- Johanson, Ted, et al.
(författare)
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Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone
- 2008
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 77:5, s. 1111-1118
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Tidskriftsartikel (refereegranskat)abstract
- Reduction of bicyclo[2.2.2]octane-2,6-dione to (1R, 4S, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one by whole cells of Saccharomyces cerevisiae was improved using an engineered recombinant strain and process design. The substrate inhibition followed a Han-Levenspiel model showing an effective concentration window between 12 and 22 g/l, in which the activity was kept above 95%. Yeast growth stage, substrate concentration and a stable pH were shown to be important parameters for effective conversion. The over-expression of the reductase gene YDR368w significantly improved diastereoselectivity compared to previously reported results. Using strain TMB4110 expressing YDR368w in batch reduction with pH control, complete conversion of 40 g/l (290 mM) substrate was achieved with 97% diastereomeric excess (de) and >99 enantiomeric excess (ee), allowing isolation of the optically pure ketoalcohol in 84% yield.
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| 7. |
- Johanson, Ted, et al.
(författare)
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Strain engineering for stereoselective bioreduction of dicarbonyl compounds by yeast reductases
- 2005
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1364 .- 1567-1356. ; 5:6-7, s. 513-525
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Forskningsöversikt (refereegranskat)abstract
- Pure chiral molecules are needed in the pharmaceutical and chemical industry as intermediates for the production of drugs or fine chemicals. Microorganisms represent an attractive alternative to chemical synthesis since they have the potential to generate single stereoisomers in high enantiomeric excess (ee). The baker's yeast Saccharomyces cerevisiae can notably reduce dicarbonyl compounds (in particular alpha- and beta-diketones and keto esters) to chiral alcohols with high ee. However, products are formed at a low rate. Moreover, large amounts of co-substrate are required for the regeneration of NADPH that is the preferred co-factor in almost all the known dicarbonyl reductions. Traditionally, better ee, reduction rate and product titre have been achieved via process engineering. The advent of recombinant DNA technology provides an alternative strategy to improve productivity and yield by strain engineering. This review discusses two aspects of strain engineering: (i) the generation of strains with higher reductase activity towards dicarbonyl compounds and (ii) the optimisation of co-substrate utilisation for NADPH cofactor regeneration. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
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| 8. |
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