| 1. |
- Katz, Michael, et al.
(författare)
-
An improved stereoselective reduction of a bicyclic diketone by Saccharomyces cerevisiae combining process optimization and strain engineering
- 2002
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 59:6, s. 641-648
-
Tidskriftsartikel (refereegranskat)abstract
- The stereoselective reduction of the bicyclic diketone bicyclo[2.2.2]octane-2,6-dione, to the ketoalcohol (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octane-2-one, was used as a model reduction to optimize parameters involved in NADPH-dependent reductions in Saccharomyces cerevisiae with glucose as co-substrate. The co-substrate yield (ketoalcohol formed/glucose consumed) was affected by the initial concentration of bicyclic diketone, the ratio of yeast to glucose, the medium composition, and the pH. The reduction of 5 g l(-1) bicyclic diketone was completed in less than 20 h in complex medium (pH 5.5) under oxygen limitation with an initial concentration of 200 g l(-1) glucose and 5 g l(-1) yeast. The co-substrate yield was further enhanced by genetically engineered strains with reduced phosphoglucose isomerase activity and with the gene encoding alcohol dehydrogenase deleted. Co-substrate yields were increased 2.3-fold and 2.4-fold, respectively, in these strains.
|
|
| 2. |
- Katz, Michael
(författare)
-
Bioreduction of Carbonyl Compounds to Chiral Alcohols by Whole Yeast Cells: Process Optimisation, Strain Design and Non-Conventional Yeast Screening.
- 2003
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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 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.
|
|
| 3. |
- Katz, Michael
(författare)
-
Bioreduction of carbonyl compounds to chiral alcohols by whole yeasts cells: process optimisation, strain design and non-conventional yeast screening
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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 nonconventional 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 activity 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 BCO2,6Dreductases 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.
|
|
| 4. |
- Katz, Michael, et al.
(författare)
-
Efficient Anaerobic Whole Cell Stereoselective Bioreduction with Recombinant Saccharomyces cerevisiae.
- 2003
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 84:5, s. 573-582
-
Tidskriftsartikel (refereegranskat)abstract
- In this study we investigate the NADPH-dependent stereoselective reduction of the bicyclic diketone bicyclo[2.2.2]octane-2,6-dione (BCO2,6D) to the chiral ketoalcohol (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octane-2-one (BCO2one6ol). Our aim was to develop a whole cell batch process for reduction of carbonyl substrates with (i) a high cosubstrate yield (formed product/consumed cosubstrate) and (ii) a high conversion rate under anaerobic conditions with Saccharomyces cerevisiae as biocatalyst and glucose as cosubstrate. Five open reading frames (ORFs), YMR226c, YDR368w, YOR120w, YGL157w, and YGL039w, encoding reductases involved in the conversion of BCO2,6D were identified using cell-free extract from strains belonging to the ExClone collection (yeast ORF expression clones; ResGen, Invitrogen Corp., UK). We report the one-step purification and characterization of three major BCO2,6D reductases, YMR226cp, YDR368wp (YPR1p), and YOR120wp (GCY1p). The reductases were overexpressed under a strong constitutive promoter and the impact on cosubstrate yield, conversion time, glucose consumption rate, and reduction rate was investigated when reductases were overexpressed either alone or in combination with low phosphoglucose isomerase activity (encoded by YBR196c). Combining overexpression of BCO2,6D reductase with reduced glycolytic rate (low phosphoglucose isomerase activity) offers a fast whole cell stereoselective bioreduction system useful for facilitated anaerobic batch conversions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 573-582, 2003.
|
|
| 5. |
- Katz, Michael, et al.
(författare)
-
Mild detergent treatment of Candida tropicalis reveals a NADPH-dependent reductase in the crude membrane fraction, which enables the production of pure bicyclic exo-alcohol
- 2004
-
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 21:15, s. 1253-1267
-
Tidskriftsartikel (refereegranskat)abstract
- This study demonstrated the occurrence of a NADPH-dependent exo-alcohol reductase in the crude membrane fraction of Candida tropicalis. Cytosolic endo-alcohol reductase activity could be separated from the membrane-bound exo-alcohol activity by means of detergent treatment, enabling the preparation of pure exo-alcohol via the enzymatic conversion of the bicyclic diketone, bicyclo[2.2.2]octane-2,6-dione. The exo-alcohol reductase is, to our knowledge, the first membrane-bound NADPH-dependent reductase accepting a xenobiotic carbonyl substrate that was not a steroid. When C. tropicalis was grown on D-sorbitol, a two-fold increase in the exo-reductase activity was observed as compared to when grown on glucose. An in silico comparison at the protein level between putative xenobiotic carbonyl reductases in Candida albicans, C. tropicalis and Saccharomyces cerevisiae was performed to explain why Candida species are often encountered when screening yeasts for novel stereoselective reduction properties. C. albicans contained more reductases with the potential to reduce xenobiotic carbonyl compounds than did S. cerevisiae. C. tropicalis had many membrane-bound reductases (predicted with the bioinformatics program, TMHMM), some of which had no counterpart in the two other organisms. The exo-reductase is suspected to be either a -hydroxysteroid dehydrogenase or a polyol dehydrogenase from either the short chain dehydrogenase family or the dihydroflavonol reductase family.
|
|
| 6. |
- Katz, Michael, et al.
(författare)
-
Screening of two complementary collections of Saccharomyces cerevisiae to identify enzymes involved in stereo-selective reductions of specific carbonyl compounds: an alternative to protein purification.
- 2003
-
Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 33:2-3, s. 163-172
-
Tidskriftsartikel (refereegranskat)abstract
- Two collections of Saccharomyces cerevisiae strains, having open reading frames (ORFs) either overexpressed or deleted, were screened to identify cytosolic NADPH-dependent reductases involved in stereo-selective reductions of specific carbonyl compounds. As model compounds diacetyl (diketone) and ethyl acetoacetate (3-oxo ester) were used. The reductases encoded by YBR149w, YDR368w, YMR226c and YOR120w were found to reduce diacetyl and YOL151w, YHR104w, YGL157w, YOR120w and YDR368w reduced ethyl acetoacetate. We cloned YBR149w, YDR368w and YMR226c using a vector with the strong constitutive GPD promotor and investigated the encoded reductases with respect to reduction of diacetyl. YBR149w, YDR368w and YMR226c were shown to encode (S)-specific diacetyl reductases capable of further reducing racemic acetoin to butanediol. Furthermore, we demonstrated that the purified (S)-diacetyl reductase (EC 1.2.1.56) most likely is the Image-arabinose dehydrogenase encoded by YBR149w (Ara1p).
|
|
