| 141. |
- Katz, Michael
(författare)
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Bioreduction of carbonyl compounds to chiral alcohols by whole yeasts cells: process optimisation, strain design and non-conventional yeast screening
- 2004
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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.
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| 142. |
- Katz, Michael, et al.
(författare)
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Efficient Anaerobic Whole Cell Stereoselective Bioreduction with Recombinant Saccharomyces cerevisiae.
- 2003
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 84:5, s. 573-582
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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.
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| 143. |
- Katz, Michael, et al.
(författare)
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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
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 21:15, s. 1253-1267
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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.
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| 144. |
- Katz, Michael, et al.
(författare)
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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
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Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 33:2-3, s. 163-172
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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).
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| 145. |
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| 146. |
- Musunuru, Kiran, et al.
(författare)
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From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus
- 2010
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 466:7307, s. 2-714
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Tidskriftsartikel (refereegranskat)abstract
- Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.
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| 147. |
- Nestor, Colm, 1977-, et al.
(författare)
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Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes
- 2012
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Ingår i: Genome Research. - : Cold Spring Harbor Laboratory Press (CSHL). - 1088-9051 .- 1549-5469. ; 22:3, s. 467-477
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Tidskriftsartikel (refereegranskat)abstract
- The discovery of substantial amounts of 5-hydroxymethylcytosine (5hmC), formed by the oxidation of 5-methylcytosine (5mC), in various mouse tissues and human embryonic stem (ES) cells has necessitated a reevaluation of our knowledge of 5mC/5hmC patterns and functions in mammalian cells. Here, we investigate the tissue specificity of both the global levels and locus-specific distribution of 5hmC in several human tissues and cell lines. We find that global 5hmC content of normal human tissues is highly variable, does not correlate with global 5mC content, and decreases rapidly as cells from normal tissue adapt to cell culture. Using tiling microarrays to map 5hmC levels in DNA from normal human tissues, we find that 5hmC patterns are tissue specific; unsupervised hierarchical clustering based solely on 5hmC patterns groups independent biological samples by tissue type. Moreover, in agreement with previous studies, we find 5hmC associated primarily, but not exclusively, with the body of transcribed genes, and that within these genes 5hmC levels are positively correlated with transcription levels. However, using quantitative 5hmC-qPCR, we find that the absolute levels of 5hmC for any given gene are primarily determined by tissue type, gene expression having a secondary influence on 5hmC levels. That is, a gene transcribed at a similar level in several different tissues may have vastly different levels of 5hmC (>20-fold) dependent on tissue type. Our findings highlight tissue type as a major modifier of 5hmC levels in expressed genes and emphasize the importance of using quantitative analyses in the study of 5hmC levels.
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| 148. |
- Ng, Bobby G, et al.
(författare)
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ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.
- 2016
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Ingår i: Human Mutation. - : Hindawi Limited. - 1059-7794.
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Tidskriftsartikel (refereegranskat)abstract
- Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over one hundred genes leading to impaired protein or lipid glycosylation. ALG1 encodes a β1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate (DLO) required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date thirteen mutations in eighteen patients from fourteen families have been described with varying degrees of clinical severity. We identified and characterized thirty-nine previously unreported cases of ALG1-CDG from thirty-two families and add twenty-six new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2 , was seen in all twenty-seven patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder. This article is protected by copyright. All rights reserved.
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| 149. |
- O Reese, Matthew, et al.
(författare)
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Consensus stability testing protocols for organic photovoltaic materials and devices
- 2011
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Ingår i: SOLAR ENERGY MATERIALS AND SOLAR CELLS. - : Elsevier Science B.V., Amsterdam.. - 0927-0248. ; 95:5, s. 1253-1267
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Tidskriftsartikel (refereegranskat)abstract
- Procedures for testing organic solar cell devices and modules with respect to stability and operational lifetime are described. The descriptions represent a consensus of the discussion and conclusions reached during the first 3 years of the international summit on OPV stability (ISOS). The procedures include directions for shelf life testing, outdoor testing, laboratory weathering testing and thermal cycling testing, as well as guidelines for reporting data. These procedures are not meant to be qualification tests, but rather generally agreed test conditions and practices to allow ready comparison between laboratories and to help improving the reliability of reported values. Failure mechanisms and detailed degradation mechanisms are not covered in this report.
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| 150. |
- Paci, Chris, et al.
(författare)
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Northern Science and Research : Postsecondary Perspectives in the Northwest Territories
- 2008
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Ingår i: Journal of Northern Studies. - Umeå : Umeå University & The Royal Skyttean Society. - 1654-5915. ; :1, s. 23-52
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Tidskriftsartikel (refereegranskat)abstract
- The International Polar Year (IPY) provides an opportunity to reflect on Northern science and research. For all Canadians, science and research should contribute to living a good life. A good life includes successfully making sense of the world within local contexts, sharing this knowledge beyond the immediate community and reconciling it with knowledge held by outsiders. Northern science and research are inherent in Traditional Dene, Inuvialuit and Metis knowledge; and they continue to be reflected in Northern governance, economy, and cultures. Alongside Aboriginal sciences are Western sciences; these are primarily disciplinary in nature and formally structure postsecondary education globally. Postsecondary science and research education is still being introduced to the Northwest Territories (NWT). Over the last forty years the territorial government has developed the capacity for educational services, funding, institutions, and authority through the Department of Education, Culture and Employment. The delivery of Northern-based postsecondary education through Aurora College provides Northerners with the capacity to generate science and research in the North. What place do science and research have in the North? (North in this paper demarcates the socially constructed geopolitical territories north of the 60th parallel that we use cautiously as a structural term for the purposes of our narrative.) What kinds of investments need to be made and will Northerners be prepared to overcome barriers and take advantage of the opportunities?
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