| 1. |
- Bengtsson, Oskar, et al.
(författare)
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Identification of common traits in improved xylose-growing Saccharomyces cerevisiae for inverse metabolic engineering.
- 2008
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 25:11, s. 835-847
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Tidskriftsartikel (refereegranskat)abstract
- Four recombinant Saccharomyces cerevisiae strains with enhanced xylose growth (TMB3400, C1, C5 and BH42) were compared with two control strains (TMB3399, TMB3001) through genome-wide transcription analysis in order to identify novel targets for inverse metabolic engineering. A subset of 13 genes with changed expression levels in all improved strains was selected for further analysis. Thirteen validation strains and two reference strains were constructed to investigate the effect of overexpressing or deleting these genes in xylose-utilizing S. cerevisiae. Improved aerobic growth rates on xylose were observed in five cases. The strains overexpressing SOL3 and TAL1 grew 19% and 24% faster than their reference strain, and the strains carrying deletions of YLR042C, MNI1 or RPA49 grew 173%, 62% and 90% faster than their reference strain.
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| 2. |
- 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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| 3. |
- Skorupa Parachin, Nadia, et al.
(författare)
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Comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli for the production of an optically pure keto alcohol.
- 2009
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84, s. 487-497
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Tidskriftsartikel (refereegranskat)abstract
- In this study, the production of enantiomerically pure (1R,4S,6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one ((-)-2) through stereoselective bioreduction was used as a model reaction for the comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli as biocatalysts. For both microorganisms, over-expression of the gene encoding the NADPH-dependent aldo-keto reductase YPR1 resulted in high purity of the keto alcohol (-)-2 (>99% ee, 97-98% de). E. coli had three times higher initial reduction rate but S. cerevisiae continued the reduction reaction for a longer time period, thus reaching a higher conversion of the substrate (95%). S. cerevisiae was also more robust than E. coli, as demonstrated by higher viability during bioreduction. It was also investigated whether the NADPH regeneration rate was sufficient to supply the over-expressed reductase with NADPH. Five strains of each microorganism with varied carbon flux through the NADPH regenerating pentose phosphate pathway were genetically constructed and compared. S. cerevisiae required an increased NADPH regeneration rate to supply YPR1 with co-enzyme while the native NADPH regeneration rate was sufficient for E. coli.
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| 4. |
- Skorupa Parachin, Nadia, et al.
(författare)
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Xylanases from Cryptococcus flavus isolate I-11: Enzymatic profile, isolation and heterologous expression of CfXYN1 in Saccharomyces cerevisiae
- 2009
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Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 59:1-3, s. 52-57
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to characterize the xylanolytic activity of Cryptococcus flavus isolate I-11. This microorganism was isolated from the Brazilian Cerrado, and enzyme plate assays showed that it also produces amylase and CMCase activity. The xylanolytic production of C. flavus isolate I-11 was improved by using a suitable combination of the carbon and nitrogen sources, reaching 130 U/mL A zymogram assay was performed showing three xylanase activity bands. The cDNA of one xylanase gene, CfXYN1, was obtained and preliminary expression analysis was performed on RNA samples collected after yeast growth on different carbon sources. This indicated that the CfXYN1 gene is transcribed in the presence of xylose, sugar cane bagasse and carboxymethyl cellulose, but not in the presence of glucose, as carbon source. The cDNA of CfXYN1 was cloned and expressed in Saccharomyces cerevisiae. The recombinant enzyme was partially characterized and showed an optimum at a pH of 3.0 and temperature of 50 degrees C. The recombinant enzyme retained 70% of its initial activity after pre-incubation for 30 min at the optimum pH and temperature. Computational analysis predicted a molecular weight of 21.2 kDa, and an isoelectric point of 7.02. The Cfxyn1p has 209 amino acids, including a signal peptide consisting of 16 amino acids. (C) 2009 Elsevier B.V. All rights reserved.
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