| 1. |
- Katzberg, Michael, et al.
(författare)
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Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example
- 2010
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 11:4, s. 1735-1758
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Tidskriftsartikel (refereegranskat)abstract
- The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the gamma-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the gamma-hydroxyketone (5S)-hydroxy-2-hexanone in high enantio-as well as diastereoselectivity (ee and de >99.5%). This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants.
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| 2. |
- Owen, J G, et al.
(författare)
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A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product biosynthesis genes from metagenome libraries.
- 2012
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Ingår i: Environmental Microbiology. - : Wiley. - 1462-2920 .- 1462-2912. ; 12:5, s. 1198-1209
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Tidskriftsartikel (refereegranskat)abstract
- The single-module non-ribosomal peptide synthetase BpsA from Streptomyces lavendulae has the unique ability to autonomously synthesize a coloured product (indigoidine) from a single substrate (l-glutamine), conditional upon activation by a 4'-phosphopantetheinyl transferase (PPTase) partner. We show that bpsA can be expressed in an entD PPTase gene deleted mutant of Escherichia coli to yield a sensitive reporter strain for recovery of PPTase genes from metagenome libraries. We also show that recombinant bpsA constructs, generated by substitution of the native peptidyl carrier protein domain followed by directed evolution to restore function, can be used to increase the diversity of PPTase genes recovered from a sample. As PPTases are essential for activation of non-ribosomal peptide synthetase and polyketide synthase enzymes, they are frequently associated with secondary metabolite gene clusters. Nearly half of the PPTases recovered in our screening of two small-insert soil metagenome libraries were genetically linked to recognizable secondary metabolite biosynthetic genes, demonstrating that PPTase-targeting functional screens can be used for efficient recovery of natural product gene clusters from metagenome libraries. The plasticity and portability of bpsA reporter genes can potentially be exploited to maximize recovery and expression of PPTase-bearing clones in a wide range of hosts.
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| 3. |
- Skorupa-Parachin, Nádia, 1982, et al.
(författare)
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A Microbial Perspective on Ethanolic Lignocellulose Fermentation
- 2011
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Ingår i: Comprehensive Biotechnology, 2nd Edition. - 9780080885049 ; 6, s. 605-614
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Bioethanol is an alternative to fossil transportation fuel. It is produced from sugar- and starch-containing crops but current efforts have turned to ethanol from agricultural and forestry waste. These materials are not expected to compete with food and feed production and net emission of carbon dioxide is lower. Several ethanol-producing microorganisms have been assessed at laboratory scale, including Gram-positive and Gram-negative bacteria, eukaryotes such as yeasts and filamentous fungi, but few have so far been used at industrial scale. In this article, the advantages and disadvantages of the different microorganisms including co-cultures are discussed with respect to ethanol production from lignocellulose raw materials. The complexity of lignocellulose materials may require development of different microorganisms for different applications, so that 'tailor-made' strains for different lignocellulose raw materials may be more efficient than one 'super-microorganism' for any raw material.
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| 4. |
- Skorupa Parachin, Nadia
(författare)
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Biocatalyst Engineering: Metabolic Engineering, Kinetic modeling and metagenomics applied to industrial biotechnology
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Industrial biotechnology has been defined as the use and application of biotechnology for the sustainable processing and production of chemicals, materials and fuels. It makes use of biocatalysts such as microbial communities, whole-cell microorganisms or purified enzymes. Although biocatalysts are considered advantageous, since they operate under mild conditions regarding temperature and pH and enable chemo-, regio-, and stereoselective reactions, their utilization on the industrial scale can be impeded by sub-optimal performance. The present study was aimed at the improvement of two biocatalytic processes: whole-cell bioreduction for the production of optically pure alcohols, and ethanol production from lignocellulosic feedstock. The reduction of the bicyclic diketone, bicyclo[2.2.2]octane-2,6-dione into 1R,4S,6S-6-hydroxy-bicyclo[2.2.2]octane-2-one (endo-alcohol) and 1S,4R,6S-6- hydroxy-bicyclo[2.2.2]octane-2-one (exo-alcohol) was used as a model bioreduction reaction. The identification and overexpression of an exo-reductase encoding gene in Candida tropicalis enabled the production of the uncommon exo-alcohol as major product. In parallel, the advantages and disadvantages of metabolically engineered Saccharomyces cerevisiae and Escherichia coli as host for whole-cell bioreduction were compared for the production of the endoalcohol. Both these microorganisms gave about the same product purity. While E. coli showed a three times higher reduction rate, higher cell viability was maintained during the bioreductions with recombinant S. cerevisiae, which resulted in higher final conversion (95%) and indicated that yeast could be recycled. Improvement of bioethanol production from xylose was achieved through the construction and use of a kinetic model as a simulation tool for metabolic engineering of recombinant S. cerevisiae strains. In parallel, novel xylose isomerases and reductases were isolated from soil metagenome libraries by sequence- and activity-based screening methods. In addition a novel indirect protocol for soil DNA extraction that enabled the isolation of environmental DNA at high yield and purity was developed. This study enabled the expansion of a biocatalyst toolbox by providing new catalysts, screening methods and generating new recombinant strains with improved properties, which can be utilized in the pharmaceutical and bioenergy sectors, and thus constitutes a step forward in the development of novel biobased processes.
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| 5. |
- Skorupa Parachin, Nádia, et al.
(författare)
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Bioreduction
- 2010
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Ingår i: Enclyclopedia of Industrial Biotechnology : Bioprocess, Bioseparation and Cell Technology - Bioprocess, Bioseparation and Cell Technology. - Hoboken, NJ, USA : John Wiley & Sons, Inc.. - 9780471799306 - 9780470054581
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Bokkapitel (refereegranskat)abstract
- Bioreduction has emerged over the years as an alternative method to organic synthesis for the generation of chiral precursors of commercial interest. Bioreductions operate under mild conditions of pH and temperature with the help of highly regio- and enantio-selective oxidoreductase enzymes.In this contribution, the different oxidoreductase families involved in bioreductions are exemplified and their main characteristics are presented. The wide spectrum of oxidoreductase substrates (including ketones, diketones, ketoesters, aldehydes, alkenes, and keto acids) is discussed and both preparative and industrial scale examples are reported. The advantages and disadvantages of using isolated enzymatic systems versus whole-cell systems for bioreduction are discussed in terms of cost, specificity, stereoselectivity, and cofactor regeneration. The contribution is also reviewing strategies for improving the biocatalyst at the cell or enzyme level, which include process engineering, metabolic engineering as well as structure-based and nonstructure-based enzyme engineering. Finally, the potential role of metagenomics for isolating novel biocatalysts from different environments is discussed.
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| 6. |
- Skorupa Parachin, Nadia, et al.
(författare)
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Flotation as a tool for indirect DNA extraction from soil
- 2010
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 87:5, s. 1927-1933
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Tidskriftsartikel (refereegranskat)abstract
- Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 mug DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences.
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| 7. |
- Skorupa Parachin, Nadia, et al.
(författare)
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Isolation of xylose isomerases by sequence- and function-based screening from a soil metagenomic library
- 2011
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- Background: Xylose isomerase (XI) catalyses the isomerisation of xylose to xylulose in bacteria and some fungi. Currently, only a limited number of XI genes have been functionally expressed in Saccharomyces cerevisiae, the microorganism of choice for lignocellulosic ethanol production. The objective of the present study was to search for novel XI genes in the vastly diverse microbial habitat present in soil. As the exploitation of microbial diversity is impaired by the ability to cultivate soil microorganisms under standard laboratory conditions, a metagenomic approach, consisting of total DNA extraction from a given environment followed by cloning of DNA into suitable vectors, was undertaken. Results: A soil metagenomic library was constructed and two screening methods based on protein sequence similarity and enzyme activity were investigated to isolate novel XI encoding genes. These two screening approaches identified the xym1 and xym2 genes, respectively. Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups. When xym1 and xym2 were overexpressed in a xylA-deficient Escherichia coli strain, similar growth rates to those in which the Piromyces XI gene was expressed were obtained. However, expression in S. cerevisiae resulted in only one-fourth the growth rate of that obtained for the strain expressing the Piromyces XI gene. Conclusions: For the first time, the screening of a soil metagenomic library in E. coli resulted in the successful isolation of two active XIs. However, the discrepancy between XI enzyme performance in E. coli and S. cerevisiae suggests that future screening for XI activity from soil should be pursued directly using yeast as a host.
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| 8. |
- Skorupa Parachin, Nadia, et al.
(författare)
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Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae.
- 2011
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176. ; 13, s. 508-517
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.
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| 9. |
- Skorupa Parachin, Nadia, et al.
(författare)
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The deletion of YLR042c improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae.
- 2010
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 27, s. 741-751
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Tidskriftsartikel (refereegranskat)abstract
- In a recent study combining transcriptome analyses of a number of recombinant laboratory and industrial S. cerevisiae strains with improved xylose utilization and their respective control strains, the ORF YLR042c was identified as a downregulated gene and it was shown that the gene deletion improved aerobic growth on xylose in the tested strain background. In the present study, the influence of deleting YLR042c on xylose fermentation was investigated in two different xylose-fermenting strains: TMB3001, which expresses genes from the initial xylose catabolizing pathway, including heterologous xylose reductase (XR) and xylitol dehydrogenase (XDH) and endogenous xylulokinase (XK); and TMB3057, which, in addition to the initial xylose catabolizing pathway, overexpresses the endogenous genes encoding the non-oxidative pentose phosphate pathway enzymes. The deletion of YLR042c led to improved aerobic growth on xylose in both strain backgrounds. However, the effect was more significant in the strain with the poorer growth rate on xylose (TMB3001). Under anaerobic conditions, the deletion of YLR042c increased the specific xylose consumption rate and the ethanol and xylitol yields. In strain TMB3057, xylose consumption was also improved at low concentrations and during co-fermentation of xylose and glucose. The effect of the gene deletion and overexpression was also tested for different carbon sources. Altogether, these results suggest that YLR042c influences xylose and the assimilation of carbon sources other than glucose, and that the effect could be at the level of sugar transport or sugar signalling. Copyright (c) 2010 John Wiley & Sons, Ltd.
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