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Light-mediated gene regulation for complex expression procedures in yeast
Abstracts / New Biotechnology 33S (2016) S1–S213
P31-3 Light-mediated gene regulation for complex expression procedures in yeast Peter Kusen 1,∗ , Georg Wandrey 2 , Christopher Probst 3 , Alexander Grünberger 3 , Martina Holz 4 , Sonja Meyer zu Berstenhorst 4 , Dietrich Kohlheyer 3 , Jochen Büchs 2 , Jörg Pietruszka 4 1
Forschungszentrum Jülich, Germany RWTH Aachen University, Germany 3 Forschungszentrum Jülich GmbH, Germany 4 Heinrich-Heine-University Düsseldorf, Germany 2
Light as a stimulus provides the capability to develop innovative regulation techniques for gene expression. The opportunities of non-invasive precise dosing with high spatiotemporal resolution and the application of high throughput technologies are given [1–3]. Hence, we are focusing on caged compounds to enable lightmediated gene regulation in the yeast Saccharomyces cerevisiae. Caged-compounds are photo-labile protected and therefore biologically inactive regulator molecules which can be reactivated by irradiation with certain light conditions. These molecules enable a photo regulation upgrade for already well established expression systems, in our case a repressable promoter and its corresponding repressor molecule. In this system, the expression is temporally repressed by irradiation and subsequent release of the active repressor molecule. Afterwards, the repressor molecule is consumed by the yeast cells leading to reactivation of gene expression. This effect was used to precisely control the repression duration by adjusting the amount of released repressor via different irradiation times. With this new expression system the regulation of complex expression procedures was achieved by combination of several repression/derepression intervals. In particular, a stepwise increase of temporally-constant expression levels, linear expression rates with variable slopes, and accurate control over the expression induction was realized. For future applications, optimization of multi enzyme reaction cascades and difficult protein expressions can be managed by light mediated flexible gene expression. References [1] Deiters A. ChemBioChem 2010;11:47–53. [2] Mayer G, Heckel A. Angew Chem Int Ed 2006;45:4900–21. [3] Kaplan JH, Forbush B, Hoffman JF. Biochemistry (Mosc) 1978;17:1929–35.
http://dx.doi.org/10.1016/j.nbt.2016.06.1373
P31-4 Study on vector elements for efficient selection, expression and secretion in HEK293 cells Ramon Roman ∗ , Joan Miret, Ivan Martinez, Aïda Roura, Antoni Casablancas, Martí Lecina, Jordi J. Cairó Autonomous University of Barcelona, UAB, Spain Mammalian cell lines are widely used to produce soluble recombinant proteins that can be secreted into the culture media, easing downstream processes. Thus, exploring the genetic elements responsible for expression and secretion is a must in the strain development process. Moreover, a proper selectable marker that allows the selection and stabilization of the producing strain is also highly desirable. In the present work, the influence of 3 different promoters (CMV, EF-1a and CAG) on protein expression and 5 signal pep-
S189
tides (Albumin, IgG, IFNa2, IL-2 and Gaussia Luciferase) on protein secretion were assessed in HEK293 cells using eGFP as a model protein. FACS and spectrofluorimetry were used to assess intra/extracellular protein expression level in terms of fluorescence. Protein expression and secretion results were eventually corroborated by electrophoretic analysis. Human CMV intermediate early promoter resulted in the strongest promoter (3-fold increase) and Human IFNa2 signal peptide achieved the highest efficiency of eGFP secretion (77%). Moreover, regulatory and economic concerns made metabolic markers an interesting alternative to traditional drug makers. Thus, we developed alternative metabolic selection markers following two different approaches. Firstly, taking advantage of HEK293 auxotrophy for Tyrosine, Phenylalanine Hydroxylase (PAH) gene was successfully used for selection of transfected cells in a tyrosinefree medium. Secondly, a thymidylate synthase (TYMS) knock-out mutant was generated using CRISPR/Cas9 system and TYMS gene was used for selection. FACS measurements showed selection efficiencies for PAH comparable to puromycin (99% eGFP + cells) with similar specific production; whereas TYMS system achieves 85% eGFP + cells enrichment in transfected cultures. http://dx.doi.org/10.1016/j.nbt.2016.06.1374
Microbial cell factories P32-1 Wood-rotting fungi lignocellulolytic enzyme overproduction for application in lignocellulose biorefinery and bioremediation Vladimir Elisashvili ∗ , Eva Kachlishvili, Tina Jokharidze, Eka Metreveli, Aza Kobakhidze, Tamar Khardziani, Violeta Berikashvili, Mikheil Asatiani Agricultural University of Georgia, Georgia A common feature of white-rot basidiomycetes (WRB) is production of powerful extracellular hydrolytic and oxidative enzymes that effectively degrade biopolymers in plant biomass. To develop commercially significant technologies of lignocellulosedeconstructing enzymes production for their industrial and environmental applications, various approaches and strategies have been exploited in submerged and solid-state fermentation of plant materials. Firstly, the screening programs revealed a wide diversity of tested fungi and resulted in selection of new organisms with tremendous synthesis of cellulase and xylanase and ligninmodifying enzymes (LME). Secondly, we established that several particular lignocellulosic growth substrates containing significant concentrations of soluble inducers play a crucial role in cellulases and ligninases production and determine the ratio of individual enzymes in final preparations. However, a fungus-specific lignocellulosic substrate should be elucidated. Thirdly, we show that some microelements enhance LME synthesis although their effect is very specific depending on fungi physiological peculiarities. Special attention was paid to the regulation of LME production in the presence of aromatic compounds and synthetic dyes; some of them provide directed synthesis of LME. Fourthly, co-culture of the compatible fungi may be an appropriate approach to increase hydrolytic and LME production. Fifthly, our study underlines that the maximum expression of WRB biosynthetic potential depends on the additive effect of several factors. This approach permitted to achieve the highest laccase (1450 U/ml) and MnP (12 U/ml) activities and as high as 140, 700, and 8 U/ml endoglucanase, xylanase,
P31-3 Light-mediated gene regulation for complex expression procedures in yeast Peter Kusen 1,∗ , Georg Wandrey 2 , Christopher Probst 3 , Alexander Grünberger 3 , Martina Holz 4 , Sonja Meyer zu Berstenhorst 4 , Dietrich Kohlheyer 3 , Jochen Büchs 2 , Jörg Pietruszka 4 1
Forschungszentrum Jülich, Germany RWTH Aachen University, Germany 3 Forschungszentrum Jülich GmbH, Germany 4 Heinrich-Heine-University Düsseldorf, Germany 2
Light as a stimulus provides the capability to develop innovative regulation techniques for gene expression. The opportunities of non-invasive precise dosing with high spatiotemporal resolution and the application of high throughput technologies are given [1–3]. Hence, we are focusing on caged compounds to enable lightmediated gene regulation in the yeast Saccharomyces cerevisiae. Caged-compounds are photo-labile protected and therefore biologically inactive regulator molecules which can be reactivated by irradiation with certain light conditions. These molecules enable a photo regulation upgrade for already well established expression systems, in our case a repressable promoter and its corresponding repressor molecule. In this system, the expression is temporally repressed by irradiation and subsequent release of the active repressor molecule. Afterwards, the repressor molecule is consumed by the yeast cells leading to reactivation of gene expression. This effect was used to precisely control the repression duration by adjusting the amount of released repressor via different irradiation times. With this new expression system the regulation of complex expression procedures was achieved by combination of several repression/derepression intervals. In particular, a stepwise increase of temporally-constant expression levels, linear expression rates with variable slopes, and accurate control over the expression induction was realized. For future applications, optimization of multi enzyme reaction cascades and difficult protein expressions can be managed by light mediated flexible gene expression. References [1] Deiters A. ChemBioChem 2010;11:47–53. [2] Mayer G, Heckel A. Angew Chem Int Ed 2006;45:4900–21. [3] Kaplan JH, Forbush B, Hoffman JF. Biochemistry (Mosc) 1978;17:1929–35.
http://dx.doi.org/10.1016/j.nbt.2016.06.1373
P31-4 Study on vector elements for efficient selection, expression and secretion in HEK293 cells Ramon Roman ∗ , Joan Miret, Ivan Martinez, Aïda Roura, Antoni Casablancas, Martí Lecina, Jordi J. Cairó Autonomous University of Barcelona, UAB, Spain Mammalian cell lines are widely used to produce soluble recombinant proteins that can be secreted into the culture media, easing downstream processes. Thus, exploring the genetic elements responsible for expression and secretion is a must in the strain development process. Moreover, a proper selectable marker that allows the selection and stabilization of the producing strain is also highly desirable. In the present work, the influence of 3 different promoters (CMV, EF-1a and CAG) on protein expression and 5 signal pep-
S189
tides (Albumin, IgG, IFNa2, IL-2 and Gaussia Luciferase) on protein secretion were assessed in HEK293 cells using eGFP as a model protein. FACS and spectrofluorimetry were used to assess intra/extracellular protein expression level in terms of fluorescence. Protein expression and secretion results were eventually corroborated by electrophoretic analysis. Human CMV intermediate early promoter resulted in the strongest promoter (3-fold increase) and Human IFNa2 signal peptide achieved the highest efficiency of eGFP secretion (77%). Moreover, regulatory and economic concerns made metabolic markers an interesting alternative to traditional drug makers. Thus, we developed alternative metabolic selection markers following two different approaches. Firstly, taking advantage of HEK293 auxotrophy for Tyrosine, Phenylalanine Hydroxylase (PAH) gene was successfully used for selection of transfected cells in a tyrosinefree medium. Secondly, a thymidylate synthase (TYMS) knock-out mutant was generated using CRISPR/Cas9 system and TYMS gene was used for selection. FACS measurements showed selection efficiencies for PAH comparable to puromycin (99% eGFP + cells) with similar specific production; whereas TYMS system achieves 85% eGFP + cells enrichment in transfected cultures. http://dx.doi.org/10.1016/j.nbt.2016.06.1374
Microbial cell factories P32-1 Wood-rotting fungi lignocellulolytic enzyme overproduction for application in lignocellulose biorefinery and bioremediation Vladimir Elisashvili ∗ , Eva Kachlishvili, Tina Jokharidze, Eka Metreveli, Aza Kobakhidze, Tamar Khardziani, Violeta Berikashvili, Mikheil Asatiani Agricultural University of Georgia, Georgia A common feature of white-rot basidiomycetes (WRB) is production of powerful extracellular hydrolytic and oxidative enzymes that effectively degrade biopolymers in plant biomass. To develop commercially significant technologies of lignocellulosedeconstructing enzymes production for their industrial and environmental applications, various approaches and strategies have been exploited in submerged and solid-state fermentation of plant materials. Firstly, the screening programs revealed a wide diversity of tested fungi and resulted in selection of new organisms with tremendous synthesis of cellulase and xylanase and ligninmodifying enzymes (LME). Secondly, we established that several particular lignocellulosic growth substrates containing significant concentrations of soluble inducers play a crucial role in cellulases and ligninases production and determine the ratio of individual enzymes in final preparations. However, a fungus-specific lignocellulosic substrate should be elucidated. Thirdly, we show that some microelements enhance LME synthesis although their effect is very specific depending on fungi physiological peculiarities. Special attention was paid to the regulation of LME production in the presence of aromatic compounds and synthetic dyes; some of them provide directed synthesis of LME. Fourthly, co-culture of the compatible fungi may be an appropriate approach to increase hydrolytic and LME production. Fifthly, our study underlines that the maximum expression of WRB biosynthetic potential depends on the additive effect of several factors. This approach permitted to achieve the highest laccase (1450 U/ml) and MnP (12 U/ml) activities and as high as 140, 700, and 8 U/ml endoglucanase, xylanase,