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Genome dynamics of the human embryonic kidney 293 (HEK293) lineage in response to cell biology manipulations
New Biotechnology · Volume 31S · July 2014
SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT
Symposium 19: Evolutionary strategies for cell factory development O19-1 Evolutionary and reverse metabolic engineering of Saccharomyces cerevisiae Jack T. Pronk Department of Biotechnology, Delft University of Technology, Delft, The Netherlands
Laboratory evolution is a powerful, versatile approach for improving and expanding the capabilities of industrial microorganisms which, in contrast to targeted genetic modification, does not require a detailed a priori understanding of the molecular basis for the trait of interest. Cultivation in bioreactors offers many options to design and implement culture conditions that maximize the selective advantage of spontaneous mutations that confer a specific, industrially relevant trait. I will briefly discuss applications of this ‘evolutionary engineering’ approach to the yeast Saccharomyces cerevisiae for optimizing its sugar fermentation kinetics and for increasing its robustness to industrially relevant stresses. Until recently, expression profiling with DNA micro-arrays was the only cost-affordable genome-wide analytical technique for analysing the molecular basis for improved performance of yeast strains derived from evolutionary engineering experiments. However, such transcriptome analyses tended to generate large numbers of targets, often without a clear lead to the responsible mutation(s). Based on recent studies from the Delft yeast group, I will illustrate how the advent of whole-genome sequencing has transformed the molecular analysis of evolved genotypes. Analysis of multiple, independent evolution experiments and integration of classical genetics approaches were shown to greatly amplify the power of whole-genome resequencing of evolved strains. http://dx.doi.org/10.1016/j.nbt.2014.05.1773
O19-2 Adaptive evolution of saccharomyces cerevisiae to early stage of an alcoholic fermentation Ana Mangado ∗ , Pilar Morales, Jordi Tronchoni, Ramon Gonzalez ICVV/CSIC, Spain
Experimental evolution was used to identify genes involved in the adaptation to the early stages of wine fermentation. Evolution experiments were performed in continuous culture for 150-250 generations, in conditions emulating the initial stages of alcoholic fermentation. We performed three independent experimental evolution experiments of a haploid laboratory strain BY4741 and one evolution with a haploid strain (not adapted to winemaking growth conditions) obtained by meiotic segregation of the wine yeast EC1118. By the end of the experiments, colonies were phenotypically characterized, and strains that showed improved initial growth rates were selected. We used next generation sequenc-
ing techniques in order to find the genetic changes. Four strains were selected from the evolution of BY4741. The mutations found, pointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the preferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target proteins participating in different cellular processes, while Bul1p is an Rsp5p substrate adaptor involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. Our results might be related to increased halftime of plasma membrane amino acid permeases. Apparently the genetic background of the laboratory strain is conditioning the result. However, we have shown the strength of this approach. The evolution of the haploid segregant of EC1118 was run for 250 generations, three adapted strains were selected. Preliminary bioinformatic analysis highlighted changes in chromosome numbers in mutant strains. We are currently testing these results by karyotyping, qPCR, flow cytometry and other techniques. http://dx.doi.org/10.1016/j.nbt.2014.05.1774
O19-3 Genome dynamics of the human embryonic kidney 293 (HEK293) lineage in response to cell biology manipulations Morgane Boone 1,2,∗ , Yao-Cheng Lin 3,4 , Leander Meuris 1,2 , Irma Lemmens 5,6 , Nadine Van Roy 7 , Arne Soete 8 , Joke Reumers 9 , Matthieu Moisse 9,10 , Stephane Plaisance 11 , Radoje Drmanac 12 , Jason Chen 12 , Frank Speleman 7 , Diether Lambrechts 9,10 , Yves Van de Peer 3,4,13 , Jan Tavernier 5,6 , Nico Callewaert 1,2 1
Unit for Medical Biotechnology, Inflammation Research Center (IRC), VIB, Belgium 2 Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, Belgium 3 Department of Plant Systems Biology, VIB, Belgium 4 Department of Plant Biotechnology and Bioinformatics, Ghent University, Belgium 5 Department of Medical Protein Research, VIB, Belgium 6 Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Belgium 7 Center for Medical Genetics, Ghent University Hospital (MRB), Belgium 8 Bioinformatics Core Facility, Inflammation Research Center (IRC), VIB, Belgium 9 Laboratory for Translational Genetics, Department of Oncology, KULeuven, Belgium 10 Vesalius Research Center, VIB, Belgium 11 VIB Bioinformatics Training and Services (BITS), VIB, Belgium 12 Complete Genomics, USA 13 Genomics Research Institute, University of Pretoria, South Africa
The HEK293 human cell lineage is widely used in cell biology and biotechnology. We used whole genome resequencing methods in six 293 cell lines to study the dynamics of this aneuploid genome in response to the cell biology manipulations that were used to generate common derivatives of 293 cells, such as transformation and stable clone generation (293T); suspension growth adaptation (293S) and cytotoxic lectin selection to isolate a glycosylation-homogenous clone (293SG). While the chromosomal structure of single 293 cells within a culture appears to be extremely diverse, our analysis suggests that standard cell culture
www.elsevier.com/locate/nbt S71
SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT
procedures (passaging and cell banking) do not affect the ‘average’ genome structure and sequence to a great extent. The extraordinary chromosomal plasticity of this genome, however, seems to be the driving adaptive force when cells are put through a bottleneck. This feature underlies a novel application for which we provide proof of concept here: selection of 293 clones surviving stringent selective conditions (eg ricin toxin), followed by whole-genome analysis of copy number alterations, can effectively pinpoint the genomic region(s) that contain the gene(s) required for adaptation to those selective conditions. Furthermore, up to the level of sensitivity afforded here (single copy plasmid insertions were easily detected), these cell lines have no inadvertent virus insertions. In terms of tools, we optimized a workflow to detect human/vector genome breakpoints, and enabled visualization of the 293 genome data both through a user-friendly visualization web page, as well as through the Integrative Genome Browser (IGV) for rich data mining. http://dx.doi.org/10.1016/j.nbt.2014.05.1775
O19-4 Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16 Steffen Gruber , Jeremias Hagen, Helmut Schwab, Petra Koefinger ∗ Graz University of Technology, Austria
The gram-negative -proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. Up to now some basic systems for targeted genetic manipulation of this bacterium were already established. However, the majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmids loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16 a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 hours. The utility of the new vector family is demonstrated by providing expression data with different model proteins. http://dx.doi.org/10.1016/j.nbt.2014.05.1776
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www.elsevier.com/locate/nbt
New Biotechnology · Volume 31S · July 2014
O19-5 Novel human kidney epithelial cell line in pharmaceutical biotechnology Lukas Fliedl 1,∗ , Matthias Wieser 1 , Gabriele Manhart 1 , Matthias P. Gerstl 1 , Florian Kast 1 , Abdulhameed Khan 2 , Renate Kunert 2 , Johannes Grillari 2 , Regina Grillari-Voglauer 2 1
ACIB, Austria Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria
2
Mammalian cells are used as model systems, products themselves and as producers of recombinant proteins and vaccines. In these different applications a variety of different cell lines are used and although all have proven valuable for their specific application, there is still room for improvement in terms of posttranslational modifications. Especially novel human cell lines are of ever increasing importance since they ideally represent the in vivo situation and might produce high quality biopharmaceuticals as similar to endogenous proteins as possible. Therefore we established a novel human continuously growing renal proximal tubular epithelial cell line (RPTEC) that has maintained many differentiated characteristics of the normal nontransduced counterpart and tested its performance in the different fields of pharmaceutical biotechnology. A complex model protein, erythropoietin, was stably produced in this cell line and the quality of the recombinant protein was compared to CHO derived product by analysis of isoforms as well as specific non-human glycopatterns. Additionally, we used our cell line to produce influenza virus and proved high capabilities of our cell line in this application. Finally, we used our cell line to get insights into nephrotoxicity induced by cisplatin, which has important implications as a chemotherapeutic drug and hypothesize that especially epithelial barrier formation and polarity of RPTECs need to be considered in toxicity models to validly predict the in vivo situation. Therefore, the here established kidney epithelial cells combine applicability in various fields of pharmaceutical biotechnology, as they are capable of production as well as of pre-clinical testing of biopharmaceuticals. http://dx.doi.org/10.1016/j.nbt.2014.05.1777
O19-6 De novo production of geranic acid with Pseudomonas putida Jens Schrader ∗ , Jia Mi, Daniela Becher, Patrice Lubuta, Markus Buchhaupt, Dirk Holtmann DECHEMA Research Institute, Germany
Production of plant terpenes by engineered microbes has become a prime example of applied synthetic biology with tremendous progress being made during the last decade. Whereas sesquiterpene titers reported have already reached g/L values in the bioreactor, efficient monoterpene production seems to be more
SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT
Symposium 19: Evolutionary strategies for cell factory development O19-1 Evolutionary and reverse metabolic engineering of Saccharomyces cerevisiae Jack T. Pronk Department of Biotechnology, Delft University of Technology, Delft, The Netherlands
Laboratory evolution is a powerful, versatile approach for improving and expanding the capabilities of industrial microorganisms which, in contrast to targeted genetic modification, does not require a detailed a priori understanding of the molecular basis for the trait of interest. Cultivation in bioreactors offers many options to design and implement culture conditions that maximize the selective advantage of spontaneous mutations that confer a specific, industrially relevant trait. I will briefly discuss applications of this ‘evolutionary engineering’ approach to the yeast Saccharomyces cerevisiae for optimizing its sugar fermentation kinetics and for increasing its robustness to industrially relevant stresses. Until recently, expression profiling with DNA micro-arrays was the only cost-affordable genome-wide analytical technique for analysing the molecular basis for improved performance of yeast strains derived from evolutionary engineering experiments. However, such transcriptome analyses tended to generate large numbers of targets, often without a clear lead to the responsible mutation(s). Based on recent studies from the Delft yeast group, I will illustrate how the advent of whole-genome sequencing has transformed the molecular analysis of evolved genotypes. Analysis of multiple, independent evolution experiments and integration of classical genetics approaches were shown to greatly amplify the power of whole-genome resequencing of evolved strains. http://dx.doi.org/10.1016/j.nbt.2014.05.1773
O19-2 Adaptive evolution of saccharomyces cerevisiae to early stage of an alcoholic fermentation Ana Mangado ∗ , Pilar Morales, Jordi Tronchoni, Ramon Gonzalez ICVV/CSIC, Spain
Experimental evolution was used to identify genes involved in the adaptation to the early stages of wine fermentation. Evolution experiments were performed in continuous culture for 150-250 generations, in conditions emulating the initial stages of alcoholic fermentation. We performed three independent experimental evolution experiments of a haploid laboratory strain BY4741 and one evolution with a haploid strain (not adapted to winemaking growth conditions) obtained by meiotic segregation of the wine yeast EC1118. By the end of the experiments, colonies were phenotypically characterized, and strains that showed improved initial growth rates were selected. We used next generation sequenc-
ing techniques in order to find the genetic changes. Four strains were selected from the evolution of BY4741. The mutations found, pointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the preferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target proteins participating in different cellular processes, while Bul1p is an Rsp5p substrate adaptor involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. Our results might be related to increased halftime of plasma membrane amino acid permeases. Apparently the genetic background of the laboratory strain is conditioning the result. However, we have shown the strength of this approach. The evolution of the haploid segregant of EC1118 was run for 250 generations, three adapted strains were selected. Preliminary bioinformatic analysis highlighted changes in chromosome numbers in mutant strains. We are currently testing these results by karyotyping, qPCR, flow cytometry and other techniques. http://dx.doi.org/10.1016/j.nbt.2014.05.1774
O19-3 Genome dynamics of the human embryonic kidney 293 (HEK293) lineage in response to cell biology manipulations Morgane Boone 1,2,∗ , Yao-Cheng Lin 3,4 , Leander Meuris 1,2 , Irma Lemmens 5,6 , Nadine Van Roy 7 , Arne Soete 8 , Joke Reumers 9 , Matthieu Moisse 9,10 , Stephane Plaisance 11 , Radoje Drmanac 12 , Jason Chen 12 , Frank Speleman 7 , Diether Lambrechts 9,10 , Yves Van de Peer 3,4,13 , Jan Tavernier 5,6 , Nico Callewaert 1,2 1
Unit for Medical Biotechnology, Inflammation Research Center (IRC), VIB, Belgium 2 Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and Microbiology, Ghent University, Belgium 3 Department of Plant Systems Biology, VIB, Belgium 4 Department of Plant Biotechnology and Bioinformatics, Ghent University, Belgium 5 Department of Medical Protein Research, VIB, Belgium 6 Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Belgium 7 Center for Medical Genetics, Ghent University Hospital (MRB), Belgium 8 Bioinformatics Core Facility, Inflammation Research Center (IRC), VIB, Belgium 9 Laboratory for Translational Genetics, Department of Oncology, KULeuven, Belgium 10 Vesalius Research Center, VIB, Belgium 11 VIB Bioinformatics Training and Services (BITS), VIB, Belgium 12 Complete Genomics, USA 13 Genomics Research Institute, University of Pretoria, South Africa
The HEK293 human cell lineage is widely used in cell biology and biotechnology. We used whole genome resequencing methods in six 293 cell lines to study the dynamics of this aneuploid genome in response to the cell biology manipulations that were used to generate common derivatives of 293 cells, such as transformation and stable clone generation (293T); suspension growth adaptation (293S) and cytotoxic lectin selection to isolate a glycosylation-homogenous clone (293SG). While the chromosomal structure of single 293 cells within a culture appears to be extremely diverse, our analysis suggests that standard cell culture
www.elsevier.com/locate/nbt S71
SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT
procedures (passaging and cell banking) do not affect the ‘average’ genome structure and sequence to a great extent. The extraordinary chromosomal plasticity of this genome, however, seems to be the driving adaptive force when cells are put through a bottleneck. This feature underlies a novel application for which we provide proof of concept here: selection of 293 clones surviving stringent selective conditions (eg ricin toxin), followed by whole-genome analysis of copy number alterations, can effectively pinpoint the genomic region(s) that contain the gene(s) required for adaptation to those selective conditions. Furthermore, up to the level of sensitivity afforded here (single copy plasmid insertions were easily detected), these cell lines have no inadvertent virus insertions. In terms of tools, we optimized a workflow to detect human/vector genome breakpoints, and enabled visualization of the 293 genome data both through a user-friendly visualization web page, as well as through the Integrative Genome Browser (IGV) for rich data mining. http://dx.doi.org/10.1016/j.nbt.2014.05.1775
O19-4 Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16 Steffen Gruber , Jeremias Hagen, Helmut Schwab, Petra Koefinger ∗ Graz University of Technology, Austria
The gram-negative -proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. Up to now some basic systems for targeted genetic manipulation of this bacterium were already established. However, the majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmids loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16 a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 hours. The utility of the new vector family is demonstrated by providing expression data with different model proteins. http://dx.doi.org/10.1016/j.nbt.2014.05.1776
S72
www.elsevier.com/locate/nbt
New Biotechnology · Volume 31S · July 2014
O19-5 Novel human kidney epithelial cell line in pharmaceutical biotechnology Lukas Fliedl 1,∗ , Matthias Wieser 1 , Gabriele Manhart 1 , Matthias P. Gerstl 1 , Florian Kast 1 , Abdulhameed Khan 2 , Renate Kunert 2 , Johannes Grillari 2 , Regina Grillari-Voglauer 2 1
ACIB, Austria Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria
2
Mammalian cells are used as model systems, products themselves and as producers of recombinant proteins and vaccines. In these different applications a variety of different cell lines are used and although all have proven valuable for their specific application, there is still room for improvement in terms of posttranslational modifications. Especially novel human cell lines are of ever increasing importance since they ideally represent the in vivo situation and might produce high quality biopharmaceuticals as similar to endogenous proteins as possible. Therefore we established a novel human continuously growing renal proximal tubular epithelial cell line (RPTEC) that has maintained many differentiated characteristics of the normal nontransduced counterpart and tested its performance in the different fields of pharmaceutical biotechnology. A complex model protein, erythropoietin, was stably produced in this cell line and the quality of the recombinant protein was compared to CHO derived product by analysis of isoforms as well as specific non-human glycopatterns. Additionally, we used our cell line to produce influenza virus and proved high capabilities of our cell line in this application. Finally, we used our cell line to get insights into nephrotoxicity induced by cisplatin, which has important implications as a chemotherapeutic drug and hypothesize that especially epithelial barrier formation and polarity of RPTECs need to be considered in toxicity models to validly predict the in vivo situation. Therefore, the here established kidney epithelial cells combine applicability in various fields of pharmaceutical biotechnology, as they are capable of production as well as of pre-clinical testing of biopharmaceuticals. http://dx.doi.org/10.1016/j.nbt.2014.05.1777
O19-6 De novo production of geranic acid with Pseudomonas putida Jens Schrader ∗ , Jia Mi, Daniela Becher, Patrice Lubuta, Markus Buchhaupt, Dirk Holtmann DECHEMA Research Institute, Germany
Production of plant terpenes by engineered microbes has become a prime example of applied synthetic biology with tremendous progress being made during the last decade. Whereas sesquiterpene titers reported have already reached g/L values in the bioreactor, efficient monoterpene production seems to be more