- Email: [email protected]
Extended and robust protein sequence annotation over conservative non hierarchical clusters. The Bologna Annotation Resource v 2.0
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[S.13] Design principles for information processing through signalling networks M. Schilling 1,∗ , V. Becker 1 , A. Raue 2 , T Maiwald 3 , D Winter 4 , W Lehmann 4 , W. Kolch 5 , J. Timmer 2 , U. Klingmueller 1 1
Division of Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ), Germany 2 Freiburg Center for Data Analysis and Modeling, University of Freiburg, Germany 3 Department of Systems Biology, Harvard Medical School, United States 4 Molecular Structure Analysis, German Cancer Research Center (DKFZ), Germany 5 Beatson Institute for Cancer Research & Institute for Biomedical and Life Sciences, University of Glasgow, United Kingdom Cells have to respond to changes in their environment. Alterations in extracellular signals are perceived by cell surface receptors and trigger the activation of complex intracellular signalling networks. Currently rather little is known regarding design principles that link pathway activation with cellular responses. The erythropoieting receptor (EpoR) is a cytokine receptor and the key regulator of erythropoiesis regulating proliferation, differentiation and survival of erythroid progenitor cells. A major signaling pathway activated by the EpoR is the extracellular signal regulated kinase (ERK) cascade. By combining quantitative data with mathematical modelling, we predicted and experimentally confirmed a distributive ERK phosphorylation mechanism in response to EpoR activation. Model analysis revealed that increasing one ERK isoform reduces activation of the other isoform, which was verified by protein overexpression. Furthermore we showed by statistical modelling that double-phosphorylated ERK1 attenuates proliferation beyond a certain activation level, while activated ERK2 enhances proliferation with saturation kinetics. Thus, we provide a quantitative link between previously unobservable signalling dynamics and cellular responses. doi:10.1016/j.jbiotec.2010.09.906
S547
thus interesting that Rhodospirillum rubrum produces high-levels of photosynthetic membranes (PM) completely independent of light, when grown semi-aerobically in the dark with a two-carbon substrate growth medium. On the basis of this cultivation process, we applied a systems approach using a combination of bioreactor cultivations, metabolomics, as well as mathematical modeling to develop R. rubrum for biotechnological applications. These efforts include the optimization of fed-batch processes to yield high cell densities to levels commonly employed in industry (Zeiger and Grammel, 2010). For improving the understanding of underlying metabolic and regulatory events, stoichiometric and dynamic computational models of metabolism and electron transfer chains were developed and applied (Klamt et al., 2008; Grammel and Ghosh, 2008). Furthermore, a metabolomic profile was obtained with different carbons sources which indicate additional flexibility in acetate assimilation pathways. This work is intended to open a new perspective for utilizing photosynthetic bacteria as producers in biotechnology. References Grammel, H., Ghosh, R., 2008. Redox state dynamics of ubiquinone-10 imply cooperative regulation of photosynthetic membrane expression in Rhodospirillum rubrum. J. Bacteriol. 190 (14), 4912–4921. Klamt, S., Grammel, H., Ghosh, R., Gilles, E.D., 2008. Modeling the Electron Transport Chain of Purple Nonsulfur Bacteria. Mol. Sys. Biol. 4, 156. Zeiger, L., Grammel, H., 2010. Model-based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions. Biotechnol Bioeng 105 (4), 729–739.
doi:10.1016/j.jbiotec.2010.09.907 [S.15] Extended and robust protein sequence annotation over conservative non hierarchical clusters. The Bologna Annotation Resource v 2.0 D. Piovesan 1,∗ , L. Bartoli 1 , P.L. Martelli 1 , P. Fariselli 1 , I. Rossi 2 , R. Casadio 1 1
Biocomputing Group, University of Bologna, Italy Biodec s.r.l., Bologna, Italy3 National Institute of Nuclear Physics, Bari, Italy Keywords: Protein functional annotation; Cross-genome comparison; Alignment coverage; Grid technology 2
[S.14] Development of a purple bacterium, Rhodospirillum rubrum, for applications in biotechnology – a systems biology approach H. Grammel 1,∗ , S. Klamt 1 , R. Ghosh 2 , E.D. Gilles 1 1
Max Planck Institute for Dynamics of Complex Technical Systems, Germany 2 University of Stuttgart, Biological Institute, Dept. of Bioenergetics, Germany Keywords: Rhodospirillum rubrum; Metabolomics; Computational Modeling; Process Development Anoxygenic photosynthetic purple bacteria are amongst the most versatile life forms and offer highly attractive opportunities for industrial applications. Potential products derived from intracytoplasmic photosynthetic membranes (ICM) are amongst others, photosynthetic pigments (porphyrines, carotenoids), coenzymes (Q10 ), biohydrogen and recombinant membrane proteins. Since high levels of ICM are formed at low-light intensities at anaerobic conditions most attempts to exploit purple bacteria for producing high value materials were so far conducted phototrophically, using light as energy source. However, mass cultivation of phototrophic bacteria is generally inefficient due to the inevitable limitation of light-supply when cell densities become very high. It is
Genome annotation is one of the most important issues in the genomic era. The exponential grow rate of newly sequenced genomes and proteomes urges the development of fast and reliable annotation methods, suited to exploit all the information available in curated data bases of protein sequences and structures. To this aim we developed BAR, the Bologna Annotation Resource that is now updated (available at http://microserf.biocomp.unibo.it/bar/). The basic notion is that sequences with high identity value to a counterpart can inherit the same function/s and structure, if available. What is totally new in our analysis is to cluster sequences with the constraint that sequence identity should be equal or higher than 40% on at least 90% of the pairwise alignment length. By this sequences are clustered in sets that can be annotated in terms of function and structure depending on the annotation level of the sequences within the cluster. Our method starts with on allagainst-all alignment of all the sequences in a GRID environment. The alignments are then regarded as an undirected graph and after the clustering procedure that constrains both the sequence identity value and the alignment length, all the connected nodes (proteins) collapse into a single group (cluster). A cluster that incorporates a UniProt entry inherits its annotations (GO terms that are statisti-
S548
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
cally validated, PDB structures, SCOP classifications, Pfam families, if available). Clusters can contain distantly related proteins that by this can be annotated with high confidence. Ultimately the method analyses a total of over 12 million protein sequences taken from 988 genomes and UniProt release 13. In this version HMM models of those clusters that contain PDB templates are also provided to the end-user for computing structural models of distantly related sequences. doi:10.1016/j.jbiotec.2010.09.908 [S.16] Synthetic biology approaches Corynebacterium glutamicum
to
carbon
utilization
of
V.F. Wendisch Chair of Genetics of Prokaryotes, Bielefeld University, Germany Amino acid production by Corynebacterium glutamicum amounts to about 2.5 million tons per year and, thus, is a proven large-scale biotechnological process. The traditional product spectrum has recently been widened by metabolic engineering approaches, e.g. for production of 3-aminopropionic acid or ethanol. The generally recognized as safe C. glutamicum has been shown to be robust against a variety of inhibitory compounds and to be able to efficiently co-utilize different carbon source mixtures. The characterization of genetic control mechanisms of carbon metabolism, which are distinct from those of the model bacteria E. coli and B. substilis, enabled strain development for improved carbon substrate utilization. In addition, pathways for access to new carbon sources have been engineered, e.g. for efficient use of glycerol, which arises in large quantities in the biodiesel process as major by-product of plant seed oil transesterification with methanol. Progress and future challenges of synthetic biology approaches to C. glutamicum as platform for the production of fine chemicals will be discussed. doi:10.1016/j.jbiotec.2010.09.909
References C.S.K. Reddy, R. Ghai, Rashmi, V.C. Kalia, Bioresour. Technol. 2003, 87, 137. Madison, L.L., Huisman, G.W., 1999. Microbiol. Mol. Biol. Rev. 63, 21. ˇ ´ Z., Bleha, T., 2004. Macromol. Biosci. 4, 601. Spitalsk y, Philip, S., Keshavarz, T., Roy, I., 2007. J. Chem. Technol. Biotechnol. 82, 233. Khanna, S., Srivastava, A.K., 2005. Process Biochem. 40, 607.
doi:10.1016/j.jbiotec.2010.09.910
[S.17]
[S.18]
Development of a comprehensive dynamic model for the fermentative production of poly(3-hydroxybutyrate) with tailor-made properties G. Penloglou 1,2 , Ch. Kiparissides 1,2,∗
In spite of the potential of PHAs, their introduction to the worldwide market is currently limited due to their increased production cost compared to their synthetic alternatives (Philip et al., 2007). Therefore, there is a growing need for the development of novel microbial processes with reduced production cost (Khanna and Srivastava, 2005). To this end, advanced mathematical models can provide the means to understand and control the biochemical phenomena, leading to the production of biopolymers with desirable properties, in a competitive way. In this work, an integrated metabolic/macroscopic kinetic model for the dynamic simulation of the Poly(3-hydroxybutytrate) (PHB) production in Alcaligenes latus was developed. The proposed approach includes a realistic description of cells’ metabolism, accounting for the effects of the medium composition, the culture aeration and the operating policy (batch/fed-batch). The model predicts the biomass growth, the key nutrients assimilation, the oxygen transfer/uptake rates, the PHB production rate and its molecular weight distribution (MWD). The mathematical model development is assisted by a comprehensive experimental study over the optimal PHB production giving emphasis on: (i) the assessment of key-process variables that control the PHB accumulation and its MWD and (ii) the investigation of the impact of a simple fed-batch policy on the PHB yield. Analysis of the influence of the post-treatment conditions on the efficient polymer recovery and polymer MWD accomplishes this study. Among various reports focusing on the high-yield production of PHB, the present study demonstrates a mathematical tool for the design of a production strategy of different PHB grades with tailor-made properties, under optimized conditions.
Chatzidoukas 2 , S.
Parouti 2 , C.
1
Department of Chemical Engineering, Aristotle University of Thessaloniki, Greece 2 Chemical Process Engineering Research Institute, Centre for Research and Technology Hellas6th klm. Charilaou-Thermi Road, P.O. Box 6036157001, Thermi, Thessaloniki, Greece Keywords: Alcaligenes latus; Biopolymers; Molecular weight distribution; Polyhydroxybutyrate (PHB) In view of the innumerable applications of plastics, biopolymers are urgently needed so that the adverse environmental effects of conventional plastics can be ameliorated (Reddy et al., 2003). Polyhydroxyalkanoates (PHAs) are biodegradable polymers, produced intracellularly by various bacteria from renewable sources (Madison and Huisman, 1999). Depending on their molecular characteristics various PHA grades with desired end-use properties can ˇ be produced (Spitalsk y´ and Bleha, 2004).
Systems biology characterization of engineered tomato fruits with improved carotenoid content provides novel insights on the interplay between pigment biosynthesis and post-harvest characteristics Gianfranco Diretto 1,3,∗ , Nicholas Schauer 2,3 , Alisdair Fernie 2,3 , Federico Scossa 1,3 , Antonio Matas 3,4 , Jocelyn Rose 3,4 1
Ente per le Nuove tecnologie, l’Energia e l’Ambiente (ENEA), Casaccia Research Center00123 Roma, Italy, Italy 2 eMax-Planck Institute for Molecular Plant Physiology, Am Muehlenberg 114476 Golm, Germany, Germany 3 cDept. of Plant Biology, Cornell University, Ithaca, New York 14853, United States 4 dBoyce Thompson Institute, Ithaca, New York 14853, United States Keywords: Tomato; Carotenoid; Systems biology; Gene ontology Carotenoids are secondary metabolites involved, in animals, in the prevention of several animal diseases including cancers and cardio-vascular pathologies. In plants, they play essential functions as photosynthetic pigments in leaves, secondary metabolites in fruits and flowers, and hormone precursors. Here, we report a global transcriptional, metabolomic and phenomic profiling of transgenic “Golden” tomato fruits and potato tubers accumulat-
[S.13] Design principles for information processing through signalling networks M. Schilling 1,∗ , V. Becker 1 , A. Raue 2 , T Maiwald 3 , D Winter 4 , W Lehmann 4 , W. Kolch 5 , J. Timmer 2 , U. Klingmueller 1 1
Division of Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ), Germany 2 Freiburg Center for Data Analysis and Modeling, University of Freiburg, Germany 3 Department of Systems Biology, Harvard Medical School, United States 4 Molecular Structure Analysis, German Cancer Research Center (DKFZ), Germany 5 Beatson Institute for Cancer Research & Institute for Biomedical and Life Sciences, University of Glasgow, United Kingdom Cells have to respond to changes in their environment. Alterations in extracellular signals are perceived by cell surface receptors and trigger the activation of complex intracellular signalling networks. Currently rather little is known regarding design principles that link pathway activation with cellular responses. The erythropoieting receptor (EpoR) is a cytokine receptor and the key regulator of erythropoiesis regulating proliferation, differentiation and survival of erythroid progenitor cells. A major signaling pathway activated by the EpoR is the extracellular signal regulated kinase (ERK) cascade. By combining quantitative data with mathematical modelling, we predicted and experimentally confirmed a distributive ERK phosphorylation mechanism in response to EpoR activation. Model analysis revealed that increasing one ERK isoform reduces activation of the other isoform, which was verified by protein overexpression. Furthermore we showed by statistical modelling that double-phosphorylated ERK1 attenuates proliferation beyond a certain activation level, while activated ERK2 enhances proliferation with saturation kinetics. Thus, we provide a quantitative link between previously unobservable signalling dynamics and cellular responses. doi:10.1016/j.jbiotec.2010.09.906
S547
thus interesting that Rhodospirillum rubrum produces high-levels of photosynthetic membranes (PM) completely independent of light, when grown semi-aerobically in the dark with a two-carbon substrate growth medium. On the basis of this cultivation process, we applied a systems approach using a combination of bioreactor cultivations, metabolomics, as well as mathematical modeling to develop R. rubrum for biotechnological applications. These efforts include the optimization of fed-batch processes to yield high cell densities to levels commonly employed in industry (Zeiger and Grammel, 2010). For improving the understanding of underlying metabolic and regulatory events, stoichiometric and dynamic computational models of metabolism and electron transfer chains were developed and applied (Klamt et al., 2008; Grammel and Ghosh, 2008). Furthermore, a metabolomic profile was obtained with different carbons sources which indicate additional flexibility in acetate assimilation pathways. This work is intended to open a new perspective for utilizing photosynthetic bacteria as producers in biotechnology. References Grammel, H., Ghosh, R., 2008. Redox state dynamics of ubiquinone-10 imply cooperative regulation of photosynthetic membrane expression in Rhodospirillum rubrum. J. Bacteriol. 190 (14), 4912–4921. Klamt, S., Grammel, H., Ghosh, R., Gilles, E.D., 2008. Modeling the Electron Transport Chain of Purple Nonsulfur Bacteria. Mol. Sys. Biol. 4, 156. Zeiger, L., Grammel, H., 2010. Model-based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions. Biotechnol Bioeng 105 (4), 729–739.
doi:10.1016/j.jbiotec.2010.09.907 [S.15] Extended and robust protein sequence annotation over conservative non hierarchical clusters. The Bologna Annotation Resource v 2.0 D. Piovesan 1,∗ , L. Bartoli 1 , P.L. Martelli 1 , P. Fariselli 1 , I. Rossi 2 , R. Casadio 1 1
Biocomputing Group, University of Bologna, Italy Biodec s.r.l., Bologna, Italy3 National Institute of Nuclear Physics, Bari, Italy Keywords: Protein functional annotation; Cross-genome comparison; Alignment coverage; Grid technology 2
[S.14] Development of a purple bacterium, Rhodospirillum rubrum, for applications in biotechnology – a systems biology approach H. Grammel 1,∗ , S. Klamt 1 , R. Ghosh 2 , E.D. Gilles 1 1
Max Planck Institute for Dynamics of Complex Technical Systems, Germany 2 University of Stuttgart, Biological Institute, Dept. of Bioenergetics, Germany Keywords: Rhodospirillum rubrum; Metabolomics; Computational Modeling; Process Development Anoxygenic photosynthetic purple bacteria are amongst the most versatile life forms and offer highly attractive opportunities for industrial applications. Potential products derived from intracytoplasmic photosynthetic membranes (ICM) are amongst others, photosynthetic pigments (porphyrines, carotenoids), coenzymes (Q10 ), biohydrogen and recombinant membrane proteins. Since high levels of ICM are formed at low-light intensities at anaerobic conditions most attempts to exploit purple bacteria for producing high value materials were so far conducted phototrophically, using light as energy source. However, mass cultivation of phototrophic bacteria is generally inefficient due to the inevitable limitation of light-supply when cell densities become very high. It is
Genome annotation is one of the most important issues in the genomic era. The exponential grow rate of newly sequenced genomes and proteomes urges the development of fast and reliable annotation methods, suited to exploit all the information available in curated data bases of protein sequences and structures. To this aim we developed BAR, the Bologna Annotation Resource that is now updated (available at http://microserf.biocomp.unibo.it/bar/). The basic notion is that sequences with high identity value to a counterpart can inherit the same function/s and structure, if available. What is totally new in our analysis is to cluster sequences with the constraint that sequence identity should be equal or higher than 40% on at least 90% of the pairwise alignment length. By this sequences are clustered in sets that can be annotated in terms of function and structure depending on the annotation level of the sequences within the cluster. Our method starts with on allagainst-all alignment of all the sequences in a GRID environment. The alignments are then regarded as an undirected graph and after the clustering procedure that constrains both the sequence identity value and the alignment length, all the connected nodes (proteins) collapse into a single group (cluster). A cluster that incorporates a UniProt entry inherits its annotations (GO terms that are statisti-
S548
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
cally validated, PDB structures, SCOP classifications, Pfam families, if available). Clusters can contain distantly related proteins that by this can be annotated with high confidence. Ultimately the method analyses a total of over 12 million protein sequences taken from 988 genomes and UniProt release 13. In this version HMM models of those clusters that contain PDB templates are also provided to the end-user for computing structural models of distantly related sequences. doi:10.1016/j.jbiotec.2010.09.908 [S.16] Synthetic biology approaches Corynebacterium glutamicum
to
carbon
utilization
of
V.F. Wendisch Chair of Genetics of Prokaryotes, Bielefeld University, Germany Amino acid production by Corynebacterium glutamicum amounts to about 2.5 million tons per year and, thus, is a proven large-scale biotechnological process. The traditional product spectrum has recently been widened by metabolic engineering approaches, e.g. for production of 3-aminopropionic acid or ethanol. The generally recognized as safe C. glutamicum has been shown to be robust against a variety of inhibitory compounds and to be able to efficiently co-utilize different carbon source mixtures. The characterization of genetic control mechanisms of carbon metabolism, which are distinct from those of the model bacteria E. coli and B. substilis, enabled strain development for improved carbon substrate utilization. In addition, pathways for access to new carbon sources have been engineered, e.g. for efficient use of glycerol, which arises in large quantities in the biodiesel process as major by-product of plant seed oil transesterification with methanol. Progress and future challenges of synthetic biology approaches to C. glutamicum as platform for the production of fine chemicals will be discussed. doi:10.1016/j.jbiotec.2010.09.909
References C.S.K. Reddy, R. Ghai, Rashmi, V.C. Kalia, Bioresour. Technol. 2003, 87, 137. Madison, L.L., Huisman, G.W., 1999. Microbiol. Mol. Biol. Rev. 63, 21. ˇ ´ Z., Bleha, T., 2004. Macromol. Biosci. 4, 601. Spitalsk y, Philip, S., Keshavarz, T., Roy, I., 2007. J. Chem. Technol. Biotechnol. 82, 233. Khanna, S., Srivastava, A.K., 2005. Process Biochem. 40, 607.
doi:10.1016/j.jbiotec.2010.09.910
[S.17]
[S.18]
Development of a comprehensive dynamic model for the fermentative production of poly(3-hydroxybutyrate) with tailor-made properties G. Penloglou 1,2 , Ch. Kiparissides 1,2,∗
In spite of the potential of PHAs, their introduction to the worldwide market is currently limited due to their increased production cost compared to their synthetic alternatives (Philip et al., 2007). Therefore, there is a growing need for the development of novel microbial processes with reduced production cost (Khanna and Srivastava, 2005). To this end, advanced mathematical models can provide the means to understand and control the biochemical phenomena, leading to the production of biopolymers with desirable properties, in a competitive way. In this work, an integrated metabolic/macroscopic kinetic model for the dynamic simulation of the Poly(3-hydroxybutytrate) (PHB) production in Alcaligenes latus was developed. The proposed approach includes a realistic description of cells’ metabolism, accounting for the effects of the medium composition, the culture aeration and the operating policy (batch/fed-batch). The model predicts the biomass growth, the key nutrients assimilation, the oxygen transfer/uptake rates, the PHB production rate and its molecular weight distribution (MWD). The mathematical model development is assisted by a comprehensive experimental study over the optimal PHB production giving emphasis on: (i) the assessment of key-process variables that control the PHB accumulation and its MWD and (ii) the investigation of the impact of a simple fed-batch policy on the PHB yield. Analysis of the influence of the post-treatment conditions on the efficient polymer recovery and polymer MWD accomplishes this study. Among various reports focusing on the high-yield production of PHB, the present study demonstrates a mathematical tool for the design of a production strategy of different PHB grades with tailor-made properties, under optimized conditions.
Chatzidoukas 2 , S.
Parouti 2 , C.
1
Department of Chemical Engineering, Aristotle University of Thessaloniki, Greece 2 Chemical Process Engineering Research Institute, Centre for Research and Technology Hellas6th klm. Charilaou-Thermi Road, P.O. Box 6036157001, Thermi, Thessaloniki, Greece Keywords: Alcaligenes latus; Biopolymers; Molecular weight distribution; Polyhydroxybutyrate (PHB) In view of the innumerable applications of plastics, biopolymers are urgently needed so that the adverse environmental effects of conventional plastics can be ameliorated (Reddy et al., 2003). Polyhydroxyalkanoates (PHAs) are biodegradable polymers, produced intracellularly by various bacteria from renewable sources (Madison and Huisman, 1999). Depending on their molecular characteristics various PHA grades with desired end-use properties can ˇ be produced (Spitalsk y´ and Bleha, 2004).
Systems biology characterization of engineered tomato fruits with improved carotenoid content provides novel insights on the interplay between pigment biosynthesis and post-harvest characteristics Gianfranco Diretto 1,3,∗ , Nicholas Schauer 2,3 , Alisdair Fernie 2,3 , Federico Scossa 1,3 , Antonio Matas 3,4 , Jocelyn Rose 3,4 1
Ente per le Nuove tecnologie, l’Energia e l’Ambiente (ENEA), Casaccia Research Center00123 Roma, Italy, Italy 2 eMax-Planck Institute for Molecular Plant Physiology, Am Muehlenberg 114476 Golm, Germany, Germany 3 cDept. of Plant Biology, Cornell University, Ithaca, New York 14853, United States 4 dBoyce Thompson Institute, Ithaca, New York 14853, United States Keywords: Tomato; Carotenoid; Systems biology; Gene ontology Carotenoids are secondary metabolites involved, in animals, in the prevention of several animal diseases including cancers and cardio-vascular pathologies. In plants, they play essential functions as photosynthetic pigments in leaves, secondary metabolites in fruits and flowers, and hormone precursors. Here, we report a global transcriptional, metabolomic and phenomic profiling of transgenic “Golden” tomato fruits and potato tubers accumulat-