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Systems biology of Pichia pastoris
New Biotechnology · Volume 31S · July 2014
SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION)
ACIB-5
ACIB-6
Esterases from Clostridium are involved in anaerobic degradation of synthetic polyester
Designing robust Saccharomyces cerevisiae strains against stresses encountered during bioethanol fermentations from lignocellulosic biomass
Veronika Perz 1,∗ , Veronika Perz 2 , Armin Baumschlager 2 , Klaus Bleymaier 2 , Andrzej Łyskowski 2 , Altijana Hromic 2 , Karl Gruber 3 , Carsten Sinkel 4 , Ulf Küper 4 , Melanie Bonnekessel 4 , Doris Ribitsch 2 , Georg Guebitz 5
Vinod Kumar 1,∗ , Darren Greetham 1 , Tithira Wimalasena 2 1
2
The University of Nottingham, United Kingdom Kingston Research Limited (BP and DuPont JV), United Kingdom
1
ACIB GmbH, Austria ACIB GmbH, Petersgasse 14, 8010 Graz, Austria 3 Institute of Molecular Biosciences, University of Graz, Austria 4 BASF SE, Carl-Bosch-Straße 38, 67056 Ludwigshafen, Germany 5 Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Austria 2
The anaerobic degradation of synthetic aliphatic–aromatic polyesters used in food packaging is of great importance during anaerobic digestion. Several studies have proven the biodegradability of PBAT (poly(butylene adipate-co-butylene terephthalate)) under aerobic conditions while there is only little information on anaerobic degradation. In this study, imaging analysis (CLSM, SEM) and quantification of degradation products indicated anaerobic hydrolysis of PBAT in biogas sludge. However, the detected hydrolysis rates are still too low for efficient PBAT degradation in industrial biogas plants. Consequently, hydrolysis of PBAT by enzymes from different anaerobic organisms (Clostridium species) was investigated. Therefore, various hydrolases from these organisms were successfully heterologously expressed in E. coli BL21-Gold(DE3). The kinetic parameters on the standard substrate p-nitrophenyl acetate were determined and revealed high activity of up to 700 U/mg (vmax ). Analysis of the crystal structure of one esterase from C. botulinum disclosed the presence of a Zn2+ metal ion that lies deep beneath the protein surface. The degradation of synthesized oligomeric and polymeric model substrates was studied in order to get a deeper insight into the reaction mechanisms of these new hydrolases. Esterases from C. hathewayi and C. botulinum were indeed able to hydrolyze aliphatic-aromatic polyesters like PBAT and different model substrates as indicated by HPLC/MS quantification of the hydrolysis products terephthalic acid, adipic acid, mono(4-hydroyxbutyl)terephthalate and bis(4hydroxybutyl)terephthalate. We could demonstrate that the esterases show activity under mild conditions as well as in biogas sludge. http://dx.doi.org/10.1016/j.nbt.2014.05.1619
During the pre-treatment of lignocellulosic biomass inhibitory compounds are released which can exert adverse effects on cellular growth, metabolism and ethanol production. In addition, osmotic stress caused by the high concentrations of available sugars and end-stage ethanol toxicity reduce overall rates of bioethanol fermentation. In previous studies, F1 hybrid segregants derived from clean lineage Saccharomyces cerevisiae strains were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Using a systems biology approach (QTLQuantitative Trait Locus) chromosomal loci conferring resistance against weak acid and osmotic stress were identified, and within those loci genes COX20 (acetic acid) and RCK2 (osmotic) were determined as important for stress response. In the present study, strains in which COX20 or RCK2 have either been deleted or inserted into on tetracycline induced vectors. Phenotypic microarray assessment (Biolog, US) of these strains under acetic acid, formic acid, furfural, HMF, vanillin and sorbitol stress have been determined and compared against appropriate controls. Results have highlighted that presence of COX20 improves tolerance to weak acids while RCK2 gene conferred resistance to osmotic stress. The presence of either gene also enhanced the tolerance against furanic compounds such as furfural and HMF. http://dx.doi.org/10.1016/j.nbt.2014.05.1620
ACIB-7 Systems biology of Pichia pastoris Brigitte Gasser Dep. of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Austria
Pichia pastoris is the most frequently used yeast system for heterologous protein production today, however, the toolbox of available genetic elements is rather limited. To enable more robust and cost-effective production processes for biopharmaceutical proteins and for industrial enzymes, it is crucial to understand the molecular physiology of the host, and the specific limitations that the product may exert on expression. Instead of classical genetic approaches, we applied systems biology tools to improve several aspects of the P. pastoris production platform. Combined transcriptomics, proteomics, metabolomics and flux data were used to investigate the interplay between protein production and cell metabolism. Thereby we gained insight in key regulatory effects exerted during heterologous protein production processes. These genome scale data were then further exploited for the identification of novel regulatory elements and
www.elsevier.com/locate/nbt S3
SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION)
New Biotechnology · Volume 31S · July 2014
the prediction of cell engineering targets for improved productivity and robustness. Additionally, the endowment of genes involved in the protein secretory pathway was analysed in a comparative genomics approach, as productivities are often limited at the level of protein folding and secretion.
ACIB-9
http://dx.doi.org/10.1016/j.nbt.2014.05.1621
Martina Baumann 1,∗ , Elisabeth Gludovacz 2 , Sabine Vcelar 1 , Nicole Borth 3
Utilization of recombinase mediated cassette exchange (RMCE) for the generation of recombinant CHO cell lines with defined expression properties
1
ACIB-8 Cross-species comparison of recombinant protein secretion in CHO cells and Pichia pastoris Nils Landes 1,∗ , Andreas Maccani 2 , Christian Leitner 3 , Michael Maurer 4 , Alexandra B. Graf 4 , Minoska Valli 2 , Clemens Gruber 5 , Gerda Modarres 4 , Friedrich Altmann 5 , Brigitte Gasser 3 , Wolfgang Ernst 3 , Renate Kunert 3 , Diethard Mattanovich 3 1
ACIB, Austria Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria 3 Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria 4 School of Bioengineering, University of Applied Sciences FH Campus Wien, Vienna, Austria 5 Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Austria 2
Chinese hamster ovary (CHO) cells are currently the workhorses in biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. Although a vast number of research studies has focused on characterization of each of the individual expression system, information on direct cross-species comparative studies are limited. Here we present a comprehensive comparison of recombinant P. pastoris strains and CHO cell lines, including bioprocess engineering aspects as well as systems biology approaches. Two model proteins of different complexity were chosen: monomeric and non-glycosylated human serum albumin and a more complex 3D6 single-chain Fv-Fc fusion antibody (3D6scFvFc), which is secreted as a homodimer and contains the Fc-specific glycosylation sites. High and low producing strains or cell lines of the two model proteins were established and characterized in lab-scale bioreactor cultivations. To evaluate the performance of each expression system, fed batch cultivations were performed. The production processes were characterized by monitoring biomass and product formation as well as product quality. The two host systems were then compared regarding their biomass specific secretion rates and space-time yields of the processes. Obtained results show that P. pastoris is the preferred host system for production of HSA, whereas CHO cells are more suited for the production of a complex molecule like the 3D6 scFv-Fc fusion protein. For transcriptomics and proteomics analysis steady state samples from chemostat cultures were taken. Similarities and differences between the investigated host systems as well as protein specific effects will be presented. http://dx.doi.org/10.1016/j.nbt.2014.05.1622
S4
www.elsevier.com/locate/nbt
ACIB, Austria BOKU, Austria 3 BOKU/ACIB, Austria 2
For diverse applications, including generation of recombinant production cell lines, stable integration of transgenes into the genome of the host cell is a pre-requisite. The precise position of the integrated transgene is a major determining factor, both for gene expression level and long-term stability, which makes the screening for a suitable cell clone very time-and labour intensive. The utilization of site-specific Recombinase Mediated Cassette Exchange (RMCE) opened up the possibility to transfer the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. In this study we developed a strategy supporting the identification of recombinant Chinese Hamster Ovary (CHO) cells with integration sites favourable to persistent high transgene expression and good gene-exchangeability. A sortable reporter gene (CD4) with a leaky start codon, flanked by heterospecific FRT (Flippase recognition targets) sequences was stably transfected into CHO cells. The leaky start codon reduces translation efficiency and allows sorting for the highest transcription rates, while the FRT sites mediate subsequent exchange of the expression cassette. Two rounds of RMCE followed by FACS sorting for top producers were performed to select for sites that allow reliable cassette exchange besides high transgene expression. The resulting master cell line can be used repeatedly for insertion of the GOI without major genetic alterations. A combination of chemical selection using alternative selection markers and FACS sorting for absence of CD4 expression as criterion for successful gene exchange enables fast establishment of recombinant cell lines with predictable expression properties. http://dx.doi.org/10.1016/j.nbt.2014.05.1623
ACIB-10 Integrated continuous refolding and precipitation of proteins in a tubular reactor Siqi Pan ∗ , Monika Zelger, Rainer Hahn Austrian Centre of Industrial Biotechnology, Austria
Large amounts of therapeutic proteins expressed in Escherichia coli as inclusion bodies have created a bottleneck in downstream processing. Process integration of continuous processes is one way to alleviate this bottleneck. In view of this consideration, laboratory scale tubular reactors for the continuous processing of recombinant proteins were developed. Benefits include faster mixing, high design flexibility and efficient heat transfer. With these advantages in mind, refolding strategies like pulsed refolding and
SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION)
ACIB-5
ACIB-6
Esterases from Clostridium are involved in anaerobic degradation of synthetic polyester
Designing robust Saccharomyces cerevisiae strains against stresses encountered during bioethanol fermentations from lignocellulosic biomass
Veronika Perz 1,∗ , Veronika Perz 2 , Armin Baumschlager 2 , Klaus Bleymaier 2 , Andrzej Łyskowski 2 , Altijana Hromic 2 , Karl Gruber 3 , Carsten Sinkel 4 , Ulf Küper 4 , Melanie Bonnekessel 4 , Doris Ribitsch 2 , Georg Guebitz 5
Vinod Kumar 1,∗ , Darren Greetham 1 , Tithira Wimalasena 2 1
2
The University of Nottingham, United Kingdom Kingston Research Limited (BP and DuPont JV), United Kingdom
1
ACIB GmbH, Austria ACIB GmbH, Petersgasse 14, 8010 Graz, Austria 3 Institute of Molecular Biosciences, University of Graz, Austria 4 BASF SE, Carl-Bosch-Straße 38, 67056 Ludwigshafen, Germany 5 Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Austria 2
The anaerobic degradation of synthetic aliphatic–aromatic polyesters used in food packaging is of great importance during anaerobic digestion. Several studies have proven the biodegradability of PBAT (poly(butylene adipate-co-butylene terephthalate)) under aerobic conditions while there is only little information on anaerobic degradation. In this study, imaging analysis (CLSM, SEM) and quantification of degradation products indicated anaerobic hydrolysis of PBAT in biogas sludge. However, the detected hydrolysis rates are still too low for efficient PBAT degradation in industrial biogas plants. Consequently, hydrolysis of PBAT by enzymes from different anaerobic organisms (Clostridium species) was investigated. Therefore, various hydrolases from these organisms were successfully heterologously expressed in E. coli BL21-Gold(DE3). The kinetic parameters on the standard substrate p-nitrophenyl acetate were determined and revealed high activity of up to 700 U/mg (vmax ). Analysis of the crystal structure of one esterase from C. botulinum disclosed the presence of a Zn2+ metal ion that lies deep beneath the protein surface. The degradation of synthesized oligomeric and polymeric model substrates was studied in order to get a deeper insight into the reaction mechanisms of these new hydrolases. Esterases from C. hathewayi and C. botulinum were indeed able to hydrolyze aliphatic-aromatic polyesters like PBAT and different model substrates as indicated by HPLC/MS quantification of the hydrolysis products terephthalic acid, adipic acid, mono(4-hydroyxbutyl)terephthalate and bis(4hydroxybutyl)terephthalate. We could demonstrate that the esterases show activity under mild conditions as well as in biogas sludge. http://dx.doi.org/10.1016/j.nbt.2014.05.1619
During the pre-treatment of lignocellulosic biomass inhibitory compounds are released which can exert adverse effects on cellular growth, metabolism and ethanol production. In addition, osmotic stress caused by the high concentrations of available sugars and end-stage ethanol toxicity reduce overall rates of bioethanol fermentation. In previous studies, F1 hybrid segregants derived from clean lineage Saccharomyces cerevisiae strains were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Using a systems biology approach (QTLQuantitative Trait Locus) chromosomal loci conferring resistance against weak acid and osmotic stress were identified, and within those loci genes COX20 (acetic acid) and RCK2 (osmotic) were determined as important for stress response. In the present study, strains in which COX20 or RCK2 have either been deleted or inserted into on tetracycline induced vectors. Phenotypic microarray assessment (Biolog, US) of these strains under acetic acid, formic acid, furfural, HMF, vanillin and sorbitol stress have been determined and compared against appropriate controls. Results have highlighted that presence of COX20 improves tolerance to weak acids while RCK2 gene conferred resistance to osmotic stress. The presence of either gene also enhanced the tolerance against furanic compounds such as furfural and HMF. http://dx.doi.org/10.1016/j.nbt.2014.05.1620
ACIB-7 Systems biology of Pichia pastoris Brigitte Gasser Dep. of Biotechnology, BOKU University of Natural Resources and Life Sciences Vienna, Austria
Pichia pastoris is the most frequently used yeast system for heterologous protein production today, however, the toolbox of available genetic elements is rather limited. To enable more robust and cost-effective production processes for biopharmaceutical proteins and for industrial enzymes, it is crucial to understand the molecular physiology of the host, and the specific limitations that the product may exert on expression. Instead of classical genetic approaches, we applied systems biology tools to improve several aspects of the P. pastoris production platform. Combined transcriptomics, proteomics, metabolomics and flux data were used to investigate the interplay between protein production and cell metabolism. Thereby we gained insight in key regulatory effects exerted during heterologous protein production processes. These genome scale data were then further exploited for the identification of novel regulatory elements and
www.elsevier.com/locate/nbt S3
SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION)
New Biotechnology · Volume 31S · July 2014
the prediction of cell engineering targets for improved productivity and robustness. Additionally, the endowment of genes involved in the protein secretory pathway was analysed in a comparative genomics approach, as productivities are often limited at the level of protein folding and secretion.
ACIB-9
http://dx.doi.org/10.1016/j.nbt.2014.05.1621
Martina Baumann 1,∗ , Elisabeth Gludovacz 2 , Sabine Vcelar 1 , Nicole Borth 3
Utilization of recombinase mediated cassette exchange (RMCE) for the generation of recombinant CHO cell lines with defined expression properties
1
ACIB-8 Cross-species comparison of recombinant protein secretion in CHO cells and Pichia pastoris Nils Landes 1,∗ , Andreas Maccani 2 , Christian Leitner 3 , Michael Maurer 4 , Alexandra B. Graf 4 , Minoska Valli 2 , Clemens Gruber 5 , Gerda Modarres 4 , Friedrich Altmann 5 , Brigitte Gasser 3 , Wolfgang Ernst 3 , Renate Kunert 3 , Diethard Mattanovich 3 1
ACIB, Austria Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria 3 Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria 4 School of Bioengineering, University of Applied Sciences FH Campus Wien, Vienna, Austria 5 Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Austria 2
Chinese hamster ovary (CHO) cells are currently the workhorses in biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. Although a vast number of research studies has focused on characterization of each of the individual expression system, information on direct cross-species comparative studies are limited. Here we present a comprehensive comparison of recombinant P. pastoris strains and CHO cell lines, including bioprocess engineering aspects as well as systems biology approaches. Two model proteins of different complexity were chosen: monomeric and non-glycosylated human serum albumin and a more complex 3D6 single-chain Fv-Fc fusion antibody (3D6scFvFc), which is secreted as a homodimer and contains the Fc-specific glycosylation sites. High and low producing strains or cell lines of the two model proteins were established and characterized in lab-scale bioreactor cultivations. To evaluate the performance of each expression system, fed batch cultivations were performed. The production processes were characterized by monitoring biomass and product formation as well as product quality. The two host systems were then compared regarding their biomass specific secretion rates and space-time yields of the processes. Obtained results show that P. pastoris is the preferred host system for production of HSA, whereas CHO cells are more suited for the production of a complex molecule like the 3D6 scFv-Fc fusion protein. For transcriptomics and proteomics analysis steady state samples from chemostat cultures were taken. Similarities and differences between the investigated host systems as well as protein specific effects will be presented. http://dx.doi.org/10.1016/j.nbt.2014.05.1622
S4
www.elsevier.com/locate/nbt
ACIB, Austria BOKU, Austria 3 BOKU/ACIB, Austria 2
For diverse applications, including generation of recombinant production cell lines, stable integration of transgenes into the genome of the host cell is a pre-requisite. The precise position of the integrated transgene is a major determining factor, both for gene expression level and long-term stability, which makes the screening for a suitable cell clone very time-and labour intensive. The utilization of site-specific Recombinase Mediated Cassette Exchange (RMCE) opened up the possibility to transfer the gene of interest (GOI) into pre-selected genomic locations with defined expression properties. In this study we developed a strategy supporting the identification of recombinant Chinese Hamster Ovary (CHO) cells with integration sites favourable to persistent high transgene expression and good gene-exchangeability. A sortable reporter gene (CD4) with a leaky start codon, flanked by heterospecific FRT (Flippase recognition targets) sequences was stably transfected into CHO cells. The leaky start codon reduces translation efficiency and allows sorting for the highest transcription rates, while the FRT sites mediate subsequent exchange of the expression cassette. Two rounds of RMCE followed by FACS sorting for top producers were performed to select for sites that allow reliable cassette exchange besides high transgene expression. The resulting master cell line can be used repeatedly for insertion of the GOI without major genetic alterations. A combination of chemical selection using alternative selection markers and FACS sorting for absence of CD4 expression as criterion for successful gene exchange enables fast establishment of recombinant cell lines with predictable expression properties. http://dx.doi.org/10.1016/j.nbt.2014.05.1623
ACIB-10 Integrated continuous refolding and precipitation of proteins in a tubular reactor Siqi Pan ∗ , Monika Zelger, Rainer Hahn Austrian Centre of Industrial Biotechnology, Austria
Large amounts of therapeutic proteins expressed in Escherichia coli as inclusion bodies have created a bottleneck in downstream processing. Process integration of continuous processes is one way to alleviate this bottleneck. In view of this consideration, laboratory scale tubular reactors for the continuous processing of recombinant proteins were developed. Benefits include faster mixing, high design flexibility and efficient heat transfer. With these advantages in mind, refolding strategies like pulsed refolding and