The Experts below are selected from a list of 54681 Experts worldwide ranked by ideXlab platform
Patrick Fickers - One of the best experts on this subject based on the ideXlab platform.
-
Integrating metabolic modeling and population heterogeneity analysis into optimizing Recombinant Protein Production by Komagataella (Pichia) pastoris
Applied Microbiology and Biotechnology, 2018Co-Authors: Chrispian W. Theron, Frank Delvigne, Julio Berrios, Patrick FickersAbstract:The methylotrophic yeast Komagataella ( Pichia ) pastoris has become one of the most utilized cell factories for the Production of Recombinant Proteins over the last three decades. This success story is linked to its specific physiological traits, i.e., the ability to grow at high cell density in inexpensive culture medium and to secrete Proteins at high yield. Exploiting methanol metabolism is at the core of most P. pastoris -based processes but comes with its own challenges. Co-feeding cultures with glycerol/sorbitol and methanol is a promising approach, which can benefit from improved understanding and prediction of metabolic response. The development of profitable processes relies on the construction and selection of efficient producing strains from less efficient ones but also depends on the ability to master the bioreactor process itself. More specifically, how a bioreactor processes could be monitored and controlled to obtain high yield of Production. In this review, new perspectives are detailed regarding a multi-faceted approach to Recombinant Protein Production processes by P. pastoris ; including gaining improved understanding of the metabolic pathways involved, accounting for variations in transcriptional and translational efficiency at the single cell level and efficient monitoring and control of methanol levels at the bioreactor level.
-
Deciphering how LIP2 and POX2 promoters can optimally regulate Recombinant Protein Production in the yeast Yarrowia lipolytica
Microbial Cell Factories, 2016Co-Authors: Hosni Sassi, Tambi Kar, Anne-Marie Crutz- Coq, Sebastien Steels, Frank Delvigne, Jean-marc Nicaud, Patrick FickersAbstract:BackgroundIn recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing Recombinant Proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for Protein Production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, Production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting Recombinant Protein Production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study’s aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources.ResultspPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any Recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic acid. Indeed, no distinct subpopulations that specialized on glucose versus oleic acid were observed; such an outcome would have led to producer and non-producer phenotypes. In medium containing both glucose and oleic acid, cells tended to directly metabolize oleic acid instead of storing it in lipid bodies.ConclusionsThis study found that pLIP2 is a promoter of choice as compared to pPOX2 to drive gene expression for Recombinant Protein Production by Y. lipolytica used as cell factory.
Francisco Valero - One of the best experts on this subject based on the ideXlab platform.
-
rationale based selection of optimal operating strategies and gene dosage impact on Recombinant Protein Production in komagataella phaffii pichia pastoris
Microbial Biotechnology, 2020Co-Authors: Miguel Angel Nietotaype, Javier Garrigosmartinez, Marc Sanchezfarrando, Francisco Valero, Xavier Garciaortega, Jose Luis MontesinosseguiAbstract:Its features as a microbial and eukaryotic organism have turned Komagataella phaffii (Pichia pastoris) into an emerging cell factory for Recombinant Protein Production (RPP). As a key step of the bioprocess development, this work aimed to demonstrate the importance of tailor designing the cultivation strategy according to the Production kinetics of the cell factory. For this purpose, K. phaffii clones constitutively expressing (PGAP ) Candida rugosa lipase 1 (Crl1) with different gene dosage were used as models in continuous and fed-batch cultures. Production parameters were much greater with a multicopy clone (MCC) than with the single-copy clone (SCC). Regarding Production kinetics, the specific product generation rate (qP ) increased linearly with increasing specific growth rate (µ) in SCC; by contrast, qP exhibited saturation in MCC. A transcriptional analysis in chemostat cultures suggested the presence of eventual post-transcriptional bottlenecks in MCC. After the strain characterization, in order to fulfil overall development of the bioprocess, the performance of both clones was also evaluated in fed-batch mode. Strikingly, different optimal strategies were determined for both models due to the different Production kinetic patterns observed as a trade-off for product titre, yields and productivity. The combined effect of gene dosage and adequate µ enables rational process development with a view to optimize K. phaffii RPP bioprocesses.
-
rational development of bioprocess engineering strategies for Recombinant Protein Production in pichia pastoris komagataella phaffii using the methanol free gap promoter where do we stand
New Biotechnology, 2019Co-Authors: Xavier Garciaortega, Pau Ferrer, Jose Luis Montesinossegui, Elena Camara, Joan Albiol, Francisco ValeroAbstract:The increasing demand for Recombinant Proteins for a wide range of applications, from biopharmaceutical Protein complexes to industrial enzymes, is leading to important growth in this market. Among the different efficient host organism alternatives commonly used for Protein Production, the yeast Pichia pastoris (Komagataella phaffii) is currently considered to be one of the most effective and versatile expression platforms. The promising features of this cell factory are giving rise to interesting studies covering the different aspects that contribute to improving the bioprocess efficiency, from strain engineering to bioprocess engineering. The numerous drawbacks of using methanol in industrial processes are driving interest towards methanol-free alternatives, among which the GAP promoter-based systems stand out. The aim of this work is to present the most promising innovative developments in operational strategies based on rational approaches through bioprocess engineering tools. This rational design should be based on physiological characterization of the producing strains under bioprocess conditions and its interrelation with specific rates. This review focuses on understanding the key factors that can enhance Recombinant Protein Production in Pichia pastoris; they are the basis for a further discussion on future industrial applications with the aim of developing scalable alternative strategies that maximize yields and productivity.
-
a step forward to improve Recombinant Protein Production in pichia pastoris from specific growth rate effect on Protein secretion to carbon starving conditions as advanced strategy
Process Biochemistry, 2016Co-Authors: Xavier Garciaortega, Pau Ferrer, J L Montesinos, Nuria Adelantado, Francisco ValeroAbstract:The Recombinant Protein Production platform based on the GAP promoter and Pichia pastoris as a host has become a very promising system from an industrial point of view. The need for highly productive bioprocesses gives grounds for the optimization of fermentation strategies maximizing yields and/or productivities, which are often associated with cell growth. Coherent with previous studies, a positive effect of high specific growth rate (μ) on the productivity was observed in carbon-limited chemostat cultivations secreting an antibody fragment. Notably, no significant impact of this factor could be observed in the balance intra- and extracellular of the product. Accordingly, fed-batch cultures operating at a constant high μ were conducted. Furthermore, short carbon-starving periods were introduced along the exponential substrate feeding phase. Strikingly, it was observed an important increase of specific Production rate (qP) during such short carbon-starving periods in relation to the exponential substrate feeding intervals. Therefore, the application of carbon-starving periods as an innovative operational strategy was proposed, resulting into increments up to 50% of both yields and total Production. The implementation of the proposed substrate feeding profiles should be complementary to cell engineering strategies to improve the relation qP vs μ, thereby enhancing the overall bioprocess efficiency.
-
combined effect of the methanol utilization mut phenotype and gene dosage on Recombinant Protein Production in pichia pastoris fed batch cultures
Journal of Biotechnology, 2005Co-Authors: Alicia Serrano, Pau Ferrer, J L Montesinos, James M Cregg, Francisco ValeroAbstract:An important number of heterologous Proteins have been produced in the methylotrophic yeast Pichia pastoris using the alcohol oxidase promoter. Two factors that drastically influence Protein Production and cultivation process development in this system are gene dosage and methanol assimilation capacity of the host strain (Mut phenotype). Using a battery of four strains which secrete a Rhizopus oryzaelipase (ROL), the combined effects of gene dosage and Mut phenotype on Recombinant Protein Production in Pichia pastoris was studied in fed-batch cultures. Regarding the effect of phenotype, the specific productivity and the YP/X were 1.29- and 2.34-fold higher for Mut s ROL single copy strain than for Mut + ROL single copy strain. On the contrary, the productivity of Mut + ROL single copy strain was 1.34-fold higher than Mut s ROL single copy strain. An increase in ROL gene dosage seems to negatively affect cell's performance in bioreactor cultures, particularly in Mut s strains. Overall, the Mut s strain may be still advantageous to use because it allows for easier process control strategies. © 2005 Elsevier B.V. All rights reserved.
-
combined effect of the methanol utilization mut phenotype and gene dosage on Recombinant Protein Production in pichia pastoris fed batch cultures
Journal of Biotechnology, 2005Co-Authors: Oriol Cos, Pau Ferrer, Alicia Serrano, J L Montesinos, James M Cregg, Francisco ValeroAbstract:An important number of heterologous Proteins have been produced in the methylotrophic yeast Pichia pastoris using the alcohol oxidase promoter. Two factors that drastically influence Protein Production and cultivation process development in this system are gene dosage and methanol assimilation capacity of the host strain (Mut phenotype). Using a battery of four strains which secrete a Rhizopus oryzae lipase (ROL), the combined effects of gene dosage and Mut phenotype on Recombinant Protein Production in Pichia pastoris was studied in fed-batch cultures. Regarding the effect of phenotype, the specific productivity and the Y(P/X) were 1.29- and 2.34-fold higher for Mut(s)ROL single copy strain than for Mut+ROL single copy strain. On the contrary, the productivity of Mut+ROL single copy strain was 1.34-fold higher than Mut(s)ROL single copy strain. An increase in ROL gene dosage seems to negatively affect cell's performance in bioreactor cultures, particularly in Mut(s) strains. Overall, the Mut(s) strain may be still advantageous to use because it allows for easier process control strategies.
Frank Delvigne - One of the best experts on this subject based on the ideXlab platform.
-
Integrating metabolic modeling and population heterogeneity analysis into optimizing Recombinant Protein Production by Komagataella (Pichia) pastoris
Applied Microbiology and Biotechnology, 2018Co-Authors: Chrispian W. Theron, Frank Delvigne, Julio Berrios, Patrick FickersAbstract:The methylotrophic yeast Komagataella ( Pichia ) pastoris has become one of the most utilized cell factories for the Production of Recombinant Proteins over the last three decades. This success story is linked to its specific physiological traits, i.e., the ability to grow at high cell density in inexpensive culture medium and to secrete Proteins at high yield. Exploiting methanol metabolism is at the core of most P. pastoris -based processes but comes with its own challenges. Co-feeding cultures with glycerol/sorbitol and methanol is a promising approach, which can benefit from improved understanding and prediction of metabolic response. The development of profitable processes relies on the construction and selection of efficient producing strains from less efficient ones but also depends on the ability to master the bioreactor process itself. More specifically, how a bioreactor processes could be monitored and controlled to obtain high yield of Production. In this review, new perspectives are detailed regarding a multi-faceted approach to Recombinant Protein Production processes by P. pastoris ; including gaining improved understanding of the metabolic pathways involved, accounting for variations in transcriptional and translational efficiency at the single cell level and efficient monitoring and control of methanol levels at the bioreactor level.
-
Deciphering how LIP2 and POX2 promoters can optimally regulate Recombinant Protein Production in the yeast Yarrowia lipolytica
Microbial Cell Factories, 2016Co-Authors: Hosni Sassi, Tambi Kar, Anne-Marie Crutz- Coq, Sebastien Steels, Frank Delvigne, Jean-marc Nicaud, Patrick FickersAbstract:BackgroundIn recent years, the non-conventional model yeast species Yarrowia lipolytica has received much attention because it is a useful cell factory for producing Recombinant Proteins. In this species, expression vectors involving LIP2 and POX2 promoters have been developed and used successfully for Protein Production at yields similar to or even higher than those of other cell factories, such as Pichia pastoris. However, Production processes involving these promoters can be difficult to manage, especially if carried out at large scales in fed-batch bioreactors, because they require hydrophobic inducers, such as oleic acid or methyl oleate. Thus, the challenge has become to reduce loads of hydrophobic substrates while simultaneously promoting Recombinant Protein Production. One possible solution is to replace a portion of the inducer with a co-substrate that can serve as an alternative energy source. However, implementing such an approach would require detailed knowledge of how carbon sources impact promoter regulation, which is surprisingly still lacking for the LIP2 and POX2 promoters. This study’s aim was thus to better characterize promoter regulation and cell metabolism in Y. lipolytica cultures grown in media supplemented with different carbon sources.ResultspPOX2 induction could be detected when glucose or glycerol was used as sole carbon source, which meant these carbon source could not prevent promoter induction. In addition, when a mixture of glucose and oleic acid was used in complex medium, pPOX2 induction level was lower that that of pLIP2. In contrast, pLIP2 induction was absent when glucose was present in the culture medium, which meant that cell growth could occur without any Recombinant gene expression. When a 40/60 mixture of glucose and oleic acid (w/w) was used, a tenfold increase in promoter induction, as compared to when an oleic-acid-only medium was observed. It was also clear that individual cells were adapting metabolically to use both glucose and oleic acid. Indeed, no distinct subpopulations that specialized on glucose versus oleic acid were observed; such an outcome would have led to producer and non-producer phenotypes. In medium containing both glucose and oleic acid, cells tended to directly metabolize oleic acid instead of storing it in lipid bodies.ConclusionsThis study found that pLIP2 is a promoter of choice as compared to pPOX2 to drive gene expression for Recombinant Protein Production by Y. lipolytica used as cell factory.
Peter Neubauer - One of the best experts on this subject based on the ideXlab platform.
-
high cell density cultivation and Recombinant Protein Production with escherichia coli in a rocking motion type bioreactor
Microbial Cell Factories, 2010Co-Authors: Julia Glazyrina, Evamaria Materne, Thomas Dreher, Dirk Storm, Stefan Junne, Thorsten Adams, Gerhard Greller, Peter NeubauerAbstract:Background Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale Production of Recombinant Proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and Recombinant Protein Production.
-
high cell density cultivation and Recombinant Protein Production with escherichia coli in a rocking motion type bioreactor
Microbial Cell Factories, 2010Co-Authors: Julia Glazyrina, Evamaria Materne, Thomas Dreher, Dirk Storm, Stefan Junne, Thorsten Adams, Gerhard Greller, Peter NeubauerAbstract:Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale Production of Recombinant Proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and Recombinant Protein Production. Cultivation of E. coli in the BIOSTAT® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD600) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD600 of 60 (20 g L-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase® Flo, OD600 of 30 (10 g L-1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase® Flo also worked well in the CultiBag RM system with a Recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for Recombinant Protein Production also in a rocking-motion-type bioreactor. Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and Recombinant Protein Production. The BIOSTAT® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.
Hongying Chen - One of the best experts on this subject based on the ideXlab platform.
-
development of a baculovirus vector carrying a small hairpin rna for suppression of sf caspase 1 expression and improvement of Recombinant Protein Production
BMC Biotechnology, 2018Co-Authors: Xiaoyue Zhang, Keyan Xu, Yanmei Ou, Xiaodong Xu, Hongying ChenAbstract:The Baculovirus expression vector system (BEVS) is a transient expression platform for Recombinant Protein Production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of Protein expression. Previous reports have demonstrated the enhancement of Protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the Recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent Protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter Proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous Proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter Proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated Protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved Protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the Production of active Proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of Protein expression in different insect cell lines that can be infected by AcMNPV.
-
Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of Recombinant Protein Production
BMC, 2018Co-Authors: Xiaoyue Zhang, Hongying ChenAbstract:Abstract Background The Baculovirus expression vector system (BEVS) is a transient expression platform for Recombinant Protein Production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of Protein expression. Previous reports have demonstrated the enhancement of Protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. Results In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the Recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent Protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter Proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous Proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter Proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated Protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved Protein folding rather than translation efficiency at the very late stage of baculovirus infection. Conclusions The baculovirus vector developed in this study would be a useful tool for the Production of active Proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of Protein expression in different insect cell lines that can be infected by AcMNPV