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Recombinant protein production by Mut+ strain of Pichia pastoris using dual carbon sources: methanol and sorbitol
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
In the studies involving other microorganisms it was shown that addition of carboxylic acids with carbon chains containing five and more carbons as substrate can induce incorporation of the respective -hydroxy acids into the PHA. In our experiments luminous bacteria were grown on media supplemented with valeric acid. PHAs synthesized by the most of studied strains of P. leiognathi contained significantly larger amounts of -hydroxyvaleric acid in such conditions. It was shown that V. fisheri, strain 2089, included up to 1.5% -hydroxyvalerate in PHAs. For V. harveyi influence of valeric acid on polymer composition was not detected. Also, no minor hydroxy acids were found in the PHAs produced by P. phosphoreum regardless of the addition of valerate. Thus, P. leiognathi and V. fisheri cells grown in the medium supplemented with valerate, which is hydroxyvalerate precursor substrate, can produce polymers containing increased amounts of this hydroxy acid. By contrast, P. phosphoreum strains were found unable to synthesize multi-component PHAs. doi:10.1016/j.nbt.2009.06.285
2.1.039 Recombinant protein production by Mut+ strain of Pichia pastoris using dual carbon sources: methanol and sorbitol P. Calik 1,∗ , E. Celik 1 , S.G. Oliver 2 1
2
Middle East Technical University, Ankara, Turkey Cambridge, United Kingdom
The methylotrophic yeast Pichia pastoris has become a popular host for the expression of recombinant proteins, due to its ability to produce foreign proteins at high levels using one of the strongest and most tightly regulated eukaryotic promoters, alcohol oxidase I (AOX1). In fermentations by P. pastoris, methanol is used not only as the carbon and energy source, but also as an inducer of the expression of recombinant proteins; and because, at high concentrations it inhibits growth, fed-batch operations have been preferred. Moreover, fed-batch strategy for Mut+ (methanol utilization positive) strain of P. pastoris using mixed substrates of glycerol and methanol has been widely used in the past decade to increase productivity, cell density, and reduce the induction time. However, the optimal level of protein expression is not achievable with mixtures of glycerol and methanol, due to a partial repression of the AOX1 promoter by glycerol, which may result in lower specific productivities of r-protein. By contrast, sorbitol is a non-repressing carbon source for AOX1 promoter, thus sorbitol accumulation during the induction phase does not affect the expression level of r-protein. In this study, batch-wise sorbitol addition as a co-substrate at the induction phase of methanol fed-batch fermentation by P. pastoris (Mut+ ) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. In the presence of sorbitol, 130 mg L−1 of recombinant human erythropoietin (rHuEPO) was produced at t = 24 hours, compared to 80 mg L−1 of rHuEPO (t = 24 hours) on methanol alone. Considering many other advantages to be presented, this S60
www.elsevier.com/locate/nbt
work demonstrates the ease and efficiency of incorporating sorbitol to the fermentations by the popular methylotrophic yeast P. pastoris, for the production of any bio-product. doi:10.1016/j.nbt.2009.06.286
2.1.040 Artificial pathway design: from enzyme discovery to designer bugs J. Eck ∗ , J. Mampel, G. Meurer BRAIN Aktiengesellschaft, 64673 Zwingenberg, Germany
Industrial ‘white’ biotechnology is regarded as a central feature of the sustainable economic future of modern industrialized societies. Highly effective enzymes, heavily engineered microorganisms and ‘designer bugs’ promise improvement for existing processes or could enable novel product ideas. For any industrial application, enzymes and biosynthetic pathways need to function sufficiently well according to several application-specific performance parameters. Instead of designing a process to fit a mediocre enzyme, it is conceivable that a comprehensive access to the microbial diversity might be used to find a suitable natural enzyme(s) that optimally fits process requirements. In view of multi-step bioconversions and the production of specialty- and bulk chemicals from renewable resources current technologies and screening strategies for the development of optimised biocatalysts from microbial biodiversity as well as from ‘metagenome’ libraries will be presented. To tap into the next generation biocatalysis using engineered ‘designer’ microorganisms for multi-step bioconversions, it is necessary to move into the construction of artificial operons and the heterologous expression of modified biosynthetic pathways. As a case study the construction of designer microorganisms for the production of 1,2-propanediol will be discussed. doi:10.1016/j.nbt.2009.06.287
2.1.041 Preliminary characterization of a beta-glucosidase from a Pichia anomala yeast strain of olive brine C. Restuccia ∗ , R. Palmeri, S. Muccilli, C.L. Randazzo, C. Caggia, G. Spagna DOFATA-Sezione Tecnologie Agroalimentari, University of Catania, Italy
An interesting field of -glucosidase (G, EC 3.2.1.21) application in food processing is production of naturally fermented table olives and olive oil. Olives have a bitter compound named oleuropein, which must be eliminated during the olive elaboration process. Nowadays, the most usual industrial debittering method consists of treating the fruits with a NaOH solution. However, these technology presents huge drawbacks due to the highly contaminant discharges generated; moreover, wastewaters originating from table olives processing pose an important environmental threat, as they are characterized by a very high organic load and
ABSTRACTS
In the studies involving other microorganisms it was shown that addition of carboxylic acids with carbon chains containing five and more carbons as substrate can induce incorporation of the respective -hydroxy acids into the PHA. In our experiments luminous bacteria were grown on media supplemented with valeric acid. PHAs synthesized by the most of studied strains of P. leiognathi contained significantly larger amounts of -hydroxyvaleric acid in such conditions. It was shown that V. fisheri, strain 2089, included up to 1.5% -hydroxyvalerate in PHAs. For V. harveyi influence of valeric acid on polymer composition was not detected. Also, no minor hydroxy acids were found in the PHAs produced by P. phosphoreum regardless of the addition of valerate. Thus, P. leiognathi and V. fisheri cells grown in the medium supplemented with valerate, which is hydroxyvalerate precursor substrate, can produce polymers containing increased amounts of this hydroxy acid. By contrast, P. phosphoreum strains were found unable to synthesize multi-component PHAs. doi:10.1016/j.nbt.2009.06.285
2.1.039 Recombinant protein production by Mut+ strain of Pichia pastoris using dual carbon sources: methanol and sorbitol P. Calik 1,∗ , E. Celik 1 , S.G. Oliver 2 1
2
Middle East Technical University, Ankara, Turkey Cambridge, United Kingdom
The methylotrophic yeast Pichia pastoris has become a popular host for the expression of recombinant proteins, due to its ability to produce foreign proteins at high levels using one of the strongest and most tightly regulated eukaryotic promoters, alcohol oxidase I (AOX1). In fermentations by P. pastoris, methanol is used not only as the carbon and energy source, but also as an inducer of the expression of recombinant proteins; and because, at high concentrations it inhibits growth, fed-batch operations have been preferred. Moreover, fed-batch strategy for Mut+ (methanol utilization positive) strain of P. pastoris using mixed substrates of glycerol and methanol has been widely used in the past decade to increase productivity, cell density, and reduce the induction time. However, the optimal level of protein expression is not achievable with mixtures of glycerol and methanol, due to a partial repression of the AOX1 promoter by glycerol, which may result in lower specific productivities of r-protein. By contrast, sorbitol is a non-repressing carbon source for AOX1 promoter, thus sorbitol accumulation during the induction phase does not affect the expression level of r-protein. In this study, batch-wise sorbitol addition as a co-substrate at the induction phase of methanol fed-batch fermentation by P. pastoris (Mut+ ) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. In the presence of sorbitol, 130 mg L−1 of recombinant human erythropoietin (rHuEPO) was produced at t = 24 hours, compared to 80 mg L−1 of rHuEPO (t = 24 hours) on methanol alone. Considering many other advantages to be presented, this S60
www.elsevier.com/locate/nbt
work demonstrates the ease and efficiency of incorporating sorbitol to the fermentations by the popular methylotrophic yeast P. pastoris, for the production of any bio-product. doi:10.1016/j.nbt.2009.06.286
2.1.040 Artificial pathway design: from enzyme discovery to designer bugs J. Eck ∗ , J. Mampel, G. Meurer BRAIN Aktiengesellschaft, 64673 Zwingenberg, Germany
Industrial ‘white’ biotechnology is regarded as a central feature of the sustainable economic future of modern industrialized societies. Highly effective enzymes, heavily engineered microorganisms and ‘designer bugs’ promise improvement for existing processes or could enable novel product ideas. For any industrial application, enzymes and biosynthetic pathways need to function sufficiently well according to several application-specific performance parameters. Instead of designing a process to fit a mediocre enzyme, it is conceivable that a comprehensive access to the microbial diversity might be used to find a suitable natural enzyme(s) that optimally fits process requirements. In view of multi-step bioconversions and the production of specialty- and bulk chemicals from renewable resources current technologies and screening strategies for the development of optimised biocatalysts from microbial biodiversity as well as from ‘metagenome’ libraries will be presented. To tap into the next generation biocatalysis using engineered ‘designer’ microorganisms for multi-step bioconversions, it is necessary to move into the construction of artificial operons and the heterologous expression of modified biosynthetic pathways. As a case study the construction of designer microorganisms for the production of 1,2-propanediol will be discussed. doi:10.1016/j.nbt.2009.06.287
2.1.041 Preliminary characterization of a beta-glucosidase from a Pichia anomala yeast strain of olive brine C. Restuccia ∗ , R. Palmeri, S. Muccilli, C.L. Randazzo, C. Caggia, G. Spagna DOFATA-Sezione Tecnologie Agroalimentari, University of Catania, Italy
An interesting field of -glucosidase (G, EC 3.2.1.21) application in food processing is production of naturally fermented table olives and olive oil. Olives have a bitter compound named oleuropein, which must be eliminated during the olive elaboration process. Nowadays, the most usual industrial debittering method consists of treating the fruits with a NaOH solution. However, these technology presents huge drawbacks due to the highly contaminant discharges generated; moreover, wastewaters originating from table olives processing pose an important environmental threat, as they are characterized by a very high organic load and