Yeasts are commonly used hosts in heterologous protein production and their cultivation in aerated fed-batch reactors is employed for large scale production to achieve high cell densities with high yields of recombinant product. Notwithstanding the advantages of fed-batch cultivation, limitations and restrictions may be encountered, mainly due to the response of the producer strain to process conditions. In our laboratory, human interleukin-1 production by two recombinant yeast strains has been accomplished in an aerated reactor operating in fed-batch mode. We have used a prototrophic non-conventional yeast, Zygosaccharomyces bailii [pZ3KlIL-1], and the auxotrophic Saccharomyces cerevisiae BY4741 [PIR4-IL1β]. In both cases, a mathematical model has been developed to both define the profile of the exponential feeding regime applied to the bioreactor and describe the time-course of the process in terms of cell density and product concentration. The theoretical and experimental approach has allowed to evidence some constraints in the use of both the strains in the aerated fed-batch reactor. In the case of Z. bailii [pZ3KlIL-1], the necessity of starting the fed-batch reactor with a fermentative inoculum has been demonstrated. The results obtained with the auxotrophic S. cerevisiae BY4741[PIR4-IL1β] have revealed that a proper auxotrophy-complementing aminoacid concentration in the medium is essential to improve the strain performance. Notwithstanding this, loss of cell viability during the process carried out with S. cerevisiae BY4741[PIR4-IL1β] has been evidenced and ascribed to the oxidative stress arising from the air forced conditions applied to the reactor. In fact, deletion of the caspase gene (YCA1) in S. cerevisiae BY4741 improves yeast viability, though dramatically raises the maintenance-energy demand of the cells. The mathematical model has represented the tool to confer the proper importance to the physiological response of the producer strains. In particular, cell death and energy demand have been considered and, once suitably expressed, inserted into the model, so to obtain a better description of the process in the view of its optimisation.

Constraints in the use of prototrophic and auxotrophic yeast strains for heterologous proteins production in aerated fed-batch reactors

PACIELLO, LUCIA;ROMANO, Vittorio Raffaele A.;PARASCANDOLA, Palma
2009

Abstract

Yeasts are commonly used hosts in heterologous protein production and their cultivation in aerated fed-batch reactors is employed for large scale production to achieve high cell densities with high yields of recombinant product. Notwithstanding the advantages of fed-batch cultivation, limitations and restrictions may be encountered, mainly due to the response of the producer strain to process conditions. In our laboratory, human interleukin-1 production by two recombinant yeast strains has been accomplished in an aerated reactor operating in fed-batch mode. We have used a prototrophic non-conventional yeast, Zygosaccharomyces bailii [pZ3KlIL-1], and the auxotrophic Saccharomyces cerevisiae BY4741 [PIR4-IL1β]. In both cases, a mathematical model has been developed to both define the profile of the exponential feeding regime applied to the bioreactor and describe the time-course of the process in terms of cell density and product concentration. The theoretical and experimental approach has allowed to evidence some constraints in the use of both the strains in the aerated fed-batch reactor. In the case of Z. bailii [pZ3KlIL-1], the necessity of starting the fed-batch reactor with a fermentative inoculum has been demonstrated. The results obtained with the auxotrophic S. cerevisiae BY4741[PIR4-IL1β] have revealed that a proper auxotrophy-complementing aminoacid concentration in the medium is essential to improve the strain performance. Notwithstanding this, loss of cell viability during the process carried out with S. cerevisiae BY4741[PIR4-IL1β] has been evidenced and ascribed to the oxidative stress arising from the air forced conditions applied to the reactor. In fact, deletion of the caspase gene (YCA1) in S. cerevisiae BY4741 improves yeast viability, though dramatically raises the maintenance-energy demand of the cells. The mathematical model has represented the tool to confer the proper importance to the physiological response of the producer strains. In particular, cell death and energy demand have been considered and, once suitably expressed, inserted into the model, so to obtain a better description of the process in the view of its optimisation.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11386/3018692
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