Interleukin-1β production by Zygosaccharomyces bailii [pZ3 KlIL-1β] in aerated fed-batch reactor: Importance of inoculum physiology and bioprocess modelling

Romano, Vittorio; Paciello, Lucia; Romano, Franca; Alteriis, Elisabetta de; Brambilla, Luca; Parascandola, Palma

doi:10.1016/j.procbio.2009.01.004

Cited by 4 publications

(5 citation statements)

References 19 publications

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“…Interleukin-1 β was determined in quadruplicate by immuno-blot analysis in Bio-Dot ® Microfiltration Apparatus (Bio-Rad, Hercules, CA, USA), as already reported [ 18 ]. All samples were analysed in triplicate and the values of standard deviation obtained varied between 1 and 2%.…”

Section: Methodsmentioning

confidence: 99%

“…With regard to this matter, only a small number of works in the literature state the importance of these process parameters during fermentation runs carried out with yeast cells [17,18]. …”

Section: Introductionmentioning

confidence: 99%

“…Fed-batch operative conditions and the design of the fermentation strategy may affect the performance of the producer strain, in particular, agitation and aeration. With regard to this matter, only a small number of works in the literature state the importance of these process parameters during fermentation runs carried out with yeast cells [ 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

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Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

et al. 2009

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BackgroundSaccharomyces cerevisiae BY4741 is an auxotrophic commonly used strain. In this work it has been used as host for the expression and secretion of human interleukin-1β (IL1β), using the cell wall protein Pir4 as fusion partner. To achieve high cell density and, consequently, high product yield, BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor, using a defined mineral medium supplemented with casamino acids as ACA (auxotrophy-complementing amino acid) source. Also the S. cerevisiae mutant BY4741 Δyca1 [PIR4-IL1β], carrying the deletion of the YCA1 gene coding for a caspase-like protein involved in the apoptotic response, was cultured in aerated fed-batch reactor and compared to the parental strain, to test the effect of this mutation on strain robustness. Viability of the producer strains was examined during the runs and a mathematical model, which took into consideration the viable biomass present in the reactor and the glucose consumption for both growth and maintenance, was developed to describe and explain the time-course evolution of the process for both, the BY4741 parental and the BY4741 Δyca1 mutant strain.ResultsOur results show that the concentrations of ACA in the feeding solution, corresponding to those routinely used in the literature, are limiting for the growth of S. cerevisiae BY4741 [PIR4-IL1β] in fed-batch reactor. Even in the presence of a proper ACA supplementation, S. cerevisiae BY4741 [PIR4-IL1β] did not achieve a high cell density. The Δyca1 deletion did not have a beneficial effect on the overall performance of the strain, but it had a clear effect on its viability, which was not impaired during fed-batch operations, as shown by the kd value (0.0045 h-1), negligible if compared to that of the parental strain (0.028 h-1). However, independently of their robustness, both the parental and the Δyca1 mutant ceased to grow early during fed-batch runs, both strains using most of the available carbon source for maintenance, rather than for further proliferation. The mathematical model used evidenced that the energy demand for maintenance was even higher in the case of the Δyca1 mutant, accounting for the growth arrest observed despite the fact that cell viability remained comparatively high.ConclusionsThe paper points out the relevance of a proper ACA formulation for the outcome of a fed-batch reactor growth carried out with S. cerevisiae BY4741 [PIR4-IL1β] strain and shows the sensitivity of this commonly used auxotrophic strain to aerated fed-batch operations. A Δyca1 disruption was able to reduce the loss of viability, but not to improve the overall performance of the process. A mathematical model has been developed that is able to describe the behaviour of both the parental and mutant producer strain during fed-batch runs, and evidence the role played by the energy demand for maintenance in the outcome of the process.

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Section: Methodsmentioning

confidence: 99%

“…With regard to this matter, only a small number of works in the literature state the importance of these process parameters during fermentation runs carried out with yeast cells [17,18]. …”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

et al. 2009

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“…[24]. As far as this, implementation of the model for the recombinant Z. bailii, [25], including microbial death kinetic, appears to be essential in the next future to optimise the bioprocess in terms of cell density and product concentration.…”

Section: Resultsmentioning

confidence: 99%

Glucoamylase by recombinant Kluyveromyces lactis cells: production and modelling of a fed batch bioreactor

Paciello

Romano

Alteriis

et al. 2009

Bioprocess Biosyst Eng

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A cultural system, aimed at the production of glucoamylase with cells of a non-conventional yeast transformed for the enzyme expression, Kluyveromyces lactis JA6-GAA was realised. Glucoamylase production was accomplished in a reactor operating in fed batch mode to avoid limitations with respect to oxygen transfer, and achieve high cell density. A mathematical model able to describe batch and fed batch operations was developed. The theoretical and experimental approach permitted to catch sight of possible physiological changes in the producer strain and set up a suitable fed-batch run to achieve a higher cell density.

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“…Ethanol production was measured with the enzymatic kit from R-Biopharm). Interleukin-1β was determined in quadruplicate by immuno-blot analysis in Bio-Dot® Microfiltration Apparatus (Bio-Rad, Hercules, CA, USA) and quantified by densitometric analysis (MultiAnalyst; BioRad) using the human EuroClone IL-1β as a standard, as previously reported (Romano et al 2009). …”

Section: Analysesmentioning

confidence: 99%

Expression of human interleukin-1β in Saccharomyces cerevisiae using PIR4 as fusion partner and production in aerated fed-batch reactor

et al. 2010

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To circumvent cell wall retention commonly associated to Saccharomyces cerevisiae when used as a host for heterologous protein production, we have created a translational fusion of human interleukin-1β (IL-1β) to the Pir4 cell wall protein, so as to drive the secretion of the recombinant product to the growth medium. The auxotrophic S. cerevisiae BY4741 was used as host to express the Pir4-IL1β fusion protein. Once it was ascertained that the fusion protein was secreted to the culture medium and behaved as a growth-linked product, S. cerevisiae BY4741 [PIR4-IL1β] was cultured in an aerated fed-batch reactor to achieve high cell density and, consequently, high product concentration in the medium. Two cultivation media were employed, a rich complex and a defined mineral medium, the latter suitably supplemented with bacto-casamino acids as ACA (auxotrophy-complementing amino acid) source. The rich complex medium allowed a good performance of the producer strain only during batch growth, but was revealed to be inadequate for long-term fed-batch operations. The defined mineral medium ensured a better performance, even though not yet satisfactory in spite of a proper ACA supplementation. The behaviour of BY4741 was attributed to an intrinsic sensitivity of the producer strain to long-term aerated fed-batch operations.

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Interleukin-1β production by Zygosaccharomyces bailii [pZ3 KlIL-1β] in aerated fed-batch reactor: Importance of inoculum physiology and bioprocess modelling

Cited by 4 publications

References 19 publications

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Glucoamylase by recombinant Kluyveromyces lactis cells: production and modelling of a fed batch bioreactor

Expression of human interleukin-1β in Saccharomyces cerevisiae using PIR4 as fusion partner and production in aerated fed-batch reactor

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