Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures

Jungo, Carmen; Marison, I. W.; Stockar, Urs von

doi:10.1016/j.jbiotec.2007.04.004

Cited by 55 publications

(33 citation statements)

References 46 publications

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“…The lipase activity and protein concentration declined when methanol concentrations were higher than 1%. This result is consistent with the previous report (Jungo et al 2007b). It may be attributed to the fact that transient methanol accumulation can lead to intoxication of cells and excessive methanol inhibits lipase production in the induction phase.…”

Section: Optimization Of Conditions In Shake Flaskssupporting

confidence: 93%

Combined Strategies for Improving the Production of Recombinant Rhizopus oryzae Lipase in Pichia pastoris

Li¹,

He²,

Li³

et al. 2013

BioResources

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We have developed a yeast Pichia pastoris system for the high-level expression of recombinant Rhizopus oryzae lipase (ROL), which is a potentially effective catalyst in the solvent-free production of biodiesel fuel. In the glycerol fed-batch phase, the combination of the dissolvedoxygen-stat and gradient-control glycerol feeding strategies resulted in a higher cell biomass in the P. pastoris culture, with shorter feed times. In the methanol fed-batch phase, a constant methanol concentration of 0.3-0.5% (v/v) was found to be optimal for high ROL activity, maximum protein concentration, and a maximum biomass. Cells were grown at 30 °C and induced by methanol at 22 °C and a pH of 6.0 with the addition of 0.5% (w/v) casein, which reduced the proteolytic degradation of ROL. Using a combined glycerol step-increasing, on-line methanol feeding strategy and these reducing proteolytic degradation methods, a maximum biomass of 96 g DCW/l, a maximum concentration of 2.0 g/L, and an activity of 1302.2 U/mL ROL were attained. The simple culture process is the highest level of ROL expression reported in P. pastoris. Our results strongly demonstrate that the efficiency of the recombinant ROL expression is directly dependent on feed strategies, lower induction temperature, maintaining the proper pH, and the addition of casein.

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Section: Optimization Of Conditions In Shake Flaskssupporting

confidence: 93%

Combined Strategies for Improving the Production of Recombinant Rhizopus oryzae Lipase in Pichia pastoris

Li¹,

He²,

Li³

et al. 2013

BioResources

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“…The observation that cells which had previously been exposed to (and simultaneously utilized) methanol are more sensitive to any additional stress factor introduced subsequently is consistent with previous results. Jungo et al (38) found that Pichia cells previously exposed to an excess of methanol have already "learned" to synthesize the enzymes of the methanol catabolism. They are therefore again able to synthesize these enzymes immediately, increasing the formation and secretion of toxic intermediates, such as formaldehyde, hydrogen peroxide, and formiate.…”

Section: Discussionmentioning

confidence: 99%

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Hyka

Züllig

Ruth

et al. 2010

Appl Environ Microbiol

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Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut ؉ (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.Changes to the product quantity and quality as well as the robustness of bioprocesses can be triggered by a number of factors which affect the physiological state of the microorganisms. The expression of a foreign gene, the processing of the recombinant protein, and the exposure of the cell to metabolites, inductors, substrates, unfavorable environmental conditions, high cell density (HCD), and/or "aging" can all result in markedly different physiological responses (17).The interpretation of bioprocess data often fails to reflect the actual state of individual cells within a population. It is based typically on performance characterization using concentration measurements and the simplifying assumption of uniform ("averaged") performance of each cell. This practice (39) provides an example of the failure to distinguish between a homogenous population of equally compromised microorganisms and a heterogeneous population of cells each performing differently. An understanding of the state of the individual cells is critical in achieving high efficiency in recombinant protein production. Only by knowing the number of (vital) cells that express the target molecule at the highest possible rate can the number of such cells within the population be maximized and the propo...

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“…The media were sterilized by filtration and supplemented with vitamins and trace elements as prescribed by Jungo [27].…”

Section: General Applicabilitymentioning

confidence: 99%

“…Experimental remained unchanged. Figure 10 shows the evolution of the estimated growth rate obtained using the proportional-integral feedback controller to maintain two setpoints of 0.1 and 0.15/h (lower values of l sp were chosen for this experiment since the l max for this strain grown on glycerol is in the order of 0.24/h [27]). Again, the PI controller was successful at maintaining the specific growth rate at the two experimental setpoints.…”

Section: General Applicabilitymentioning

confidence: 99%

Simple control of specific growth rate in biotechnological fed-batch processes based on enhanced online measurements of biomass

Dabros

Schuler

Marison

2010

Bioprocess Biosyst Eng

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Reliable control of the specific growth rate (μ) in fed-batch fermentations depends on the availability of accurate online estimations of the controlled variable. Due to difficulties in measuring biomass, μ is typically estimated using reference models relating measurements of substrate consumption or oxygen uptake rate to biomass growth. However, as culture conditions vary, these models are adapted dynamically, resulting in complex algorithms that lack the necessary robustness for industrial applicability. A simpler approach is presented where biomass is monitored using dielectric spectroscopy. The measurements are subjected to online balances and reconciled in real time against metabolite concentrations and off-gas composition. The reconciled biomass values serve to estimate the growth rate and a simple control scheme is implemented to maintain the desired value of μ. The methodology is developed with the yeast Kluyveromyces marxianus, tested for disturbance rejection and validated with two other strains. It is applicable to other cellular systems with minor modifications.

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Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures

Cited by 55 publications

References 46 publications

Combined Strategies for Improving the Production of Recombinant Rhizopus oryzae Lipase in Pichia pastoris

Combined Strategies for Improving the Production of Recombinant Rhizopus oryzae Lipase in Pichia pastoris

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Simple control of specific growth rate in biotechnological fed-batch processes based on enhanced online measurements of biomass

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