Fermentation strategies for improved heterologous expression of laccase in <i>Pichia pastoris</i>

Hong, Feng; Meinander, Nina Q.; Jönsson, Leif J.

doi:10.1002/bit.10297

Cited by 199 publications

(133 citation statements)

References 46 publications

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“…anol or intermediates formed during methanol catabolism (29,41,46). 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.…”

Section: Discussionsupporting

confidence: 90%

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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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Section: Discussionsupporting

confidence: 90%

“…The optimum pH value for Pichia growth is approximately 5 (6), but the pH value is commonly reduced to counteract proteolysis (9,32) or to increase both the stability and productivity of heterologous proteins (5,29,56). However, a pH value of 4 or lower can compromise cells during the production process (27).…”

mentioning

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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“…Our results support the positive effect of low temperatures (20°C) on laccase production. Low temperatures can benefit protein folding, as they reduce cell growth rates, allowing non-rate-limiting protein folding (27). Although the S. cerevisiae strain used in this work is protease deficient, reduced activity of residual proteases at low temperature cannot be ruled out.…”

Section: Discussionmentioning

confidence: 99%

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

Camarero

Pardo

Cañas

et al. 2012

Appl Environ Microbiol

123

173

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While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the ␣-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved ␣-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k cat . While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (ϳ23 mg/ liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).

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“…Lower cultivation temperature can also influence yields of recombinant protein, possibly due to poor stability of the recombinant protein at higher temperatures, release of more proteases from dead cells, and folding problems at higher temperatures [48]. Li and co-workers [64] have shown that lowering the process temperature (from 30 • C to 23 • C) increased the yield of herring antifreeze proteins from 5.3 mg/l to 18.0 mg/l, and also increased cell viability.…”

Section: Cultivation-level Strategiesmentioning

confidence: 99%

“…Li and co-workers [64] have shown that lowering the process temperature (from 30 • C to 23 • C) increased the yield of herring antifreeze proteins from 5.3 mg/l to 18.0 mg/l, and also increased cell viability. Hong et al [48] achieved higher laccase activity by decreasing the cultivation temperature (from 30 • C to 20 • C) and by reducing methanol concentration (from 1.0% to 0.5%). Jahic et al [54] obtained much higher concentration of a fusion protein by applying a temperature-limited fed-batch (TLFB) technique compared with the traditional methanol fed-batch technique.…”

Section: Cultivation-level Strategiesmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,096

633

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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris

Cited by 199 publications

References 46 publications

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

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

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

Heterologous protein production using the Pichia pastoris expression system

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