Stress in recombinant protein producing yeasts

Mattanovich, Diethard; Gasser, Brigitte; Hohenblum, Hubertus; Sauer, Michael

doi:10.1016/j.jbiotec.2004.04.035

Cited by 221 publications

(174 citation statements)

References 105 publications

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“…A distinct advantage of eukaryotic expression hosts is their capacity to facilitate the post-translation modification of proteins [16]. Additionally, extracellular protein secretion from Pichia tends to be low, reducing the competition for secretion machinery during heterologous expression [17]. A wide variety of proteins have been expressed by this system with varying degrees of success with respect to product yield [14].…”

Section: Introductionmentioning

confidence: 99%

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Gaffney

Carberry

Doyle

et al. 2009

Enzyme and Microbial Technology

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a b s t r a c tA xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100• C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.

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Section: Introductionmentioning

confidence: 99%

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Gaffney

Carberry

Doyle

et al. 2009

Enzyme and Microbial Technology

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“…This demands mechanisms of quality control and coordinated regulation, in order to avoid (or reduce) cellular stress that can result in reduced cell growth and protein secretion (1,2) or even apoptosis and cell death (3,4). Misfolded proteins are detected and removed via the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway (5), the ubiquitin-proteasome system (UPS) (5), the autophagy pathway (6), or the unfolded protein response (UPR) pathway (7).…”

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confidence: 99%

Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

Liu

Österlund

Hou

et al. 2013

Appl Environ Microbiol

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bIn this study, we focus on production of heterologous ␣-amylase in the yeast Saccharomyces cerevisiae under anaerobic conditions. We compare the metabolic fluxes and transcriptional regulation under aerobic and anaerobic conditions, with the objective of identifying the final electron acceptor for protein folding under anaerobic conditions. We find that yeast produces more amylase under anaerobic conditions than under aerobic conditions, and we propose a model for electron transfer under anaerobic conditions. According to our model, during protein folding the electrons from the endoplasmic reticulum are transferred to fumarate as the final electron acceptor. This model is supported by findings that the addition of fumarate under anaerobic (but not aerobic) conditions improves cell growth, specifically in the ␣-amylase-producing strain, in which it is not used as a carbon source. Our results provide a model for the molecular mechanism of anaerobic protein secretion using fumarate as the final electron acceptor, which may allow for further engineering of yeast for improved protein secretion under anaerobic growth conditions.

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“…The batch medium may have an osmolarity around 1,000 mosmol/liter, which induces osmotic stress in yeasts (20). The K. lactis cells overexpressing KlSOD1 showed an enhanced production of heterologous proteins and were more resistant to additional osmotic and oxidative stresses, two gainful industrial features.…”

Section: Discussionmentioning

confidence: 99%

SOD1, a New Kluyveromyces lactis Helper Gene for Heterologous Protein Secretion

Raimondi

Zanni

Talora

et al. 2008

Appl Environ Microbiol

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Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O 2 ؊ ) into oxygen and hydrogen peroxide. It is a Cu 2؉ /Zn 2؉ metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans. In addition, KlSOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing KlSOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses.

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Stress in recombinant protein producing yeasts

Cited by 221 publications

References 105 publications

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

SOD1, a New Kluyveromyces lactis Helper Gene for Heterologous Protein Secretion

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