The impact of respiration and oxidative stress response on recombinant α-amylase production by Saccharomyces cerevisiae

Martínez, José Luis; Meza, Eugenio; Petranović, Dina; Nielsen, Jens

doi:10.1016/j.meteno.2016.06.003

Cited by 29 publications

(22 citation statements)

References 36 publications

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“…The mitochondrial staining method was described in a previous publication (49). Briefly, the cells were cultivated in SD-2xSCAA medium using tube fermentation and harvested when the OD 600 was Ϸ1.…”

Section: Methodsmentioning

confidence: 99%

Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

Bao

Huang

Petranović

et al. 2017

Appl Environ Microbiol

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The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous ␣-amylase, a model protein used for studying the protein secretory pathway in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased ␣-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous ␣-amylase production was reduced. A genome-wide expression analysis indicated decreased endoplasmic reticulum stress in the strain that moderately overexpressed SEC16, which was consistent with a decreased volume of the endoplasmic reticulum. Additionally, fewer mitochondria were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-␣-glucosidase, indicating that this mechanism is of general relevance.IMPORTANCE There is an increasing demand for recombinant proteins to be used as enzymes and pharmaceuticals. The yeast Saccharomyces cerevisiae is a cell factory that is widely used to produce recombinant proteins. Our study revealed that moderate overexpression of SEC16 increased recombinant protein secretion in S. cerevisiae. This new strategy can be combined with other targets to engineer cell factories to efficiently produce protein in the future. KEYWORDS protein secretion, mitochondria, ERES, ROS, SEC16T he yeast Saccharomyces cerevisiae is a widely used cell factory for the production of recombinant proteins (1). The advantages of S. cerevisiae are fast growth, easy cultivation, and ability to perform posttranslational modifications and secrete proteins to the extracellular medium, which facilitates the purification of the protein (2, 3). However, S. cerevisiae naturally secretes only a few proteins, such as aspartyl protease, invertase, and ␣-factor pheromone (4, 5); therefore, it has evolved a secretory pathway with a relatively low capacity. Many different strategies have been used to engineer this pathway with the objective of improving heterologous protein production (6-8). When expressing a protein that has a secretory signal peptide, the protein is cotranslationally translocated into the endoplasmic reticulum (ER) lumen, where disulfide bond formation occurs with the initial glycosylation (9). The secretion of recombinant proteins is affected by their signal peptide sequences (10). A synthetic leader sequence increases human insulin precursor production, whereas the use of the ␣-factor leader sequence increased ␣-amylase production (7). Excessive mannose glycosylation of recombinant proteins can reduce the efficiency of protein secretion, and it was recently found that

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Section: Methodsmentioning

confidence: 99%

Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

Bao

Huang

Petranović

et al. 2017

Appl Environ Microbiol

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“…Normally, heme biosynthesis is under tight control of the transcription factor Rox1p, which prevents cellular heme accumulation via repression of HEM13. Rox1p itself is controlled by Hap1p activity, which in turn is activated by high heme levels (Martínez et al 2016). Interestingly, overexpression of HAP1 gene has recently been shown to have a profound effect on growth and to some extent protein expression (Martínez et al 2015) putatively by induction of oxidative stress response and expression of enzymes involved in respiration.…”

Section: Verification Of Phenotypes Using Defined Deletions Of Targetmentioning

confidence: 99%

Screening for novel genes ofSaccharomyces cerevisiaeinvolved in recombinant antibody production

Ruijter

Jurgens

Frey

2016

FEMS Yeast Research

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Cost-effective manufacturing of biopharmaceuticals in non-mammalian hosts still requires tremendous efforts in strain development. In order to expedite identification of novel leads for strain engineering, we used a transposon-mutagenized yeast genomic DNA library to create a collection of Saccharomyces cerevisiae deletion strains expressing a full-length IgG antibody. Using a high-throughput screening, transformants with either significantly higher or lower IgG expression were selected. The integration site of the transposon in three of the selected strains was located by DNA sequencing. The inserted DNA lay within the VPS30 and TAR1 open reading frame, and upstream of the HEM13 open reading frame. The complete coding sequence of these genes was deleted in the wild-type strain background to confirm the IgG expression phenotypes. Production of recombinant antibody was increased 2-fold in the Δvps30 strain, but only mildly affected secretion levels in the Δtar1 strain. Remarkably, expression of endogenous yeast acid phosphatase was increased 1.7- and 2.4-fold in Δvps30 and Δtar1 strains. The study confirmed the power of genome-wide high-throughput screens for strain development and highlights the importance of using the target molecule during the screening process.

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“…It was shown that free radicals and reactive species are linked to endoplasmic reticulum (ER) stress and the unfolded protein responses (UPR) in different cells, which activate a set of oxidative stress response genes, mainly those associated with protein folding. These responses mitigate the negative effects of ROS accumulation and hence increase the production capacity 20 . The plasma jet produces a variety of ions and free radicals in the gas phase.…”

Section: Discussionmentioning

confidence: 99%

The effect of non-thermal atmospheric plasma on the production and activity of recombinant phytase enzyme

Farasat

Arjmand

Siadat

et al. 2018

Sci Rep

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Atmospheric pressure cold plasma (ACP) is introduced as a useful tool in a variety of biological applications. Proteins are the most abundant macromolecules in living systems with a central role in all biological processes. These organic molecules are modified by ACP exposure that is responsible for many of ACP’s biological effects. This study evaluated the effect of ACP on the production of recombinant phytase in yeast Pichia pastoris (P. pastoris) as well as the structure and function of the phytase enzyme. The results indicated that yeast cells treated with ACP, directly or indirectly, produced higher amounts of recombinant phytase, which was associated with the time of ACP treatment. The exposure of commercial phytase solution with ACP caused a significant increase in the enzyme activity (125%) after 4 hours. Evaluation of the phytase solution by far- and near-UV circular dichroism (CD) and fluorescence analysis indicated that this protein maintained its secondary structure when exposed to ACP while the tertiary structure was slightly unfolded. The effects of heat and H2O2 on the phytase structure and function were compared with the effect of ACP treatment. The modification of Cys, Tyr and Trp amino acids upon reactive oxygen/nitrogen spices was simulated using a molecular dynamics approach. RMSF and RMSD analysis suggested that this structural alteration occurs owing to changes made by reactive species in accessible amino acids.

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The impact of respiration and oxidative stress response on recombinant α-amylase production by Saccharomyces cerevisiae

Cited by 29 publications

References 36 publications

Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

Screening for novel genes ofSaccharomyces cerevisiaeinvolved in recombinant antibody production

The effect of non-thermal atmospheric plasma on the production and activity of recombinant phytase enzyme

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