Three gene expression vector sets for concurrently expressing multiple genes in<i>Saccharomyces cerevisiae</i>

Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Mizutani, Fumio; Kondo, Akihiko

doi:10.1111/1567-1364.12138

Cited by 33 publications

(26 citation statements)

References 21 publications

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“…Fluorescence microscopy observation was performed as described previously53. The harvested cells were washed and resuspended in distilled water; the cell suspensions were then observed using a BIOREVO BZ-9000 fluorescence microscope equipped with a 60× objective lens (Keyence, Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

Kaishima

Ishii

Matsuno

et al. 2016

Sci Rep

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Green fluorescent protein (GFP), which was originally isolated from jellyfish, is a widely used tool in biological research, and homologs from other organisms are available. However, researchers must determine which GFP is the most suitable for a specific host. Here, we expressed GFPs from several sources in codon-optimized and non-codon-optimized forms in the yeast Saccharomyces cerevisiae, which represents an ideal eukaryotic model. Surprisingly, codon-optimized mWasabi and mNeonGreen, which are typically the brightest GFPs, emitted less green fluorescence than did the other five codon-optimized GFPs tested in S. cerevisiae. Further, commercially available GFPs that have been optimized for mammalian codon usage (e.g., EGFP, AcGFP1 and TagGFP2) unexpectedly exhibited extremely low expression levels in S. cerevisiae. In contrast, codon-optimization of the GFPs for S. cerevisiae markedly increased their expression levels, and the fluorescence intensity of the cells increased by a maximum of 101-fold. Among the tested GFPs, the codon-optimized monomeric mUkG1 from soft coral showed the highest levels of both expression and fluorescence. Finally, the expression of this protein as a fusion-tagged protein successfully improved the reporting system’s ability to sense signal transduction and protein–protein interactions in S. cerevisiae and increased the detection rates of target cells using flow cytometry.

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Section: Methodsmentioning

confidence: 99%

Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

Kaishima

Ishii

Matsuno

et al. 2016

Sci Rep

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“…The usefulness of FC in combination with fluorescent proteins has previously been demonstrated for measuring the activity of a number of different yeast expression systems [24], [47]. Also, FC have been used to measure fluorescent proteins correlated to other user-specified properties, for example for measuring intracellular pH [49] or for monitoring cell growth and cell membrane robustness [7].…”

Section: Introductionmentioning

confidence: 99%

Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae

Knudsen

Johanson

Lantz

et al. 2015

Biotechnology Reports

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A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by placing it in strains with different ability to reoxidise NADH, and applying different environmental conditions. Flow cytometric analysis of reporter strains expressing green fluorescent protein (GFP) under the control of the GPD2 promoter was used to determine the promoter activity at the single-cell level. When placed in a gpd1Δgpd2Δ strain background, the GPD2 promoter displayed a 2-fold higher activity as compared to the strong constitutive glyceraldehyde-3-phosphate dehydrogenase (TDH3). In contrast, the GPD2 promoter was found to be inactive when cells were cultivated in continuous mode at a growth rate of 0.3 h−1 and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the gpd1Δgpd2Δ strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression.

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“…Yeast is also tolerant to low pH and robust against autolysis, thereby reducing the risk of contamination and permitting long-term repeated or continuous fermentation. These attributes are expected to contribute to cost reduction and to facilitate commercial viability [29]. Therefore, the co-displaying of β-mannanase and β-mannosidase on the yeast cell surface is a promising strategy applicable to yeast fermentations designed to produce other (non-ethanol) biofuels and biochemicals in metabolically engineered S. cerevisiae [30–32].…”

Section: Discussionmentioning

confidence: 99%

From mannan to bioethanol: cell surface co-display of β-mannanase and β-mannosidase on yeast Saccharomyces cerevisiae

Ishii

Okazaki

Djohan

et al. 2016

Biotechnol Biofuels

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BackgroundMannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source.ResultsParental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate.ConclusionsWe successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0600-4) contains supplementary material, which is available to authorized users.

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Three gene expression vector sets for concurrently expressing multiple genes inSaccharomyces cerevisiae

Cited by 33 publications

References 21 publications

Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae

From mannan to bioethanol: cell surface co-display of β-mannanase and β-mannosidase on yeast Saccharomyces cerevisiae

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