Production of heterologous proteins using the fission‐yeast (<i>Schizosaccharomyces pombe</i>) expression system

Takegawa, Kaoru; Tohda, Hideki; Sasaki, Michihito; Idiris, Alimjan; Ohashi, Takao; Mukaiyama, Hiroyuki; Giga-Hama, Yuko; Kumagai, Hiromichi

doi:10.1042/ba20090048

Cited by 59 publications

(36 citation statements)

References 81 publications

(82 reference statements)

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“…S. pombe is particularly attractive, having served as a model organism for the development of metabolic engineering strategies to produce foreign gene products and certain metabolites (Giga-Hama et al 1994;Isoai et al 2002;Ikeda et al 2004;Giga-Hama et al 2007;Takegawa et al 2009;Idiris et al 2006Idiris et al , 2010a. Also, its fatty acid composition is simple.…”

Section: Introductionmentioning

confidence: 98%

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe

Holic̆

Yazawa

Kumagai

et al. 2012

Appl Microbiol Biotechnol

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In an effort to produce ricinoleic acid (12-hydroxy-octadeca-cis-9-enoic acid: C18:1-OH) as a petrochemical replacement in a variety of industrial processes, we introduced Claviceps purpurea oleate ∆12-hydroxylase gene (CpFAH12) to Schizosaccharomyces pombe, putting it under the control of inducible nmt1 promoter. Since Fah12p is able to convert oleic acid to ricinoleic acid, we thought that S. pombe, in which around 75% of total fatty acid (FA) is oleic acid, would accordingly be an ideal microorganism for high production of ricinoleic acid. Unfortunately, at the normal growth temperature of 30 °C, S. pombe cells harboring CpFAH12 grew poorly when the CpFAH12 gene expression was induced, perhaps implicating ricinoleic acid as toxic in S. pombe. However, in line with a likely thermoinstability of Fah12p, there was almost no growth inhibition at 37 °C or, by contrast with 30 °C and lower temperatures, ricinoleic acid accumulation. Accordingly, various optimization steps led to a regime with preliminary growth at 37 °C followed by a 5-day incubation at 20 °C, and the level of ricinoleic acid reached 137.4 μg/ml of culture that corresponded to 52.6% of total FA.

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Section: Introductionmentioning

confidence: 98%

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe

Holic̆

Yazawa

Kumagai

et al. 2012

Appl Microbiol Biotechnol

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“…Biodiesel production worldwide has grown exponentially over the past several years, which could lead to glycerol becoming a valuable feedstock for heterologous protein production. Therefore, the metabolic regulation of glycerol utilization in model and industrial microorganisms, including S. pombe (Giga-Hama et al 2007;Takegawa et al 2009), is a matter of growing importance.…”

Section: Introductionmentioning

confidence: 99%

Promotion of glycerol utilization using ethanol and 1-propanol in Schizosaccharomyces pombe

Matsuzawa

Hara

Tohda

et al. 2012

Appl Microbiol Biotechnol

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The fission yeast Schizosaccharomyces pombe does not grow in media containing glycerol as a sole carbon source but uses glycerol in the presence of ethanol. Ethanol, but not glycerol, triggered upregulation of gld1+ and fbp1+ during glucose starvation even though gld1+ and fbp1+ are essential for growth on glycerol. This upregulation occurred at a very low concentration of ethanol. The transcriptional regulation of gld1+ was tested in the presence of various alcohols, and both ethanol and 1-propanol were found to induce gld1+ and to support growth in glycerol-containing media. We suggest that S. pombe has a novel ethanol and/or 1-propanol recognition mechanism that upregulates glycerol utilization during glucose starvation.

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“…Thus, the use of S. pombe as a host for expression of secreted mammalian proteins may be likely to provide a protein that is close to its native form. For these reasons, S. pombe has emerged to be an attractive host for the expression and secretion of heterologous proteins that are therapeutic and commercially relevant [25].…”

Section: Introductionmentioning

confidence: 99%

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

Naumann¹,

Küttner

Bureik³

2010

Appl Biochem Biotechnol

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There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv-GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv-GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv-GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion.

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Production of heterologous proteins using the fission‐yeast (Schizosaccharomyces pombe) expression system

Cited by 59 publications

References 81 publications

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe

Promotion of glycerol utilization using ethanol and 1-propanol in Schizosaccharomyces pombe

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

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