Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe

Idiris, Alimjan; Tohda, Hideki; Bi, Ke-wei; Isoai, Atsushi; Kumagai, Hiromichi; Giga-Hama, Yuko

doi:10.1007/s00253-006-0489-0

Cited by 47 publications

(17 citation statements)

References 44 publications

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“…The S. pombe strain ARC010-1 (h − leu1-32 ura4-D18) (Idiris et al 2006) was used for this study. S. pombe strains were grown in EMMS or EMMS dropout media depending on the selective pressure required to maintain the plasmids (Alfa et al 1993).…”

Section: Strains Media and Growthmentioning

confidence: 99%

“…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%

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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: Strains Media and Growthmentioning

confidence: 99%

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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“…One promising approach to overcome this difficulty is the creation of appropriate protease-deficient mutants. For example, the production of protease-sensitive human growth hormone or human transferrin was found to be significantly enhanced in S. pombe strains that lack single or multiple proteases [46][47][48][49]. In addition, it has been previously reported that modification of the growth medium conditions clearly improved the secretion efficiency [37,50].…”

Section: Discussionmentioning

confidence: 94%

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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“…In the fission yeast Schizosaccharomyces pombe , disruption of multiple protease genes, including ppp80 / atg4 , enhances the productivity of human growth hormone (hGH) [35]. However, the atg4 gene was one of seven protease genes that were disrupted based on a search for such genes in the S. pombe genomic database, but the functional effects of atg4 disruption on hGH production were not examined.…”

Section: Discussionmentioning

confidence: 99%

Enhanced Production of Bovine Chymosin by Autophagy Deficiency in the Filamentous Fungus Aspergillus oryzae

et al. 2013

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Aspergillus oryzae has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, A. oryzae often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production. In the present study, we examined the production of heterologous protein in several autophagy (Aoatg) gene disruptants of A. oryzae. We transformed A. oryzae gene disruptants of Aoatg1, Aoatg13, Aoatg4, Aoatg8, or Aoatg15, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the Aoatg gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed Aoatg gene-conditional expression strains in which the promoter region of the Aoatg gene was replaced with the thiamine-controllable thiA promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the Aoatg gene disruptants. Consequently, we succeeded in the construction of A. oryzae strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing heterologous protein production in A. oryzae.

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Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe

Cited by 47 publications

References 44 publications

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe

Engineered high content of ricinoleic acid in fission yeast Schizosaccharomyces pombe

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

Enhanced Production of Bovine Chymosin by Autophagy Deficiency in the Filamentous Fungus Aspergillus oryzae

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