Skip to main content
SpringerLink
Log in

Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeast Schizosaccharomyces pombe

  • Applied genetics and molecular biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

We previously succeeded to obtain a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we identified plg7, encoding phospholipase A2, as a multicopy suppressor that restored the growth defect by removing RA from phospholipids and induced secretion of a part of the released free RA into culture media. In this study, we extended our analysis and examined the effect of triglyceride (TG) lipase overexpression on the tolerance to RA toxicity and RA productivity. S. pombe has three TG lipase genes, ptl1, ptl2, and ptl3, which have high protein sequence similarities to each other and to Saccharomyces cerevisiae counterparts TGL3, TGL4, and TGL5, but only ptl2 overexpression suppressed the growth defect induced by RA production, and the culture grown at 20 °C secreted free RA into media like plg7 overexpression. Suppression by ptl2 was independent of plg7, and a large amount of free RA was accumulated in the cells concomitant with the decrease in RA moieties in phospholipids. Furthermore, the suppression by ptl2 was attenuated by bromoenol lactone (BEL), a phospholipase A2 specific inhibitor, suggesting that Ptl2p may have phospholipase activity. Simultaneous overexpression of ptl2 and plg7 in the FAH12 integrant increased secretion and intracellular accumulation of RA 1.2- and 1.3-fold, respectively, compared to those with single overexpression of plg7 on day 10 at 20 °C.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Ackermann EJ, Conde-Frieboes K, Dennis EA (1995) Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J Biol Chem 270:445–450

    Article  PubMed  CAS  Google Scholar 

  • Alfa C, Fantes P, Hyams J, McLeod M, Warbrick E (1993) Experiments with fission yeast: a laboratory course manual. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Balsinde J, Bianco ID, Ackermann EJ, Conde-Frieboes K, Dennis EA (1995) Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages. Proc Natl Acad Sci U S A 92:8527–8531

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Basi G, Schmid E, Maundrell K (1993) TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123:131–136

    Article  PubMed  CAS  Google Scholar 

  • Beopoulos A, Verbeke J, Bordes F, Guicherd M, Bressy M, Marty A, Nicaud JM (2014) Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 98:251–262

    Article  PubMed  CAS  Google Scholar 

  • Broun P, Somerville C (1997) Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsis plants that express a fatty acyl hydroxylase cDNA from castor bean. Plant Physiol 113:933–942

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Broun P, Boddupalli S, Somerville C (1998) A bifunctional oleate 12-hydroxylase: desaturase from Lesquerella fendleri. Plant J 13:201–210

    Article  PubMed  CAS  Google Scholar 

  • Foulks JM, Weyrich AS, Zimmerman GA, McIntyre TM (2008) A yeast PAF acetylhydrolase ortholog suppresses oxidative death. Free Radic Biol Med 45:434–442

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Galliard T, Stumpf PK (1966) Fat metabolism in higher plants. 30 Enzymatic synthesis of RA by a microsomal preparation from developing Ricinus communis seeds. J Biol Chem 241:5806–5812

    PubMed  CAS  Google Scholar 

  • Hazen SL, Zupan LA, Weiss RH, Getman DP, Gross RW (1991) Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2. J Biol Chem 266:7227–7232

    PubMed  CAS  Google Scholar 

  • Holic R, Yazawa H, Kumagai H, Uemura H (2012) Engineered high content of RA in fission yeast Schizosaccharomyces pombe. Appl Microbiol Biotechnol 95:179–187

    Article  PubMed  CAS  Google Scholar 

  • Idiris A, Tohda H, Bi KW, Isoai A, Kumagai H, Giga-Hama Y (2006) Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe. Appl Microbiol Biotechnol 73:404–420

    Article  PubMed  CAS  Google Scholar 

  • Kainou K, Kamisaka Y, Kimura K, Uemura H (2006) Isolation of Δ12 and ω3-fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and α-linolenic acids in Saccharomyces cerevisiae. Yeast 23:605–612

    Article  PubMed  CAS  Google Scholar 

  • Kumar R, Wallis JG, Skidmore C, Browse J (2006) A mutation in Arabidopsis cytochrome b5 reductase identified by high-throughput screening differentially affects hydroxylation and desaturation. Plant J 48:920–932

    Article  PubMed  CAS  Google Scholar 

  • Lu C, Fulda M, Wallis JG, Browse J (2006) A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. Plant J 45:847–856

    Article  PubMed  CAS  Google Scholar 

  • Matsuyama A, Arai R, Yashiroda Y, Shirai A, Kamata A, Sekido S, Kobayashi Y, Hashimoto A, Hamamoto M, Hiraoka Y, Horinouchi S, Yoshida M (2006) ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol 24:841–847

    Article  PubMed  CAS  Google Scholar 

  • Maundrell K (1993) Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123:127–130

    Article  PubMed  CAS  Google Scholar 

  • Meesapyodsuk D, Qiu X (2008) An oleate hydroxylase from the fungus Claviceps purpurea: cloning, functional analysis, and expression in Arabidopsis. Plant Physiol 147:1325–1333

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Nakamura T, Nakamura-Kubo M, Hirata A, Shimoda C (2001) The Schizosaccharomyces pombe spo3 + gene is required for assembly of the forespore membrane and genetically interacts with psy1 +-encoding syntaxin-like protein. Mol Biol Cell 12:3955–3972

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Rajakumari S, Daum G (2010) Multiple functions as lipase, steryl ester hydrolase, phospholipase, and acyltransferase of Tgl4p from the yeast Saccharomyces cerevisiae. J Biol Chem 285:15769–15776

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Ratledge C, Evans CT (1989) Lipids and their metabolism. In: Rose AH, Harrison JS (eds) The yeast, 2nd edn. Academic, London, pp 367–455

    Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Scharnewski M, Pongdontri P, Mora G, Hoppert M, Fulda M (2008) Mutants of Saccharomyces cerevisiae deficient in acyl-CoA synthetases secrete fatty acids due to interrupted fatty acid recycling. FEBS J 275:2765–2778

    Article  PubMed  CAS  Google Scholar 

  • Smith MA, Moon H, Chowrira G, Kunst L (2003) Heterologous expression of a fatty acid hydroxylase gene in developing seeds of Arabidopsis thaliana. Planta 217:507–516

    Article  PubMed  CAS  Google Scholar 

  • Tanaka K, Yonekawa T, Kawasaki Y, Kai M, Furuya K, Iwasaki M, Murakami H, Yanagida M, Okayama H (2000) Fission yeast Eso1p is required for establishing sister chromatid cohesion during S phase. Mol Cell Biol 20:3459–3469

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • van de Loo FJ, Broun P, Turner S, Somerville C (1995) An oleate 12-hydroxylase from Ricinus communis L is a fatty acyl desaturase homolog. Proc Natl Acad Sci U S A 92:6743–6747

    Article  PubMed  PubMed Central  Google Scholar 

  • Yazawa H, Kumagai H, Uemura H (2012) Characterization of triglyceride lipase genes of fission yeast Schizosaccharomyces pombe. Appl Microbiol Biotechnol 96:981–991

    Article  PubMed  CAS  Google Scholar 

  • Yazawa H, Holic R, Kumagai H, Uemura H (2013a) Toxicity of ricinoleic acid production in fission yeast Schizosaccharomyces pombe is suppressed by the overexpression of plg7, a phospholipase A2 of a platelet-activating factor (PAF) family homolog. Appl Microbiol Biotechnol 97:8193–8203

    Article  PubMed  CAS  Google Scholar 

  • Yazawa H, Kumagai H, Uemura H (2013b) Secretory production of ricinoleic acid in fission yeast Schizosaccharomyces pombe. Appl Microbiol Biotechnol 97:8663–8671

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Genomic DNA clones of S. pombe were provided by the National Bio-Resource Project (NBRP) of the MEXT, Japan. This work was partly supported by research grants from Noda Institute for Scientific Research and the New Energy and Industrial Technology Development Organization (NEDO) to HU.

Author information

Authors and Affiliations

  1. Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, 305-8566, Japan

    Hisashi Yazawa, Masayo Ogiso & Hiroshi Uemura

  2. Asahi Glass Co. Ltd., Yokohama, Kanagawa, 221-8755, Japan

    Hiromichi Kumagai

Authors
  1. Hisashi Yazawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masayo Ogiso
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hiromichi Kumagai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiroshi Uemura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hiroshi Uemura.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yazawa, H., Ogiso, M., Kumagai, H. et al. Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeast Schizosaccharomyces pombe . Appl Microbiol Biotechnol 98, 9325–9337 (2014). https://doi.org/10.1007/s00253-014-6006-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-6006-y

Keywords

Search

Navigation