Functional analysis of very long-chain fatty acid elongase gene, HpELO2, in the methylotrophic yeast Hansenula polymorpha
- 118 Downloads
We describe the cloning and functional characterization of the fatty acid elongase gene HpELO2, a homologue of the HpELO1 gene required for the production of C24:0 in the yeast Hansenula polymorpha. The open reading frame (ORF) of HpELO2 consists of 1,035 bp, encoding 344 amino acids, sharing about 65% identity with that of Saccharomyces cerevisiae Elo2. Expression of HpELO2 rescued the lethality of the S. cerevisiae elo2Δ elo3Δ double disruptant. An accumulation of C18:0 and a significant increase and decrease in the levels of C24:0 and C26:0, respectively, were observed in the Hpelo2Δ disruptant. These results supported an idea that HpELO2 encodes a fatty acid elongase involved in the elongation of C18:0 to very long-chain fatty acids. The Hpelo1Δ Hpelo2Δ double disruption was nonviable, suggesting that HpELO1 and HpELO2 are the only two genes necessary for the biosynthesis in H. polymorpha. Interestingly, transcription of HpELO2 and HpELO1 were found to be transiently up-regulated by exogenous long-chain fatty acids; however, this up-regulation was not observed with HpELO1 and HpELO2 genes driven by the constitutively expressed promoter of the HpACT gene, suggesting that exogenous fatty acids specifically trigger the transcriptional induction of HpELO1 and HpELO2 through their promoter regions.
KeywordsMethylotrophic yeast Hansenula polymorpha Elongase for very long-chain fatty acids Transient transcriptional induction
We would like to thank Y. Sakai for providing plasmid pREMI-Z, M. Veenhuis for providing plasmid pHIPA4, and H. A. Kang for providing plasmid pHACT850-HyL.
- Amberg DC, Burke DJ, Stratern JN (eds) (2005) Methods in yeast genetics: PCR-mediated gene disruption. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 155–160Google Scholar
- Gasch AP, Spellman PT, Kao CM, Carnel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11:4241–4257Google Scholar
- Haan GJ, van Dijk R, Kiel JAKW, Veenhuis M (2001) Characterization of the Hansenula polymorpha PUR7 gene and its use as selectable marker for targeted chromosomal integration. FEMS Yeast Res 2:17–24Google Scholar
- Kaneko Y, Rueksomtawin K, Maekawa T, Harashima S (2003) Structural and functional analysis of the FAS1 gene encoding fatty acid synthase β-subunit in methylotrophic yeast Hansenula polymorpha. In: Murata N, Yamada M, Nishida I, Okuyama H, Sekiya J, Wada H (eds) Advanced research on plant lipids. Kluwer, London, pp 61–64Google Scholar
- Mcdonough VM, Stukey JE, Martin CE (1992) Specificity of unsaturated fatty acid-regulated expression of the Saccharomyces cerevisiae OLE1 gene. J Biol Chem 267:5931–5936Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory: Cold Spring Harbor, New YorkGoogle Scholar
- Schweizer M (2004) Lipids and membranes. In: Dickinson JR, Schweizer M (eds) The metabolism and molecular physiology of Saccharomyces cerevisiae. CRC LLC, New York, pp 140–223Google Scholar
- Stock SD, Hama H, Radding JA, Young DA, Takemoto JY (2000) Syringomycin E inhibition of Saccharomyces cerevisiae: requirement for biosynthesis of sphingolipids with very-long-chain fatty acids and mannose- and phosphoinositol-containing head groups. Antimicrob Agents Chemother 44:1174–1180CrossRefGoogle Scholar
- Wijeyaratne SC, Ohta K, Chavanich S, Mahamontri V, Ninubol N, Hayashida S (1986) Lipid composition of a thermotolerant yeast, Hansenula polymorpha. Agric Biol Chem 50:827–832Google Scholar
- Zeidel ML (1996) Low permeabilities of apical membranes of barrier epithelia: what makes watertight membranes watertight? Am J Physiol 271:F243–F245Google Scholar