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Archives of Microbiology

, Volume 195, Issue 12, pp 843–852 | Cite as

Ketoacyl synthase domain is a major determinant for fatty acyl chain length in Saccharomyces cerevisiae

  • Juthaporn Sangwallek
  • Yoshinobu Kaneko
  • Minetaka Sugiyama
  • Hisayo Ono
  • Takeshi Bamba
  • Eiichiro Fukusaki
  • Satoshi Harashima
Original Paper

Abstract

Yeast fatty acid synthase (Fas) comprises two subunits, α6 and β6, encoded by FAS2 and FAS1, respectively. To determine features of yeast Fas that control fatty acyl chain length, chimeric genes were constructed by combining FAS sequences from Saccharomyces cerevisiae (ScFAS) and Hansenula polymorpha (HpFAS), which mostly produces C16 and C18 fatty acids, respectively. The C16/C18 ratios decreased from 2.2 ± 0.1 in wild-type S. cerevisiae to 1.0 ± 0.1, 0.5 ± 0.2 and 0.8 ± 0.1 by replacement of ScFAS1, ScFAS2 and ScFAS1 ScFAS2 with HpFAS1, HpFAS2 and HpFAS1 HpFAS2, respectively, suggesting that the α, but not β subunits play a major role in determining fatty acyl chain length. Replacement of phosphopantetheinyl transferase (PPT) domain with the equivalent region from HpFAS2 did not affect C16/C18 ratio. Chimeric Fas2 containing half N-terminal ScFas2 and half C-terminal HpFas2 carrying H. polymorpha ketoacyl synthase (KS) and PPT gave a remarkable decrease in C16/C18 ratio (0.6 ± 0.1), indicating that KS plays a major role in determining chain length.

Keywords

Fatty acid synthase Hansenula polymorpha FAS1 FAS2 

Abbreviations

Fas

Fatty acid synthase

LCSFA

Long-chain saturated fatty acid

ACP

Acyl carrier protein

KR

Ketoacyl reductase

KS

Ketoacyl synthase

PPT

Phosphopantetheinyl transferase

AT

Acetyl transferase

ER

Enoyl reductase

DH

Dehydratase

MPT

Malonyl/palmitoyl transferase

Supplementary material

203_2013_933_MOESM1_ESM.tif (194 kb)
Fig. S1 Construction of HpFAS1 (A) and HpFAS2 (B) subunit replacement cassettes. HpFAS replacement cassettes were amplified as two DNA fragments: ScFAS promoter + first-half of HpFAS and second-half of HpFAS + ScFAS terminator. In the first PCR, FAS DNA fragments I, II, III and IV (FAS1-I, FAS1-II, FAS1-III and FAS1-IV or FAS2-I, FAS2-II, FAS2-III and FAS2-IV designated collectively as FAS) were amplified separately, and subsequently, FAS-I and FAS-II, and FAS-III and FAS-IV were joined in the second PCR (TIFF 194 kb)
203_2013_933_MOESM2_ESM.tif (242 kb)
Fig. S2 Southern hybridization analysis for structural verification of the HpFAS1 replacement strain (A) and HpFAS2 replacement strains (B). In order to confirm the HpFAS1 replacement, an upstream segment of the HpFAS1 ORF and CgLEU2 were used as probes. PstI digestion of genomic DNA from the HpFAS1 replacement candidate yielded a 6.2-kb band, while Scfas1∆::CgLEU2 yielded a 5.1-kb band. To verify the HpFAS2 replacement, a 561-bp downstream segment of ScFAS2 ORF was used as a probe. EcoRI and PstI digestion of genomic DNA from the HpFAS2 replacement candidates generated bands of 3.1- and 1.3-kb in size, respectively, while generating 2.1 and 1.1-kb bands for Scfas2∆::CgHIS3 (TIFF 241 kb)
203_2013_933_MOESM3_ESM.tif (162 kb)
Fig. S3 Construction of a ScFAS2-PPT domain-specific disruption and HpFAS2-PPT replacement. A 1.6-kb disruption cassette was prepared by fusion of a 219-bp upstream segment of ScFAS2-PPT, ScURA3 gene and a 233-bp downstream fragment of ScFAS2-PPT to create Scfas2-ppt∆::ScURA3 (B). A 0.9-kb replacement cassette named Sc(ACP-KR-KS) Hp(PPT) was prepared by fusion of the 219-bp upstream segment of ScFAS2-PPT, HpFAS2-PPT and the 233-bp downstream fragment of ScFAS2-PPT (C) (TIFF 161 kb)
203_2013_933_MOESM4_ESM.tif (145 kb)
Fig. S4 Construction of a chimeric FAS2 cassette containing HpFAS2-KS PPT [Sc(ACP-KR) Hp(KS-PPT)] obtained by fusion PCR. Fragment I carrying approximately 0.4-kb upstream of FAS2 and the ScFAS2-ACP-KR region and fragment II harboring the HpFAS2-KS PPT region and 0.1-kb downstream of FAS2 were amplified in a first PCR. Fragments I and II were fused in a second round of PCR generating Sc(ACP-KR) Hp(KS-PPT) cassette (B) (TIFF 145 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Juthaporn Sangwallek
    • 1
  • Yoshinobu Kaneko
    • 1
  • Minetaka Sugiyama
    • 1
  • Hisayo Ono
    • 1
  • Takeshi Bamba
    • 1
  • Eiichiro Fukusaki
    • 1
  • Satoshi Harashima
    • 1
  1. 1.Department of Biotechnology, Graduate School of EngineeringOsaka UniversityOsakaJapan

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