Engineering the Saccharomyces cerevisiae β-oxidation pathway to increase medium chain fatty acid production as potential biofuel

PLoS One. 2014 Jan 21;9(1):e84853. doi: 10.1371/journal.pone.0084853. eCollection 2014.

Abstract

Fatty acid-derived biofuels and biochemicals can be produced in microbes using β-oxidation pathway engineering. In this study, the β-oxidation pathway of Saccharomyces cerevisiae was engineered to accumulate a higher ratio of medium chain fatty acids (MCFAs) when cells were grown on fatty acid-rich feedstock. For this purpose, the haploid deletion strain Δpox1 was obtained, in which the sole acyl-CoA oxidase encoded by POX1 was deleted. Next, the POX2 gene from Yarrowia lipolytica, which encodes an acyl-CoA oxidase with a preference for long chain acyl-CoAs, was expressed in the Δpox1 strain. The resulting Δpox1 [pox2+] strain exhibited a growth defect because the β-oxidation pathway was blocked in peroxisomes. To unblock the β-oxidation pathway, the gene CROT, which encodes carnitine O-octanoyltransferase, was expressed in the Δpox1 [pox2+] strain to transport the accumulated medium chain acyl-coAs out of the peroxisomes. The obtained Δpox1 [pox2+, crot+] strain grew at a normal rate. The effect of these genetic modifications on fatty acid accumulation and profile was investigated when the strains were grown on oleic acids-containing medium. It was determined that the engineered strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] had increased fatty acid accumulation and an increased ratio of MCFAs. Compared to the wild-type (WT) strain, the total fatty acid production of the strains Δpox1 [pox2+] and Δpox1 [pox2+, crot+] were increased 29.5% and 15.6%, respectively. The intracellular level of MCFAs in Δpox1 [pox2+] and Δpox1 [pox2+, crot+] increased 2.26- and 1.87-fold compared to the WT strain, respectively. In addition, MCFAs in the culture medium increased 3.29-fold and 3.34-fold compared to the WT strain. These results suggested that fatty acids with an increased MCFAs ratio accumulate in the engineered strains with a modified β-oxidation pathway. Our approach exhibits great potential for transforming low value fatty acid-rich feedstock into high value fatty acid-derived products.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl Coenzyme A / metabolism*
  • Acyl-CoA Oxidase / deficiency
  • Acyl-CoA Oxidase / genetics
  • Biofuels
  • Carnitine Acyltransferases / genetics
  • Carnitine Acyltransferases / metabolism
  • Cytoplasm / enzymology
  • Fatty Acids / biosynthesis*
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Metabolic Engineering*
  • Oxidation-Reduction
  • Peroxisomes / enzymology
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins / genetics
  • Transgenes
  • Yarrowia / chemistry
  • Yarrowia / enzymology

Substances

  • Acyl Coenzyme A
  • Biofuels
  • Fatty Acids
  • Isoenzymes
  • Saccharomyces cerevisiae Proteins
  • Acyl-CoA Oxidase
  • POX1 protein, S cerevisiae
  • Carnitine Acyltransferases
  • carnitine octanoyltransferase

Grant support

National Research Foundation, Singapore. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.