Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation

Abstract

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.

FIG. 1

DNA construct used to express the PHAC1 synthase of P. aeruginosa in S. cerevisiae. Only the portion of the construct containing the gene (open box) and regulatory elements (shaded box) is shown. The partial amino acid sequence of the C terminus of the PHA synthase-isocitrate lyase (ICL) fusion is indicated using the one-letter symbols, with the amino acids derived from the PHAC1 protein given in plain letters, novel amino acids created at the fusion junction italicized, and the last 34 amino acids derived from the B. napus isocitrate lyase underlined. CTA-Pr, CTA1 promoter; CTA-Tr, CTA1 terminator; E, EcoRI; H, HindIII.

FIG. 2

Western blot analysis of PHA synthase expression in S. cerevisiae. Wild-type (A) or recombinant yeast transformed with the PHA synthase (B to H) was grown for 24 h in media containing 1% glucose (B) or 0.1% glucose, 2% Pluronic-127, and 0.1% oleic acid (A and C) or for 72 h in media containing 0.1% glucose (D); 0.1% glucose and 0.5% Tween 80 (E); 0.1% glucose and 2% Pluronic-127 (F); 0.1% glucose, 0.5% Tween 80, and 0.1% oleic acid (G); or 0.1% glucose, 2% Pluronic-127, and 0.1% oleic acid (H). Molecular mass marker (in kilodaltons) is indicated on the left.

FIG. 3

GC-MS analysis of PHA produced in transgenic yeast expressing the PHA synthase. Yeast cells transformed with the control vector pYE352-CTA1 (A) or the plasmid pYE352-PHA harboring the PHA synthase gene (B) were grown for 3 days in media containing 2% Pluronic-127, 0.1% glucose, and 0.1% oleic acid, and the PHA was analyzed as described in Materials and Methods. Only ions with a mass-to-charge ratio of 103 are shown. The various 3-hydroxy acids are identified with the prefix H. The y axes in panels A and B are on the same scale.

FIG. 4

Time course of the accumulation of PHA in S. cerevisiae. Recombinant yeast was used to inoculate media containing 0.1% glucose, 2% Pluronic-127, and 0.1% oleic acid. PHA content (▪) in cells and the concentration of oleic acid (●) present in the media were monitored over 6 days. Values represent the mean and standard deviation of four measurements. w/dwt, weight/dwt; v/v, vol/vol.

FIG. 5

Analysis of PHA inclusions in S. cerevisiae. The wild-type (A) or recombinant yeast expressing the PHA synthase gene (B to D) was used to inoculate media containing 0.1% glucose, 2% Pluronic-127, and 0.1% oleic acid and was grown for 4 days before being processed for TEM. Panels C and D are close-up views of panel B. Arrows indicate the presence of PHA inclusions within membrane-bound organelles. ob, oil body. Bars indicate 1 μm (A and B) and 0.5 μm (C and D).