Modification of the monomer composition of polyhydroxyalkanoate synthesized in Saccharomyces cerevisiae expressing variants of the beta-oxidation-associated multifunctional enzyme.
Expression by Saccharomyces cerevisiae of a polyhydroxyalkanoate (PHA) synthase modified at the carboxy end by the addition of a peroxisome targeting signal derived from the last 34 amino acids of the Brassica napus isocitrate lyase (ICL) and containing the terminal tripeptide Ser-Arg-Met resulted in the synthesis of PHA. The ability of the terminal peptide Ser-Arg-Met and of the 34-amino-acid peptide from the B. napus ICL to target foreign proteins to the peroxisome of S. cerevisiae was demonstrated with green fluorescent protein fusions. PHA synthesis was found to be dependent on the presence of both the enzymes generating the beta-oxidation intermediate 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-[CoA]) and the peroxin-encoding PEX5 gene, demonstrating the requirement for a functional peroxisome and a beta-oxidation cycle for PHA synthesis. Using a variant of the S. cerevisiae beta-oxidation multifunctional enzyme with a mutation inactivating the B domain of the R-3-hydroxyacyl-CoA dehydrogenase, it was possible to modify the PHA monomer composition through an increase in the proportion of the short-chain monomers of five and six carbons.
Marchesini
Silvia
S
Laboratoire de Biotechnologie Végétale, Département de Biologie Moléculaire Végétale, Université de Lausanne, CH-1015 Lausanne, Switzerland.
Erard
Nadine
N
Glumoff
Tuomo
T
Hiltunen
J Kalervo
JK
Poirier
Yves
Y
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000235:genetics*; D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D004746:Enoyl-CoA Hydratase / Q000235:genetics*; D005227:Fatty Acids / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods; D009097:Multienzyme Complexes / Q000235:genetics*; D009154:Mutation; D010084:Oxidation-Reduction; D020675:Peroxisomes / Q000201:enzymology; D011091:Polyesters / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D005227:Fatty Acids; D009097:Multienzyme Complexes; D011091:Polyesters; D015094:3-Hydroxyacyl CoA Dehydrogenases; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D004746:Enoyl-CoA Hydratase