Poly(beta-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers whi ch offer an environmentally sustainable alternative to petroleum-based plas tics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(beta-hydro xybutyrate) (PHB) has previously been produced in leaves of Arabinopsis tha liana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91. 12760-12764). Howev er, Brassica napus oilseed may provide a better system for PHB production b ecause acetyl-CoA, the substrate required in the first step of PHB biosynth esis, is prevalent during fatty acid biosynthesis. Three enzymatic activiti es are needed to synthesize PHB: a beta-ketothiolase, an aceto-acetyl-CoA r eductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha en coding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(beta-hydroxybutyrate) acc umulated in leukoplasts to levels as high as 7.7% fresh seed weight of matu re seeds. Electronmicroscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to p olymer accumulation.