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

Abstract

Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(beta-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760-12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a beta-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding 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) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation.

Hybrid fiber production: a wood and plastic combination in transgenic rice and Tamarix made by accumulating poly-3-hydroxybutyrate

“…The values obtained in this study were in line with other studies that looked at different plants, though the transformed genes and targeting techniques we used were not the same: 20-100 mg gFW Ϫ1 in Arabidopsis (Poirier et al 1992), 14% DW in Arabidopsis (Nawrath et al 1994), less than 10 mg gFW Ϫ1 in tobacco (Nakashita et al 1999), 0-7.7% FW in Brassica (Houmiel et al 1999), 4% FW in Arabidopsis (Bohmert et al 2000), 2 to 8 ppm DW in tobacco (Nakashita et al 2001), maximal values of 3.2 mg gDW Ϫ1 in tobacco, 0.09 mg gDW Ϫ1 in potato, and 132 mg gDW Ϫ1 in Arabidopis (Bohmart et al 2002), 1.7% DW in tobacco (Lössl et al 2003), 55 mg gDW Ϫ1 in sugar beet (Menzel et al 2003), and 4.62 mg gFW Ϫ1 in flax (Wróbel et al 2004). It has been reported that growth retardation often accompanies the transformation, as is the case in tobacco (Lössl et al 2003).…”

“…These genes are found in many species of bacteria (see review by Madison and Huisman 1999), and the transfer of genes from bacteria to plant cells has been demonstrated (Poirier et al 1992). Although there are many reports about PHB production in transgenic plants, the levels of PHB accumulation have fallen short of expectations: 14% DW is the highest level reported in Arabidopsis (Nawrath et al 1994), Ͻ10 mg gFW Ϫ1 in tobacco (Nakashita et al 1999), 1-1.7% DW in Brassica napus (Houmiel et al 1999), and 4-5 mg gFW Ϫ1 in flax (Wróbel et al 2004). In addition, plant growth has reportedly been inhibited because of depletion of the available pool of acetyl-CoA (Lössl et al 2003).…”

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs

“…The use of transgenic plants for bulk PHA accumulation seems attractive because of the expected low cost of production compared to microbial fermentative PHA production (Nawrath et al 1995). However, until now, most of the reports describing the production of PHAs in plants have focussed on polyhydroxybutyrate (PHB) biosynthesis (Poirier et al 1992;Nawrath et al 1994;John and Keller 1996;Hahn et al 1999;Houmiel et al 1999;Nakashita et al 1999;Slater et al 1999) whose range of applications is limited compared to that of mcl-PHAs. Until recently, knowledge of mcl-PHA biosynthesis was incomplete, which hampered the identification of the minimum gene-set required for its production in recombinant hosts.…”

Analysis of bulked and redundant accessions of Brassica germplasm using assignment tests of microsatellite markers

“…At present, they are also used to produce biodiesel, as well as rubber and plastics (Kimber and McGregor 1995;Houmiel et al 1999). Brassica rapa (2n = 20, AA) is believed to be one of the diploid progenitor species of the amphidiploid B. napus (2n = 38, AACC).…”