Production of polyhydroxyalkanoates (PHAs) in transgenic potato

摘要: Polyhydroxyalkanoates (PHAs) represent a large class of microbial polyesters which are widely distributed in prokaryotes. Because the current environmental concerns related to use mineral-oil-based plastics, PHAs gained considerable interest for their possible as biodegradable polymers. In recent years fields application have broadened and number novel uses rubber material, coating binder paints several medical applications been developed. However, high costs fermentative production natural or recombinant microorganisms hampered on scale expansion utilisation into society. The transgenic plants has considered an excellent elegant strategy accumulate bulk amounts at low costs. this thesis, possibility produce potato was investigated. A metabolic engineering strategies accumulation lines achieved. PHA is one those traits that require coordinate expression genes. minimum gene set required not completely known beginning experiments described flexible system gene-transfer genome order avoid time-consuming technically difficult approaches (multiple genes plant vector construction, crossing, re-transformation). It advantageous introduce simultaneously all delivered via separate DNA molecules. This allowed easy combination different sets establish gene-set potato. Although Agrobacterium -mediated transformation well established technique deliver alien potato, our preliminary analyses showed it suitable simultaneous co-transformation using plasmids. Thus, we developed particle bombardment-mediated procedure from plasmids linear fragments. bombardment protocol during course project proved be very efficient comparable with transformation. We observed obtained by relatively simple patterns integration transgenes independent sites genome. also applicable cassettes, i.e. fragments comprising promoter - terminator. Particle subsequently used involved biosynthetic pathways. Four methods were Method 1. homopolymer polyhydroxybutyrate (PHB) achieved after Ralstonia eutrophaphb B phb C coding NADPH-dependent acetoacetyl-CoA reductase PHB-polymerase, respectively, expressed lines. PHB documented when started, seemed simplest model test ability PHAs. Indeed, formation small observed. Subsequently, attempted heteropolymer medium-chain-length (mcl)-PHA 2. To study Pseudomonas oleovorans mcl-PHA-polymerase polymerase first cytoplasm, where no post-transcriptional modification polypeptide needed. Proper mcl-PHA indeed selected because cytoplasm does contain substrates (mcl-hydroxy fatty acids), substrate feeding performed. These resulted mcl-PHA. Methods 3 4. Afterwards, plastids, re-directing hydroxy acid precursors FAB biosynthesis. truncated form E. coli thioesterase-I ( tes gene. 3) P. putida ACP-CoA-transacylase pha G 4) Pha-C1 polymerase. All heterologous proteins targeted plastid N terminal fusion Rubisco transit peptide. two combinations (Pha-C1 plus thioesterase, transacylase) theoretically deriving FAB. double transformants did few b -oxidation steps may provide proper precursor result synthesis leaves intact plants. routes achieve based endogenous pool acetyl-CoA, cell suspension cultures derived de novo improvements these systems still increase yields PHAs, results open new prospects Final conclusions future development overviewed. Advantages disadvantages fermentation-based-PHAs, plant-based-PHAs, other polymers oil-based-plastics compared. Novel interesting productions, described.