|Title||Production of polyhydroxyalkanoates (PHAs) in transgenic potato|
|Source||Wageningen University. Promotor(en): L.H.W. van der Plas; R.G.F. Visser; H. Mooibroek. - S.l. : [s.n.] - ISBN 9789058087263 - 149|
Laboratory of Plant Physiology
|Publication type||Dissertation, internally prepared|
|Keyword(s)||solanum tuberosum - polymeren - polyesters - transgene planten - genetische transformatie - genetische modificatie - gentransfer - polyhydroxyalkanoaten - solanum tuberosum - polymers - polyesters - transgenic plants - genetic transformation - genetic engineering - gene transfer - polyhydroxyalkanoates|
|Categories||Genetic Engineering / Plant Physiology / Molecular Genetics|
Polyhydroxyalkanoates (PHAs) represent a large class of microbial polyesters which are widely distributed in prokaryotes. Because of the current environmental concerns related to the use of mineral-oil-based plastics, PHAs gained a considerable interest for their possible use as biodegradable polymers. In recent years the possible fields of application for PHAs have broadened and a considerable number of novel uses of PHAs as rubber material, coating material, binder in paints and several medical applications have been developed. However, high costs related to the fermentative production of PHAs in natural or recombinant microorganisms hampered the production of PHAs on large scale and the expansion of the utilisation of PHAs into society. The use of transgenic plants for the production of PHAs has been considered an excellent and elegant strategy to accumulate bulk amounts of PHAs at low costs. In this thesis, the possibility to produce PHAs in transgenic potato was investigated. A number of metabolic engineering strategies has been considered and the accumulation of low amounts of PHAs in transgenic potato lines has been achieved.
PHA production is one of those traits that require the coordinate expression of several genes. Because the minimum gene set required for PHA accumulation was not completely known at the beginning of the experiments described in this thesis, a flexible system was required for gene-transfer into the potato genome in order to avoid time-consuming or technically difficult approaches (multiple genes plant vector construction, crossing, re-transformation). It was advantageous to introduce simultaneously into the potato genome all the genes of interest delivered via separate DNA molecules. This allowed the easy combination of different sets of genes to establish the minimum gene-set required for PHA accumulation in potato. Although Agrobacterium -mediated transformation is a well established technique to deliver alien genes into potato, our preliminary analyses showed that it was not suitable for simultaneous co-transformation using separate plasmids. Thus, we developed a particle bombardment-mediated procedure to introduce simultaneously into the potato genome several genes from separate plasmids or separate linear DNA fragments. The particle bombardment protocol established during the course of this project proved to be very efficient and comparable with Agrobacterium -mediated transformation. We observed that transgenic potato plants obtained by particle bombardment showed relatively simple patterns of integration of the transgenes and integration of different transgenes at independent sites into the plant genome. The system also proved applicable for co-transformation of potato using gene cassettes, i.e. DNA fragments comprising promoter - gene - terminator.
Particle bombardment-mediated co-transformation was subsequently used to introduce the genes involved in the PHA biosynthetic pathways. Four methods were considered for the production of PHAs in potato.
Method 1. The accumulation of the homopolymer polyhydroxybutyrate (PHB) was achieved after Ralstonia eutrophaphb B and phb C genes coding for an NADPH-dependent acetoacetyl-CoA reductase and for the PHB-polymerase, respectively, were expressed in transgenic potato lines. Because the minimum gene set for PHB production in plants was well documented when this project started, the possible production of PHB seemed to be the simplest model to test the ability of potato to accumulate PHAs. Indeed, PHB formation in small amounts was observed.
Subsequently, we attempted the production of the heteropolymer medium-chain-length (mcl)-PHA in potato.
Method 2. To study the expression of the Pseudomonas oleovorans mcl-PHA-polymerase in potato, the polymerase was first expressed in the cytoplasm, where no post-transcriptional modification of the polypeptide is needed. Proper expression of the mcl-PHA polymerase was indeed observed in selected transgenic lines. However, because the cytoplasm of potato does not contain suitable substrates for the polymerase (mcl-hydroxy fatty acids), substrate feeding experiments were performed. These experiments resulted in the accumulation of low amounts of mcl-PHA.
Methods 3 and 4. Afterwards, mcl-PHA accumulation was attempted in plastids, by re-directing hydroxy fatty acid precursors from FAB to mcl-PHA biosynthesis. A truncated form of E. coli thioesterase-I ( tes A gene. Method 3) and P. putida ACP-CoA-transacylase ( pha G gene. Method 4) were expressed in combination with the Pha-C1 polymerase. All heterologous proteins were targeted to the plastid by N terminal fusion with the Rubisco transit peptide. The two combinations of genes (Pha-C1 plus thioesterase, and Pha-C1 plus transacylase) represent theoretically the minimum gene set for mcl-PHA accumulation deriving precursors from FAB. The double transformants which expressed the Pha-C1 polymerase and the thioesterase-I did not accumulate mcl-PHA because a fewb-oxidation steps may be required to provide the proper precursor to the polymerase. However, simultaneous expression of the Pha-C1 polymerase and the ACP-CoA-transacylase did result in the synthesis of low amounts of mcl-PHA in leaves of intact plants.
We established several possible routes to achieve the accumulation of PHAs in transgenic potato, based on the use of the endogenous pool of acetyl-CoA, on feeding approaches of cell suspension cultures or on using precursors derived from de novo FAB. Although improvements in these systems are still required in order to increase the yields of PHAs, the results open new prospects for the accumulation of PHAs in potato. Final conclusions on the future development of production of PHAs in plants are overviewed. Advantages and disadvantages of fermentation-based-PHAs, plant-based-PHAs, other biodegradable polymers and oil-based-plastics are compared. Novel and future possible interesting applications of PHAs not based on bulk productions, are also described.