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Exploitable results
Sustainable production of biodegradable polyesters in starch-storing crop plants (PHASTICS, EU-FAIR Project CT96-1780) Introduction Within an existing consortium between the Wageningen University and Research Center, Agrotechnological Research Institute (WU-ATO, NL), Eidgenössische Technische Hochschule (ETHz, CH), Westfälische Wilhelms Universität Münster (WWUm, DE) and John Innes Centre (JIC, GB) an EU- sponsored project is in progress aiming at the development of a collection of novel bacterial polyesters (poly-[3-hydroxyalkanoates]) and their production in oleaginous yeasts and ultimately in starch-storing crop plants. The project involves the identification, isolation and characterization of bacterial genes involved in the biosynthesis of different medium chain length (mcl, C6-C16) PHAs and PHB. In addition, genes that mediate the supply of sufficient and adequate precursors both from bacterial and plant origin are similarly being isolated and characterized. In different combinations these genes are being and have been introduced, firstly into bacteria and yeasts in order to identify the minimum gene set required for plants, and then introduced into low-starch potato and pea. PHAs to be produced will be extracted, processed, characterized and subjected to application tests as shown in the example of figure 1. Work on bacteria A number of approaches for work on bacteria concerns: (i) heterologous expression of mcl-PHA synthase genes in the already available ß-oxidation mutants from R. eutropha, (ii) the production of 4HV-containing polyesters, (iii) the catabolism of levulinic acid in R. eutropha, (iv) the application of different E. coli fad-mutants and Pseudomonas transposon mutants, (v) identification and analysis of required gene(s) for the production of mcl-PHAs from non-related carbon sources, such as glucose, and (vi) the production, extraction and characterization of PHAs (e.g. molecular weight determination, glass transition temperature, melting temperature) from recombinant E. coli strains. Considerable progress has been achieved in the isolation and characterization of bacterial genes involved in the biosynthesis of different types of PHAs, including e.g. 3HB, 3HV and 4HV-PHA-containing polyesters. Mutants have been generated and corresponding genes are being characterized to further elucidate pathways involved in the biosynthesis of PHAs. Also recombinant bacteria have been constructed able to produce a number of PHAs. Recently, P. putida phaG and truncated E. coli 'tesA genes have been isolated and characterized which are involved in de novo mcl-PHA synthesis. This type of genes is considered essential for PHA synthesis in plants as part of the minimum gene set required. Plant work A number of these bacterial genes involved in mcl-PHA and PHB synthesis have been introduced into potato (and pea). Transgenic lines are now under study. Low-starch potato varieties and pea (rug) mutants are available for further transformation experiments. For the introduction of several essential genes into potato and pea multiple transformations and crossing of single transgenics is envisaged. Potato transformants containing and expressing the P. oleovorans phaC2 gene (in pET100) in microtubers have been generated, but no PHA has yet been detected, suggesting that the supply of precursors is insufficient or inadequate (or that plant transformation vectors need optimization). A microtuber induction system has been optimized for potato cultivars used. Also the P. oleovorans phaC1 and Alcaligenes eutrophus (renamed Ralstonia eutropha) phb-genes have been cloned into plant transformation vectors and used for transformation. A second marker (bar) system is available, which will allow re-transformation of available and coming primary potato transformants for Bialaphos-resistance and the introduction of genes for enhanced precursor supply. Within the framework of a Madam Curie Fellowship (FAIR-CT98-5036) a promising approach of simultaneous introduction of a number of different genes via Particle Bombardment is addressed. For pea the work has been initiated with vector constructions also based on bar selection, using the R. eutropha phbA, phbB and phbC genes and plastidial targeting sequences. Initial transformation experiments have yielded PPT resistant plant material that is currently being characterized. Similarly, phaC1, phaC2 and phaG genes are being inserted into vectors for pea transformation. Further research, also based on literature, will focus on the identification and isolation of bacterial and plant genes involved in the supply of PHA precursors and de novo PHA synthesis. These genes will be introduced first into the oleaginous yeast Cryptococcus curvatus, which already contains considerable amounts of fatty acids (50% DW) as storage compounds and also into plants by re-transformation, crossing and/or particle bombardment. The detection of PHAs in plants will firstly be performed following routine methods involving Nile Blue and Nile Red staining and later by more sophisticated techniques like in situ natural abundance 13C NMR. Once PHAs will have been detected methods for their extraction, processing, characterization and their applications will be addressed. Applications In order to generate a firm base of knowledge on the PHA polymer and material characteristics, and consequently to speed up the development of novel applications, ample research has been performed on bacterially produced PHAs (in parallel with PHA production in plants). Applications have already been developed in different areas that cover packaging, hygienic, agricultural and biomedical products. Recent applications of mcl-PHAs developed at ATO range from high solid alkyd-like paints to biodegradable cheese coatings and biodegradable rubbers. Many of these rubbers are used in applications which are difficult to recycle, such as consumables, diapers, back covers of carpets, construction moulds, and surgical devices and gloves. In the manufacture of coatings and paints, resins consisting of synthetic polymers are commonly used. Most of these synthetic binders are diluted with organic solvents to afford an optimal applicability and performance to the final paint formulation. The application of mcl-PHAs in paints has several important advantages. In contrast to the commercial synthetic alkyd resins, mcl-PHA binders are produced biologically from relatively cheap renewable resources. Furthermore, the amount of organic solvents needed in mcl-PHA based paints is very low. This significantly reduces the health and environmental burden by the use of organic solvents (Patent pending). Moreover, the development of water-based mcl-PHA paints is now in progress. In general, as demonstrated in these examples, the prospects for commercial applications based on PHAs are highly promising. Especially, once mcl-PHAs can be produced efficiently in plants at a large scale prices will also drop accordingly.
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