Plant endophytes as Novel Bioresource

Natural biodiversity represents a nearly unlimited pool of biostructures and therefore it is the major source for obtaining enzymes and metabolites to be used in various industrial, medical, agricultural and environmental applications. Especially the microbial world reflects a huge genetic and metabolic biodiversity, which is to a great extent not yet explored, but offers a very high application potential. However, only a very small percentage of microbial species can be cultivated but in the last years methods to explore the uncultivable microbial world have been developed. One major fact determining the available pool of biostructures is the specific habitat of a microbial community which determines the composition of the available enzymatic and metabolic activities. One such interesting habitat are endophytic microbial populations of plants. This habitat reflects intense interactions between the microbial and the plant biological activities. Therefore it can be expected that it contains very specific biostructures, both at the enzymatic and the metabolic level. This project intends to explore plant endophytic habitats with a focus on obtaining a new diversity of biostructures, having a high potency for biocatalytic and agricultural applications. Metagenomic libraries of the microbial community present in such specific habitats will be set up to create a novel bioresource at the metagenome level. Though this project focusses on recruiting interesting new enzymes, the basic setup of metagenomic libraries will address both, creating a new resource for enzymes as well as for metabolites. Expression libraries of small fragments will be generated on the one hand to serve as a source for novel enzymes. On the other hand the creation of libraries which contain large genomic fragments will allow also to explore the metabolic diversity. Libraries will be established from two interesting habitats, one being a wild plant known to be able to efficiently grow at low temperatures (Crocus albiflorus) and the other being a plant of high agricultural interest (potato). One problem in exploring metagenomic populations for novel enzymes is functional expression in heterologous hosts. Therefore suitable host-vector systems which in addition to E.coli should serve as potent expression systems will be established, focussing on a Gram-positive Bacillus system and on a Streptomyces system. Established libraries will be screened by high-throughput techniques for novel enzymes with a focus on hydrolytic enzymes. Identified hits will be further explored for biocatalytic features and a comparison characterized at the molecular level. In addition, libraries are screened for ACC terminases serving as a basis for agricultural and environmental applications The combination of the specific expertises at the ARC Seibersdorf in exploring endophytes and of the Institute of Molecular Biotechnology of the TU Graz in molecular enzymology and screening will provide the basis to reach the major outcome of this project which will be: - the availability of metagenomic libraries from specific microbial communities - established tools for screening expression libraries using different hosts - sets of new enzymes being of high potential for biocatalytic and agricultural or environmental applications - extended scientific knowledge on the diversity of the endophytic communities of plants, including uncultivable species