PUB - Publikationen an der Universität Bielefeld

Actinoplanes sp. SE50/110 is a natural producer of acarviosyl-maltose (acarbose), which is used in the treatment of diabetes mellitus. The genome, transcriptome and proteome have been extensively studied in the last decades and methods for targeted genome editing were established. In this work, the acarviosyl-maltose metabolism is studied by gene deletion and overexpression. Since a suitable system for the overexpression of singular genes did not exist before, different strategies were tested and evaluated in this work, which led to the development of a novel expression system, called pSETT4. In order to improve the acarviose-synthesis, three different strategies were followed: increasing of the gene dose of acb genes to enhance the flux through the acarbose biosynthesis, deployment of precursors and reducing of the metabolic burden. Approaches of all of these strategies led to an improved acarbose formation: By overexpression of the acb gene encoding the dTDP-D-glucose-4,6-dehydratase AcbB, the final acarbose concentration was significantly increased by r. a. 50 %. In order to improve supply of the precursor glucose-1P, the uridyltransferase GtaB was overexpressed yielding into 8.5 % more acarbose. By functional deletion of the small carbohydrate protein Cgt in order to reduce the metabolic burden, the acarbose formation was significantly enhanced to 8-16 %, which was robust over long time periods and in different cultivation settings. Furthermore, a putative bottleneck reaction within the acarbose biosynthesis was unraveled by overexpression of the gene coding for the C7-cyclitol synthase AcbC. Besides, growth experiments of the wild type and a regulator mutant ∆merR exposed to and hidden from light were conducted, by which a negative influence of light-induced stress and the carotenoid formation on the acarbose production was observed. Since maltose is an important building block of the acarviosyl-maltose biosynthesis and a central carbon source, this work focused on the maltose metabolism of Actinoplanes sp. SE50/110. By growth analyses and enzymatic assays, strain specific peculiarities like the lack of a phosphohydrolase MalP and the central role of the maltase AmlE were unraveled. This led to the first reconstruction of the maltose/maltodextrin system in a member of the family Micromonosporaceae. Furthermore, a complex transcriptional regulation was investigated, which connects the maltose assimilation to the presence of the degradation product glucose via the local transcriptional repressor AmlR and to global “life-switch-mechanisms” via the nucleotide messenger c-di-GMP. Research in this area provides a great potential for the future strain development in order to improve the quantity and quality of the fine-chemical acarbose.