PUB - Publikationen an der Universität Bielefeld

The alpha-glucosidase inhibitor acarbose is used for treatment of diabetes mellitus type 2, and is manufactured industrially with overproducing derivatives of _Actinoplanes_ sp. SE50/110. This strain was reportedly optimized through step-by-step conventional mutagenesis procedures in the past, however this strategy seems to reach its limits by now. Despite of high industrial significance, only limited information exists regarding acarbose metabolism, function and regulation of these processes, due to the absence of proper genetic engineering methods and tools developed for this strain. In this work, a full toolkit and set of methods for genetic engineering of _Actinoplanes_ sp. SE50/110 were developed. A standardized protocol for a DNA transfer through _E. coli_ - _Actinoplanes_ conjugation was adjusted and applied for the transfer of phiC31, phiBT1 and VWB actinophage-based integrative vectors and pSG5-based replicative vector. Integration sites, occurring once per genome for all integrative vectors, were sequenced and characterized for the first time in _{Actinoplanes_ sp. SE50/110. Notably, the studied plasmids were proven to be stable and neutral with respect to strain morphology and acarbose production, enabling future use for genetic manipulations of _Actinoplanes_ sp. SE50/110. To further broaden the spectrum of available tools, a GUS reporter system, was established in _Actinoplanes_ sp. SE50/110. The set of different methods for gene knockouts was tested, which included integrative and replicative vector based knockouts, ReDirect system based knockouts and CRISPR-Cas9 genetic engineering. ReDirect system was further used to create a library of _Actinoplanes_ single knockout strains. Two of the strains, _Actinoplanes_ _acbD_ and _Actinoplanes_ _cadC_ knockout mutants were further characterized in detail regarding their phenotype.
The developed gene cloning system offers multiple possibilities to solve fundamental questions regarding acarbose production, in particular, formulation and verification of the complete acarbose metabolism model, as well as the rational design of acarbose overproducing strains.