PUB - Publikationen an der Universität Bielefeld

Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the global incidence of diabetes is rapidly rising, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation, which were continuously optimized by mutagenesis and screening experiments. However, being applied for over 20 years, this conventional optimization strategy has now reached its limits and is generally superseded by modern genetic engineering approaches, which require the genome sequence of the organism. Hence, the first part of this Ph. D. thesis dealt with the sequencing, assembly and annotation of the complete genome sequence of Actinoplanes sp. SE50/110, the first publicly available genome of the genus Actinoplanes. Due to its high GC-content of 71.32% and the formation of stable secondary structures that hindered the sequencing process, adapted protocols were developed which allowed the establishment of the complete sequence. The final genome consists of a single circular chromosome with a size of 9.24 Mb hosting about 8,400 genes. Besides the known acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase and hybrid-clusters were identified on the Actinoplanes genome. Another key finding represents the discovery of a functional actinomycete integrative and conjugative element, which might pose an elegant way of genetically accessing the organism. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae, whereas Actinoplanes utahensis was found to be the closest species based on 16S rDNA comparison. The second part of this work complemented the genomic information with transcriptome experiments using RNA-sequencing technology. These analyses resulted in the discovery of non-coding RNAs, novel protein coding sequences, and antisense transcripts to known genes, which lead to an improved annotation of the Actinoplanes sp. SE50/110 genome. Moreover, genome wide expression quantification provided – for the first time – insights into the transcriptional landscape of the acarbose producer. In this regard, differential expression testing between three different Actinoplanes cultivations were also performed in order to elucidate the changes in gene expression in response to varying growth-media compositions. It was found that the different media had significant impact on growth rate and acarbose productivity, which was clearly reflected on the transcriptional level. In particular, the acarbose biosynthesis gene cluster happened to be highly up-regulated in maltose-containing media and almost silent in the glucose-containing medium. Additionally, one of the identified non-ribosomal peptide synthetase gene clusters showed high expression, which resembled the expressional pattern of the acarbose cluster across the analyzed conditions.