High-resolution genome and transcriptome analysis of Gluconobacter oxydans 621H and growth-improved strains by next-generation sequencing

The acetic acid bacterium Gluconobacter oxydans is an important organism used in industrial biotechnology. It is characterized by its exceptional ability to regio- and stereoselectively oxidize a broad range of substrates in the periplasm. However, a disadvantage of this bacterium is the low final biomass yield on sugar-containing complex media. Recently, metabolic engineering allowed construction of strain IK003.1 with a 60% increased biomass yield on glucose. Modifying metabolism in such a way may affect the genome stability and may cause suppressor mutations. Therefore, one aim of this thesis was the use of next-generation and nanopore sequencing to sequence the genomes of engineered and reference strains in order to detect mutations. Except for the introduced genetic alterations and one mobile element insertion, no further mutations were found in strain IK003.1 in comparison to the reference strains. This suggests that the constructed strain is quite stable and therefore well suited for further metabolic engineering efforts. Furthermore, the new sequencing results were used to update the reference genome sequence of G. oxydans 621H. The second part of this thesis dealt with comprehensive RNAseq analysis to characterize the transcriptional landscapes of G. oxydans. This resulted in the detection of 2,449 transcription start sites (TSSs) and allowed to define the -10 region “nATnnn” and the -35 region “ttGnnn” as promoter consensus sequences. Analysis of 5´-UTRs also showed that 5% of all transcripts with an identified TSS are leaderless and 43% are longer than 100 nt. Furthermore, 971 potential novel transcripts were identified. 1,144 genes (41%) were found to be expressed monocistronically, whereas 1,634 genes (59%) belonged to 571 operons. Also, TSSs within operons indicated expression of 720 genes in 341 sub-operons. The stability of mRNAs plays an important role in the post-transcriptional regulation of gene expression and can influence the production rate of proteins and growth of bacteria. Using DNA microarrays, we determined mRNA half-lives for 2,500 genes (95%) and analysed them based on a functional categorization. Furthermore, we observed instability of the 23S rRNA. Next-generation sequencing of rRNA isolated from enriched ribosomes revealed a distinct fragmentation pattern and indicated the presence of three fragmentation positions in three 23S rRNAs and four fragmentation positions in one 23S rRNA of G. oxydans.