| dc.description.abstracteng | Bacillus licheniformis has been employed as industrial workhorse since several decades, especially for the production of alkaline serine proteases like Subtilisin Carlsberg, which is used as additive inhousehold detergents. Since the progress in second-generation sequencing technologies facilitated the determination of whole bacterial transcriptomes by RNA-Seq approaches, in-depth analyses of different stages of a fermentation process are now possible and allow new insights into bacterial regulation to accomplish optimization of production strains and bioprocesses.
In total, 15 samples of B. licheniformis DSM13 were derived from industrial-oriented fermentations, aiming at the production of Subtilisin Carlsberg. The samples were taken at five different time points during three independent fermentation processes. Whole transcriptome analysis yielded 2.4x107 to 4.3x107 RNA-Seq reads per sample. Analysis of these results and the utilization of public databases enabled the complete reannotation of the genome of B. licheniformis. Subsequently, the development of suitable prediction algorithms, led to the identification of 2798 untranslated regions and 461 non-coding RNAs in the generated transcriptome data sets. Thorough examination showed that 14% and 89% of these RNA features, respectively, are located in antisense orientation to a gene on the opposite strand, revealing a previously not expected wealth of putative antisense transcription-based regulation. Furthermore, the analyses allowed the confirmation and new identification of cis-regulatory elements, and the determination of several new RNAs, which are either expressed independently or located in intergenic regions. Cluster analysis of the transcriptome data allowed the assignment of differentially expressed protein and ncRNA genes to distinct expression patterns.
Moreover, differential RNA-Seq analysis of five of the aforementioned samples allowed the identification of 1500 transcription start sites. 408 of these are not located in a common promoter region, pinpointing additional regulatory sites. The integration of dRNA-Seq and whole transcriptome RNA-Seq data made it possible to generate the first experimentally verified operon map of B. licheniformis.
Proteome analysis of the fermentation samples, based on 2D gel fractionation and subsequent assignment by mass spectrometry, yielded 367 protein spots representing 260 different proteins. Together with the obtained gene expression values, these data enabled the thorough examination of carbon and nitrogen metabolism, stress responses, sporulation, secretion capacities and cell differentiation, in order to provide an overview on the complex dynamics of B. licheniformis during an industry-oriented fermentation process.
The comprehensive analysis of all generated data facilitated the identification of putative targets for bioprocess and strain optimization approaches. These targets comprise, amongst others, the Tat-secretory pathway, the cell differentiation cascade and the antisense transcript located opposite to apr, which encodes the native Subtilisin Carlsberg preprotein.
Finally, the accomplished complete sequencing and annotation of the 3.6 Mb genome of Geobacillus sp. GHH01, a thermophilic, lipase-secreting member of the family Bacillaceae, grants access to a new possible industrial production platform for high-temperature applications. | de |