Schwientek, Patrick: Genomics and transcriptomics of the industrial acarbose producer Actinoplanes sp. SE50/110. 2012
Inhalt
- 1 Introduction
- 1.1 The genus Actinoplanes
- 1.2 The strain Actinoplanes sp. SE50/110
- 1.3 The secondary metabolite acarbose, its relevance, and mode of action
- 1.4 The biosynthesis of acarbose in Actinoplanes sp. SE50/110
- 1.5 Industrial development and fermentation of acarbose
- 1.6 Bacterial genome sequencing approaches
- 1.7 Bacterial genome annotation strategies
- 1.8 Means of bacterial transcriptome analysis
- 1.9 Motivation and aims of this thesis
- 2 Materials and Methods
- 2.1 Acquisition of the strain Actinoplanes sp. SE50/110
- 2.2 Genomic DNA-sequencing methods
- 2.2.1 Cultivation of Actinoplanes sp. SE50/110 for DNA-sequencing
- 2.2.2 Isolation of genomic DNA from Actinoplanes sp. SE50/110
- 2.2.3 Pyrosequencing of the Actinoplanes sp. SE50/110 genomic DNA on the Genome Sequencer FLX
- 2.3 Genome assembly and mapping techniques
- 2.4 Genome finishing methods
- 2.4.1 Construction of a fosmid library for the Actinoplanes sp. SE50/110 genome finishing
- 2.4.2 Polymerase chain reactions
- 2.4.3 Sanger sequencing of PCR products and terminal insert sequences from the Actinoplanes sp. SE50/110 fosmid library
- 2.4.4 Finishing of the Actinoplanes sp. SE50/110 genome sequence by manual assembly
- 2.5 Computational genome annotation
- 2.5.1 Prediction of coding sequences on the Actinoplanes sp. SE50/110 genome sequence
- 2.5.2 Functional annotation of the identified CDS on the Actinoplanes sp. SE50/110 genome
- 2.5.3 Phylogenetic analyses
- 2.6 RNA-sequencing and analysis
- 2.6.1 Cultivation of Actinoplanes sp. SE50/110 for RNA-sequencing
- 2.6.2 Total RNA isolation from Actinoplanes sp. SE50/110
- 2.6.3 Preparation of cDNA libraries and high-throughput sequencing
- 2.6.4 Determination of cell dry weights of Actinoplanes cultures
- 2.6.5 Quantification of acarbose in the supernatant of Actinoplanes cultures by HPLC and UV detection
- 2.6.6 Bioinformatic analysis of RNA-seq results
- 2.7 Gas-chromatographic analysis of the anti-self-annealing additive
- 3 Results
- 3.1 Solving the high-GC problem for Actinoplanes sp. SE50/110 genome sequencing
- 3.1.1 Analysis of gap regions resulted from standard PE sequencing
- 3.1.2 The gaps in the Actinoplanes sp. SE50/110 acarbose gene cluster are due to an extremely low read coverage
- 3.1.3 The gaps in the acarbose gene cluster are characterized by secondary structure formation
- 3.1.4 Adapted sequencing conditions solved the high-GC problem
- 3.2 The complete genome sequence of Actinoplanes sp. SE50/110
- 3.2.1 Assembly of the Actinoplanes sp. SE50/110 draft genome sequence
- 3.2.2 Finishing of the draft genome sequence
- 3.2.3 Annotation of the complete genome sequence
- 3.3 Discoveries of the Actinoplanes sp. SE50/110 genome
- 3.3.1 General features of the Actinoplanes sp. SE50/110 genome
- 3.3.2 Phylogenetic analysis of the Actinoplanes sp. SE50/110 16S rDNA reveals highest similarity to Actinoplanes utahensis
- 3.3.3 Comparative genome analysis indicates 50% singletons in the Actinoplanes sp. SE50/110 genome.
- 3.3.4 The high quality genome sequence of Actinoplanes sp. SE50/110 corrects the previously sequenced acarbose cluster.
- 3.3.5 Several genes of the acarbose gene cluster are also found in other locations of the genome.
- 3.3.6 Trehalose synthesis in Actinoplanes sp. SE50/110
- 3.3.7 The Actinoplanes sp. SE50/110 genome hosts an integrative and conjugative element
- 3.3.8 Four putative antibiotic production gene clusters were found in the Actinoplanes sp. SE50/110 genome sequence
- 3.4 RNA-sequencing of the Actinoplanes sp. SE50/110 transcriptome
- 4 Discussion
- 4.1 Establishment of the complete Actinoplanes sp. SE50/110 genome sequence
- 4.2 Annotation of the Actinoplanes sp. SE50/110 genome sequence
- 4.3 New insights related to the acarbose metabolism
- 4.3.1 Acarbose re-import after exclusion of acbHFG
- 4.3.2 Putative formation of component C by trehalose synthases
- 4.4 The actinomycete integrative and conjugative element pACPL
- 4.5 The putative antibiotic gene clusters of Actinoplanes sp. SE50/110
- 4.6 Transcriptome analyses of Actinoplanes sp. SE50/110
- 5 Conclusions and Outlook
- Bibliography
- A Appendix