Schaffert, Lena ; Schneiker-Bekel, Susanne; Gierhake, Jessica; Droste, Julian; Persicke, Marcus; Rosen, Winfried; Pühler, Alfred ; Kalinowski, Jörn: Absence of the highly expressed small carbohydrate-binding protein Cgt improves the acarbose formation in Actinoplanes sp. SE50/110. In: Applied Microbiology and Biotechnology. Jg.104. 2020, S. 5395–5408 ff.
Inhalt
- Bioinformatics
- Media and growth conditions of Actinoplanes sp. SE50/110
- Preparation of spore solutions of Actinoplanes sp. SE50/110
- Preparation of media
- Shake flask cultivation
- Miniaturized cultivation in the BioLector system of m2p-labs GmbH (Baesweiler, Germany)
- Acarbose quantification from the supernatant by high-performance liquid chromatography
- Starch-binding assay
- SDS-polyacrylamide gel electrophoresis
- Matrix-assisted laser desorption ionization-time of flight-mass spectrometry
- Recombinant DNA work
- Conjugal transfer to Actinoplanes sp. SE50/110 and plasmid curing
- Screening experiments in the Biolog® OmniLog Phenotypic Microarray System
- Sampling and RNA isolation
- Reverse transcription quantitative PCR
- Results
- Distribution of single-domain CBM-20 proteins in the eubacterial world
- Confirmation of the starch-binding function by an invitro assay
- Analysis of cgt expression during growth on different carbon sources
- The gene deletion mutant ∆cgt has no apparent growth phenotype on different carbon sources or under carbon-limited conditions
- Cgt has no impact on osmolality- or pH-tolerance
- The ∆cgt mutant displays an improved acarbose formation on maltose minimal medium
- Deletion of cgt has no impact on the expression of acarbose biosynthesis genes
- Discussion
- References