Schaffert, Lena; März, Camilla; Burkhardt, Lisa; Droste, Julian; Brandt, David; Busche, Tobias; Rosen, Winfried; Schneiker-Bekel, Susanne; Persicke, Marcus; Pühler, Alfred; Kalinowski, Jörn: Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110. In: Microbial Cell Factories. Jg.18 H. 1. 2019
Inhalt
- Results and discussion
- Unintended chromosomal integration of pKC1139-based vectors by homologous recombination in Actinoplanes sp. SE50110
- Gene duplication by use of the integrative pSET152 vector system did not lead to enhanced expression of genes
- Evaluation of heterologous and homologous promoters by screening experiments
- Transfer of promising promoters to the pSET152-expression system leads to the overexpression of the acbC gene
- Overexpression of the acbC gene leads to accumulation of a phosphorylated intermediate of acarbose biosynthesis
- Conclusion
- Methods
- Media and cultivation conditions of Actinoplanes sp. SE50110
- Preparation of glycerol stocks and spore solutions of Actinoplanes sp. SE50110
- Preparation of minimal medium
- Shake flask cultivation
- Acarbose quantification from the supernatant by high performance liquid chromatography measurement (HPLC)
- Recombinant DNA work
- Construction of pKC1139 expression system
- Construction of pSET152 expression system by use of native promoters
- Construction of plasmids for the gusA reporter system
- Construction of pSET152 overexpression system with strong promoters
- Conjugal transfer to Actinoplanes sp. SE50110
- Promoter screening experiment by spectrophotometric measurement of the glucuronidase activity
- RNA isolation and reverse transcription quantitative PCR
- Liquid chromatography-mass spectrometry (LC–MS) measurements
- LC–ESI–MS
- Acknowledgements
- References