期刊
CHEMBIOCHEM
卷 23, 期 6, 页码 -出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.202100702
关键词
directed evolution; Hly enhancer fragment; improved secretion efficiency; protein; protein secretion
资金
- CLIB Competence Center Biotechnology (CKB) - European Regional Development Fund ERDF [34.EFRE-0300096]
- Deutsche Forschungsgemeinschaft [CRC 1208]
- Projekt DEAL
In this study, the secretion efficiency of the hemolysin T1SS of Escherichia coli was optimized. Mutations in the hemolysin enhancer fragment resulted in increased secretion levels. Additionally, the inclusion of a native terminator region further enhanced secretion levels.
Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.
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