Details

Autor(en) / Beteiligte

• Pourhassan N., Zohreh
• Cui, Haiyang
• Khosa, Sakshi
• Davari, Mehdi D.
• Jaeger, Karl‐Erich
• Smits, Sander H. J.
• Schwaneberg, Ulrich
• Schmitt, Lutz

Titel

Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

Ist Teil von

• Chembiochem : a European journal of chemical biology, 2022-03, Vol.23 (6), p.e202100702-n/a

Ort / Verlag

Germany: Wiley Subscription Services, Inc

Erscheinungsjahr

2022

Link zum Volltext

Quelle

Wiley Online Library - AutoHoldings Journals

Beschreibungen/Notizen

• 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. Secretion efficiency of the Hly secretion system was optimized through directed evolution of the Hly enhancer fragment using KnowVolution strategy. The structural analysis of wildtype and the developed enhancer fragment suggested that the improved secretion is due to a different affinity of the novel enhancer fragment towards the S1 ribosomal protein. This was further supported by a RNA pulldown assay.