Cargando…
Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region
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 upstrea...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9306574/ https://www.ncbi.nlm.nih.gov/pubmed/35062047 http://dx.doi.org/10.1002/cbic.202100702 |
_version_ | 1784752569744097280 |
---|---|
author | Pourhassan N., Zohreh Cui, Haiyang Khosa, Sakshi Davari, Mehdi D. Jaeger, Karl‐Erich Smits, Sander H. J. Schwaneberg, Ulrich Schmitt, Lutz |
author_facet | Pourhassan N., Zohreh Cui, Haiyang Khosa, Sakshi Davari, Mehdi D. Jaeger, Karl‐Erich Smits, Sander H. J. Schwaneberg, Ulrich Schmitt, Lutz |
author_sort | Pourhassan N., Zohreh |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-9306574 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-93065742022-07-28 Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region Pourhassan N., Zohreh Cui, Haiyang Khosa, Sakshi Davari, Mehdi D. Jaeger, Karl‐Erich Smits, Sander H. J. Schwaneberg, Ulrich Schmitt, Lutz Chembiochem Research Articles 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. John Wiley and Sons Inc. 2022-02-08 2022-03-18 /pmc/articles/PMC9306574/ /pubmed/35062047 http://dx.doi.org/10.1002/cbic.202100702 Text en © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Research Articles Pourhassan N., Zohreh Cui, Haiyang Khosa, Sakshi Davari, Mehdi D. Jaeger, Karl‐Erich Smits, Sander H. J. Schwaneberg, Ulrich Schmitt, Lutz Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region |
title | Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region |
title_full | Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region |
title_fullStr | Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region |
title_full_unstemmed | Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region |
title_short | Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region |
title_sort | optimized hemolysin type 1 secretion system in escherichia coli by directed evolution of the hly enhancer fragment and including a terminator region |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9306574/ https://www.ncbi.nlm.nih.gov/pubmed/35062047 http://dx.doi.org/10.1002/cbic.202100702 |
work_keys_str_mv | AT pourhassannzohreh optimizedhemolysintype1secretionsysteminescherichiacolibydirectedevolutionofthehlyenhancerfragmentandincludingaterminatorregion AT cuihaiyang optimizedhemolysintype1secretionsysteminescherichiacolibydirectedevolutionofthehlyenhancerfragmentandincludingaterminatorregion AT khosasakshi optimizedhemolysintype1secretionsysteminescherichiacolibydirectedevolutionofthehlyenhancerfragmentandincludingaterminatorregion AT davarimehdid optimizedhemolysintype1secretionsysteminescherichiacolibydirectedevolutionofthehlyenhancerfragmentandincludingaterminatorregion AT jaegerkarlerich optimizedhemolysintype1secretionsysteminescherichiacolibydirectedevolutionofthehlyenhancerfragmentandincludingaterminatorregion AT smitssanderhj optimizedhemolysintype1secretionsysteminescherichiacolibydirectedevolutionofthehlyenhancerfragmentandincludingaterminatorregion AT schwanebergulrich optimizedhemolysintype1secretionsysteminescherichiacolibydirectedevolutionofthehlyenhancerfragmentandincludingaterminatorregion AT schmittlutz optimizedhemolysintype1secretionsysteminescherichiacolibydirectedevolutionofthehlyenhancerfragmentandincludingaterminatorregion |