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Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases
[Image: see text] Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and he...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10127261/ https://www.ncbi.nlm.nih.gov/pubmed/36920242 http://dx.doi.org/10.1021/acssynbio.2c00512 |
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author | Gelder, Kristen Van Oliveira-Filho, Edmar R. García-García, Jorge Donato Hu, You Bruner, Steven D. Hanson, Andrew D. |
author_facet | Gelder, Kristen Van Oliveira-Filho, Edmar R. García-García, Jorge Donato Hu, You Bruner, Steven D. Hanson, Andrew D. |
author_sort | Gelder, Kristen Van |
collection | PubMed |
description | [Image: see text] Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes (k(cat) < 1 h(–1)). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Seven beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants. |
format | Online Article Text |
id | pubmed-10127261 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-101272612023-04-26 Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases Gelder, Kristen Van Oliveira-Filho, Edmar R. García-García, Jorge Donato Hu, You Bruner, Steven D. Hanson, Andrew D. ACS Synth Biol [Image: see text] Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes (k(cat) < 1 h(–1)). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Seven beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants. American Chemical Society 2023-03-15 /pmc/articles/PMC10127261/ /pubmed/36920242 http://dx.doi.org/10.1021/acssynbio.2c00512 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Gelder, Kristen Van Oliveira-Filho, Edmar R. García-García, Jorge Donato Hu, You Bruner, Steven D. Hanson, Andrew D. Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases |
title | Directed Evolution
of Aerotolerance in Sulfide-Dependent
Thiazole Synthases |
title_full | Directed Evolution
of Aerotolerance in Sulfide-Dependent
Thiazole Synthases |
title_fullStr | Directed Evolution
of Aerotolerance in Sulfide-Dependent
Thiazole Synthases |
title_full_unstemmed | Directed Evolution
of Aerotolerance in Sulfide-Dependent
Thiazole Synthases |
title_short | Directed Evolution
of Aerotolerance in Sulfide-Dependent
Thiazole Synthases |
title_sort | directed evolution
of aerotolerance in sulfide-dependent
thiazole synthases |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10127261/ https://www.ncbi.nlm.nih.gov/pubmed/36920242 http://dx.doi.org/10.1021/acssynbio.2c00512 |
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