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Assessing and harnessing updated polyketide synthase modules through combinatorial engineering

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases constructed using the recently updated module boundary have been shown to outperform those using the traditional boundary, larger syntha...

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Autores principales: Ray, Katherine A., Lutgens, Joshua D., Bista, Ramesh, Zhang, Jie, Desai, Ronak R., Hirsch, Melissa, Miyazawa, Takeshi, Cordova, Antonio, Keatinge-Clay, Adrian T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Journal Experts 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10402262/
https://www.ncbi.nlm.nih.gov/pubmed/37546965
http://dx.doi.org/10.21203/rs.3.rs-3157617/v1
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author Ray, Katherine A.
Lutgens, Joshua D.
Bista, Ramesh
Zhang, Jie
Desai, Ronak R.
Hirsch, Melissa
Miyazawa, Takeshi
Cordova, Antonio
Keatinge-Clay, Adrian T.
author_facet Ray, Katherine A.
Lutgens, Joshua D.
Bista, Ramesh
Zhang, Jie
Desai, Ronak R.
Hirsch, Melissa
Miyazawa, Takeshi
Cordova, Antonio
Keatinge-Clay, Adrian T.
author_sort Ray, Katherine A.
collection PubMed
description The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases constructed using the recently updated module boundary have been shown to outperform those using the traditional boundary, larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases from the updated modules of the Pikromycin synthase. Every combinatorial possibility of modules 2–6 inserted between the first and last modules of the native synthase was constructed and assayed. Anticipated products were observed from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. Ketosynthase gatekeeping and module-skipping were determined to be the principal impediments to obtaining functional synthases. The platform was also used to create functional hybrid synthases through the incorporation of modules from the Erythromycin, Spinosyn, and Rapamycin assembly lines. The relaxed gatekeeping observed from a ketosynthase in the Rapamycin synthase is especially encouraging in the quest to produce designer polyketides.
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spelling pubmed-104022622023-08-05 Assessing and harnessing updated polyketide synthase modules through combinatorial engineering Ray, Katherine A. Lutgens, Joshua D. Bista, Ramesh Zhang, Jie Desai, Ronak R. Hirsch, Melissa Miyazawa, Takeshi Cordova, Antonio Keatinge-Clay, Adrian T. Res Sq Article The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases constructed using the recently updated module boundary have been shown to outperform those using the traditional boundary, larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases from the updated modules of the Pikromycin synthase. Every combinatorial possibility of modules 2–6 inserted between the first and last modules of the native synthase was constructed and assayed. Anticipated products were observed from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. Ketosynthase gatekeeping and module-skipping were determined to be the principal impediments to obtaining functional synthases. The platform was also used to create functional hybrid synthases through the incorporation of modules from the Erythromycin, Spinosyn, and Rapamycin assembly lines. The relaxed gatekeeping observed from a ketosynthase in the Rapamycin synthase is especially encouraging in the quest to produce designer polyketides. American Journal Experts 2023-07-28 /pmc/articles/PMC10402262/ /pubmed/37546965 http://dx.doi.org/10.21203/rs.3.rs-3157617/v1 Text en https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) , which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
spellingShingle Article
Ray, Katherine A.
Lutgens, Joshua D.
Bista, Ramesh
Zhang, Jie
Desai, Ronak R.
Hirsch, Melissa
Miyazawa, Takeshi
Cordova, Antonio
Keatinge-Clay, Adrian T.
Assessing and harnessing updated polyketide synthase modules through combinatorial engineering
title Assessing and harnessing updated polyketide synthase modules through combinatorial engineering
title_full Assessing and harnessing updated polyketide synthase modules through combinatorial engineering
title_fullStr Assessing and harnessing updated polyketide synthase modules through combinatorial engineering
title_full_unstemmed Assessing and harnessing updated polyketide synthase modules through combinatorial engineering
title_short Assessing and harnessing updated polyketide synthase modules through combinatorial engineering
title_sort assessing and harnessing updated polyketide synthase modules through combinatorial engineering
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10402262/
https://www.ncbi.nlm.nih.gov/pubmed/37546965
http://dx.doi.org/10.21203/rs.3.rs-3157617/v1
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