Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast

Summary Directed evolution is a powerful method to optimize proteins and metabolic reactions towards user‐defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence‐spaces...

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Autores principales:	Emil D. Jensen, Francesca Ambri, Marie B. Bendtsen, Alex A. Javanpour, Chang C. Liu, Michael K. Jensen, Jay D. Keasling
Formato:	article
Lenguaje:	EN
Publicado:	Wiley 2021
Materias:	Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/2dcfe0c268f74d13888f38a9b161fae9
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spelling	oai:doaj.org-article:2dcfe0c268f74d13888f38a9b161fae92021-11-18T15:39:53ZIntegrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast1751-791510.1111/1751-7915.13774https://doaj.org/article/2dcfe0c268f74d13888f38a9b161fae92021-11-01T00:00:00Zhttps://doi.org/10.1111/1751-7915.13774https://doaj.org/toc/1751-7915Summary Directed evolution is a powerful method to optimize proteins and metabolic reactions towards user‐defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence‐spaces is a labour‐intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high‐throughput using a transcription factor‐based biosensor. We demonstrate the strengths of this workflow by evolving a rate‐limiting enzymatic reaction of the biosynthetic pathway for cis,cis‐muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high‐throughput sorting of best‐performing variants using a transcription factor‐based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13‐fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.Emil D. JensenFrancesca AmbriMarie B. BendtsenAlex A. JavanpourChang C. LiuMichael K. JensenJay D. KeaslingWileyarticleBiotechnologyTP248.13-248.65ENMicrobial Biotechnology, Vol 14, Iss 6, Pp 2617-2626 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biotechnology TP248.13-248.65
spellingShingle	Biotechnology TP248.13-248.65 Emil D. Jensen Francesca Ambri Marie B. Bendtsen Alex A. Javanpour Chang C. Liu Michael K. Jensen Jay D. Keasling Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast
description	Summary Directed evolution is a powerful method to optimize proteins and metabolic reactions towards user‐defined goals. It usually involves subjecting genes or pathways to iterative rounds of mutagenesis, selection and amplification. While powerful, systematic searches through large sequence‐spaces is a labour‐intensive task, and can be further limited by a priori knowledge about the optimal initial search space, and/or limits in terms of screening throughput. Here, we demonstrate an integrated directed evolution workflow for metabolic pathway enzymes that continuously generate enzyme variants using the recently developed orthogonal replication system, OrthoRep and screens for optimal performance in high‐throughput using a transcription factor‐based biosensor. We demonstrate the strengths of this workflow by evolving a rate‐limiting enzymatic reaction of the biosynthetic pathway for cis,cis‐muconic acid (CCM), a precursor used for bioplastic and coatings, in Saccharomyces cerevisiae. After two weeks of simply iterating between passaging of cells to generate variant enzymes via OrthoRep and high‐throughput sorting of best‐performing variants using a transcription factor‐based biosensor for CCM, we ultimately identified variant enzymes improving CCM titers > 13‐fold compared with reference enzymes. Taken together, the combination of synthetic biology tools as adopted in this study is an efficient approach to debottleneck repetitive workflows associated with directed evolution of metabolic enzymes.
format	article
author	Emil D. Jensen Francesca Ambri Marie B. Bendtsen Alex A. Javanpour Chang C. Liu Michael K. Jensen Jay D. Keasling
author_facet	Emil D. Jensen Francesca Ambri Marie B. Bendtsen Alex A. Javanpour Chang C. Liu Michael K. Jensen Jay D. Keasling
author_sort	Emil D. Jensen
title	Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast
title_short	Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast
title_full	Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast
title_fullStr	Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast
title_full_unstemmed	Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast
title_sort	integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast
publisher	Wiley
publishDate	2021
url	https://doaj.org/article/2dcfe0c268f74d13888f38a9b161fae9
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Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast

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