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CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli

Protein engineering plays an increasingly important role in developing new and optimizing existing metabolic pathways for biosynthesis. Conventional screening approach of libraries of gene and enzyme variants is often done using a host strain under conditions not relevant to the cultivation or intra...

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Autores principales: Chen, Minliang, Chen, Lin, Zeng, An-Ping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520568/
https://www.ncbi.nlm.nih.gov/pubmed/31193188
http://dx.doi.org/10.1016/j.mec.2019.e00094
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author Chen, Minliang
Chen, Lin
Zeng, An-Ping
author_facet Chen, Minliang
Chen, Lin
Zeng, An-Ping
author_sort Chen, Minliang
collection PubMed
description Protein engineering plays an increasingly important role in developing new and optimizing existing metabolic pathways for biosynthesis. Conventional screening approach of libraries of gene and enzyme variants is often done using a host strain under conditions not relevant to the cultivation or intracellular conditions of the later production strain. This does not necessarily result in the identification of the best enzyme variant for in vivo use in the production strain. In this work, we propose a method which integrates CRISPR/Cas9-facilitated engineering of the target gene(s) with growth-coupled and sensor-guided in vivo screening (CGSS) for protein engineering and pathway optimization. The efficiency of the method is demonstrated for engineering 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase AroG, a key enzyme in the chorismate pathway for the synthesis of aromatic amino acids (AAAs), to obtain variants of AroG (AroG(fbr)) with increased resistance to feedback inhibition of Phe. Starting from a tryptophan (Trp)-producing E. coli strain (harboring a reported Phe-resistant AroG variant AroG(S180F)), the removal of all the endogenous DAHP synthases makes the growth of this strain dependent on the activity of an introduced AroG variant. The different catalytic efficiencies of AroG variants lead to different intracellular concentration of Trp which is sensed by a Trp biosensor (TnaC-eGFP). Using the growth rate and the signal strength of the biosensor as criteria, we successfully identified several novel Phe-resistant AroG variants (including the best one AroG(D6G−D7A)) which exhibited higher specific enzyme activity than that of the reference variant AroG(S180F) at the presence of 40 mM Phe. The replacement of AroG(S180F) with the newly identified AroG(D6G−D7A) in the Trp-producing strain significantly improved the Trp production by 38.5% (24.03 ± 1.02 g/L at 36 h) in a simple fed-batch fermentation.
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spelling pubmed-65205682019-05-23 CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli Chen, Minliang Chen, Lin Zeng, An-Ping Metab Eng Commun Article Protein engineering plays an increasingly important role in developing new and optimizing existing metabolic pathways for biosynthesis. Conventional screening approach of libraries of gene and enzyme variants is often done using a host strain under conditions not relevant to the cultivation or intracellular conditions of the later production strain. This does not necessarily result in the identification of the best enzyme variant for in vivo use in the production strain. In this work, we propose a method which integrates CRISPR/Cas9-facilitated engineering of the target gene(s) with growth-coupled and sensor-guided in vivo screening (CGSS) for protein engineering and pathway optimization. The efficiency of the method is demonstrated for engineering 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase AroG, a key enzyme in the chorismate pathway for the synthesis of aromatic amino acids (AAAs), to obtain variants of AroG (AroG(fbr)) with increased resistance to feedback inhibition of Phe. Starting from a tryptophan (Trp)-producing E. coli strain (harboring a reported Phe-resistant AroG variant AroG(S180F)), the removal of all the endogenous DAHP synthases makes the growth of this strain dependent on the activity of an introduced AroG variant. The different catalytic efficiencies of AroG variants lead to different intracellular concentration of Trp which is sensed by a Trp biosensor (TnaC-eGFP). Using the growth rate and the signal strength of the biosensor as criteria, we successfully identified several novel Phe-resistant AroG variants (including the best one AroG(D6G−D7A)) which exhibited higher specific enzyme activity than that of the reference variant AroG(S180F) at the presence of 40 mM Phe. The replacement of AroG(S180F) with the newly identified AroG(D6G−D7A) in the Trp-producing strain significantly improved the Trp production by 38.5% (24.03 ± 1.02 g/L at 36 h) in a simple fed-batch fermentation. Elsevier 2019-05-06 /pmc/articles/PMC6520568/ /pubmed/31193188 http://dx.doi.org/10.1016/j.mec.2019.e00094 Text en © 2019 Published by Elsevier B.V. on behalf of International Metabolic Engineering Society. http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Chen, Minliang
Chen, Lin
Zeng, An-Ping
CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli
title CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli
title_full CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli
title_fullStr CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli
title_full_unstemmed CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli
title_short CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in E. coli
title_sort crispr/cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening of enzyme variants for a more efficient chorismate pathway in e. coli
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520568/
https://www.ncbi.nlm.nih.gov/pubmed/31193188
http://dx.doi.org/10.1016/j.mec.2019.e00094
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