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Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids

ABSTRACT: Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae ha...

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Autores principales: Pöschel, Laura, Guevara-Martínez, Mónica, Hörnström, David, van Maris, Antonius J. A., Buchhaupt, Markus
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10560148/
https://www.ncbi.nlm.nih.gov/pubmed/37572123
http://dx.doi.org/10.1007/s00253-023-12691-1
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author Pöschel, Laura
Guevara-Martínez, Mónica
Hörnström, David
van Maris, Antonius J. A.
Buchhaupt, Markus
author_facet Pöschel, Laura
Guevara-Martínez, Mónica
Hörnström, David
van Maris, Antonius J. A.
Buchhaupt, Markus
author_sort Pöschel, Laura
collection PubMed
description ABSTRACT: Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M. extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M. extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches. KEY POINTS: • Substitutions at position F35 of YciAHI changed the productivity of YciA-based release of carboxylic acid products in M. extorquens AM1 and E. coli. • YciAHI F35N and F35L are improved variants for dicarboxylic production of 2-methylsuccinic acid and mesaconic acid with M. extorquens AM1. • In vitro enzyme assays did not reveal superior properties of the optimized protein variants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-023-12691-1.
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spelling pubmed-105601482023-10-09 Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids Pöschel, Laura Guevara-Martínez, Mónica Hörnström, David van Maris, Antonius J. A. Buchhaupt, Markus Appl Microbiol Biotechnol Biotechnologically Relevant Enzymes and Proteins ABSTRACT: Acyl-CoA-thioesterases, which hydrolyze acyl-CoA-esters and thereby release the respective acid, have essential functions in cellular metabolism and have also been used to produce valuable compounds in biotechnological processes. Thioesterase YciA originating from Haemophilus influenzae has been previously used to produce specific dicarboxylic acids from CoA-bound intermediates of the ethylmalonyl CoA pathway (EMCP) in Methylorubrum extorquens. In order to identify variants of the YciA enzyme with the capability to hydrolyze so far inaccessible CoA-esters of the EMCP or with improved productivity, we engineered the substrate-binding region of the enzyme. Screening a small semi-rational mutant library directly in M. extorquens yielded the F35L variant which showed a drastic product level increase for mesaconic acid (6.4-fold) and 2-methylsuccinic acid (4.4-fold) compared to the unaltered YciA enzyme. Unexpectedly, in vitro enzyme assays using respective M. extorquens cell extracts or recombinantly produced thioesterases could not deliver congruent data, as the F35L variant showed strongly reduced activity in these experiments. However, applied in an Escherichia coli production strain, the protein variant again outperformed the wild-type enzyme by allowing threefold increased 3-hydroxybutyric acid product titers. Saturation mutagenesis of the codon for position 35 led to the identification of another highly efficient YciA variant and enabled structure-function interpretations. Our work describes an important module for dicarboxylic acid production with M. extorquens and can guide future thioesterase improvement approaches. KEY POINTS: • Substitutions at position F35 of YciAHI changed the productivity of YciA-based release of carboxylic acid products in M. extorquens AM1 and E. coli. • YciAHI F35N and F35L are improved variants for dicarboxylic production of 2-methylsuccinic acid and mesaconic acid with M. extorquens AM1. • In vitro enzyme assays did not reveal superior properties of the optimized protein variants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-023-12691-1. Springer Berlin Heidelberg 2023-08-12 2023 /pmc/articles/PMC10560148/ /pubmed/37572123 http://dx.doi.org/10.1007/s00253-023-12691-1 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Biotechnologically Relevant Enzymes and Proteins
Pöschel, Laura
Guevara-Martínez, Mónica
Hörnström, David
van Maris, Antonius J. A.
Buchhaupt, Markus
Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
title Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
title_full Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
title_fullStr Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
title_full_unstemmed Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
title_short Engineering of thioesterase YciA from Haemophilus influenzae for production of carboxylic acids
title_sort engineering of thioesterase ycia from haemophilus influenzae for production of carboxylic acids
topic Biotechnologically Relevant Enzymes and Proteins
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10560148/
https://www.ncbi.nlm.nih.gov/pubmed/37572123
http://dx.doi.org/10.1007/s00253-023-12691-1
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