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The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production

Amino acid transport systems perform important physiological functions; their role should certainly be considered in microbial production of amino acids. Typically, in the context of metabolic engineering, efforts are focused on the search for and application of specific amino acid efflux pumps. How...

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Autores principales: Hook, Christine, Eremina, Natalya, Zaytsev, Petr, Varlamova, Daria, Stoynova, Nataliya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8948829/
https://www.ncbi.nlm.nih.gov/pubmed/35336222
http://dx.doi.org/10.3390/microorganisms10030647
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author Hook, Christine
Eremina, Natalya
Zaytsev, Petr
Varlamova, Daria
Stoynova, Nataliya
author_facet Hook, Christine
Eremina, Natalya
Zaytsev, Petr
Varlamova, Daria
Stoynova, Nataliya
author_sort Hook, Christine
collection PubMed
description Amino acid transport systems perform important physiological functions; their role should certainly be considered in microbial production of amino acids. Typically, in the context of metabolic engineering, efforts are focused on the search for and application of specific amino acid efflux pumps. However, in addition, importers can also be used to improve the industrial process as a whole. In this study, the protein CycA, which is known for uptake of nonpolar amino acids, was characterized from the viewpoint of regulating its expression and range of substrates. We prepared a cycA-overexpressing strain and found that it exhibited high sensitivity to branched-chain amino acids and their structural analogues, with relatively increased consumption of these amino acids, suggesting that they are imported by CycA. The expression of cycA was found to be dependent on the extracellular concentrations of substrate amino acids. The role of some transcription factors in cycA expression, including of Lrp and Crp, was studied using a reporter gene construct. Evidence for the direct binding of Crp to the cycA regulatory region was obtained using a gel-retardation assay. The enhanced import of named amino acids due to cycA overexpression in the l-isoleucine-producing strain resulted in a significant reduction in the generation of undesirable impurities. This work demonstrates the importance of uptake systems with respect to their application in metabolic engineering.
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spelling pubmed-89488292022-03-26 The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production Hook, Christine Eremina, Natalya Zaytsev, Petr Varlamova, Daria Stoynova, Nataliya Microorganisms Article Amino acid transport systems perform important physiological functions; their role should certainly be considered in microbial production of amino acids. Typically, in the context of metabolic engineering, efforts are focused on the search for and application of specific amino acid efflux pumps. However, in addition, importers can also be used to improve the industrial process as a whole. In this study, the protein CycA, which is known for uptake of nonpolar amino acids, was characterized from the viewpoint of regulating its expression and range of substrates. We prepared a cycA-overexpressing strain and found that it exhibited high sensitivity to branched-chain amino acids and their structural analogues, with relatively increased consumption of these amino acids, suggesting that they are imported by CycA. The expression of cycA was found to be dependent on the extracellular concentrations of substrate amino acids. The role of some transcription factors in cycA expression, including of Lrp and Crp, was studied using a reporter gene construct. Evidence for the direct binding of Crp to the cycA regulatory region was obtained using a gel-retardation assay. The enhanced import of named amino acids due to cycA overexpression in the l-isoleucine-producing strain resulted in a significant reduction in the generation of undesirable impurities. This work demonstrates the importance of uptake systems with respect to their application in metabolic engineering. MDPI 2022-03-17 /pmc/articles/PMC8948829/ /pubmed/35336222 http://dx.doi.org/10.3390/microorganisms10030647 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hook, Christine
Eremina, Natalya
Zaytsev, Petr
Varlamova, Daria
Stoynova, Nataliya
The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production
title The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production
title_full The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production
title_fullStr The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production
title_full_unstemmed The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production
title_short The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production
title_sort escherichia coli amino acid uptake protein cyca: regulation of its synthesis and practical application in l-isoleucine production
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8948829/
https://www.ncbi.nlm.nih.gov/pubmed/35336222
http://dx.doi.org/10.3390/microorganisms10030647
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