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Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6
Cytochrome P450 monooxygenases (CYPs) are important tools for regio- and stereoselective oxidation of target molecules or engineering of metabolic pathways. Functional heterologous expression of eukaryotic CYPs is often problematic due to their dependency on the specific redox partner and the necess...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer International Publishing
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9135824/ https://www.ncbi.nlm.nih.gov/pubmed/35416604 http://dx.doi.org/10.1007/s11248-022-00305-x |
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author | Sheludko, Y. V. Gerasymenko, I. M. Herrmann, F. J. Warzecha, H. |
author_facet | Sheludko, Y. V. Gerasymenko, I. M. Herrmann, F. J. Warzecha, H. |
author_sort | Sheludko, Y. V. |
collection | PubMed |
description | Cytochrome P450 monooxygenases (CYPs) are important tools for regio- and stereoselective oxidation of target molecules or engineering of metabolic pathways. Functional heterologous expression of eukaryotic CYPs is often problematic due to their dependency on the specific redox partner and the necessity of correct association with the membranes for displaying enzymatic activity. Plant hosts offer advantages of accessibility of reducing partners and a choice of membranes to insert heterologous CYPs. For the evaluation of plant systems for efficient CYP expression, we established transplastomic plants and hairy root cultures of Nicotiana tabacum carrying the gene encoding human CYP2D6 with broad substrate specificity. The levels of CYP2D6 transcript accumulation and enzymatic activity were estimated and compared with the data of CYP2D6 transient expression in N. benthamiana. The relative level of CYP2D6 transcripts in transplastomic plants was 2–3 orders of magnitude higher of that observed after constitutive or transient expression from the nucleus. CYP2D6 expressed in chloroplasts converted exogenous synthetic substrate loratadine without the need for co-expression of the cognate CYP reductase. The loratadine conversion rate in transplastomic plants was comparable to that in N. benthamiana plants transiently expressing a chloroplast targeted CYP2D6 from the nucleus, but was lower than the value reported for transiently expressed CYP2D6 with the native endoplasmic reticulum signal-anchor sequence. Hairy roots showed the lowest substrate conversion rate, but demonstrated the ability to release the product into the culture medium. The obtained results illustrate the potential of plant-based expression systems for exploiting the enzymatic activities of eukaryotic CYPs with broad substrate specificities. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11248-022-00305-x. |
format | Online Article Text |
id | pubmed-9135824 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Springer International Publishing |
record_format | MEDLINE/PubMed |
spelling | pubmed-91358242022-05-28 Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6 Sheludko, Y. V. Gerasymenko, I. M. Herrmann, F. J. Warzecha, H. Transgenic Res Original Paper Cytochrome P450 monooxygenases (CYPs) are important tools for regio- and stereoselective oxidation of target molecules or engineering of metabolic pathways. Functional heterologous expression of eukaryotic CYPs is often problematic due to their dependency on the specific redox partner and the necessity of correct association with the membranes for displaying enzymatic activity. Plant hosts offer advantages of accessibility of reducing partners and a choice of membranes to insert heterologous CYPs. For the evaluation of plant systems for efficient CYP expression, we established transplastomic plants and hairy root cultures of Nicotiana tabacum carrying the gene encoding human CYP2D6 with broad substrate specificity. The levels of CYP2D6 transcript accumulation and enzymatic activity were estimated and compared with the data of CYP2D6 transient expression in N. benthamiana. The relative level of CYP2D6 transcripts in transplastomic plants was 2–3 orders of magnitude higher of that observed after constitutive or transient expression from the nucleus. CYP2D6 expressed in chloroplasts converted exogenous synthetic substrate loratadine without the need for co-expression of the cognate CYP reductase. The loratadine conversion rate in transplastomic plants was comparable to that in N. benthamiana plants transiently expressing a chloroplast targeted CYP2D6 from the nucleus, but was lower than the value reported for transiently expressed CYP2D6 with the native endoplasmic reticulum signal-anchor sequence. Hairy roots showed the lowest substrate conversion rate, but demonstrated the ability to release the product into the culture medium. The obtained results illustrate the potential of plant-based expression systems for exploiting the enzymatic activities of eukaryotic CYPs with broad substrate specificities. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11248-022-00305-x. Springer International Publishing 2022-04-13 2022 /pmc/articles/PMC9135824/ /pubmed/35416604 http://dx.doi.org/10.1007/s11248-022-00305-x Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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 | Original Paper Sheludko, Y. V. Gerasymenko, I. M. Herrmann, F. J. Warzecha, H. Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6 |
title | Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6 |
title_full | Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6 |
title_fullStr | Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6 |
title_full_unstemmed | Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6 |
title_short | Evaluation of biotransformation capacity of transplastomic plants and hairy roots of Nicotiana tabacum expressing human cytochrome P450 2D6 |
title_sort | evaluation of biotransformation capacity of transplastomic plants and hairy roots of nicotiana tabacum expressing human cytochrome p450 2d6 |
topic | Original Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9135824/ https://www.ncbi.nlm.nih.gov/pubmed/35416604 http://dx.doi.org/10.1007/s11248-022-00305-x |
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