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An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation

Polyethylene terephthalate (PET) is a prevalent synthetic polymer that is known to contaminate marine and terrestrial environments. Currently, only a limited number of PET-active microorganisms and enzymes (PETases) are known. This is in part linked to the lack of highly sensitive function-based scr...

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Autores principales: Dierkes, Robert F., Wypych, Alan, Pérez-García, Pablo, Danso, Dominik, Chow, Jennifer, Streit, Wolfgang R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9888244/
https://www.ncbi.nlm.nih.gov/pubmed/36507653
http://dx.doi.org/10.1128/aem.01603-22
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author Dierkes, Robert F.
Wypych, Alan
Pérez-García, Pablo
Danso, Dominik
Chow, Jennifer
Streit, Wolfgang R.
author_facet Dierkes, Robert F.
Wypych, Alan
Pérez-García, Pablo
Danso, Dominik
Chow, Jennifer
Streit, Wolfgang R.
author_sort Dierkes, Robert F.
collection PubMed
description Polyethylene terephthalate (PET) is a prevalent synthetic polymer that is known to contaminate marine and terrestrial environments. Currently, only a limited number of PET-active microorganisms and enzymes (PETases) are known. This is in part linked to the lack of highly sensitive function-based screening assays for PET-active enzymes. Here, we report on the construction of a fluorescent biosensor based on Comamonas thiooxidans strain S23. C. thiooxidans S23 transports and metabolizes TPA, one of the main breakdown products of PET, using a specific tripartite tricarboxylate transporter (TTT) and various mono- and dioxygenases encoded in its genome in a conserved operon ranging from tphC-tphA1. TphR, an IclR-type transcriptional regulator is found upstream of the tphC-tphA1 cluster where TPA induces transcription of tphC-tphA1 up to 88-fold in exponentially growing cells. In the present study, we show that the C. thiooxidans S23 wild-type strain, carrying the sfGFP gene fused to the tphC promoter, senses TPA at concentrations as low as 10 μM. Moreover, a deletion mutant lacking the catabolic genes involved in TPA degradation thphA2-A1 (ΔtphA2A3BA1) is up to 10,000-fold more sensitive and detects TPA concentrations in the nanomolar range. This is, to our knowledge, the most sensitive reporter strain for TPA and we demonstrate that it can be used for the detection of enzymatic PET breakdown products. IMPORTANCE Plastics and microplastics accumulate in all ecological niches. The construction of more sensitive biosensors allows to monitor and screen potential PET degradation in natural environments and industrial samples. These strains will also be a valuable tool for functional screenings of novel PETase candidates and variants or monitoring of PET recycling processes using biocatalysts. Thereby they help us to enrich the known biodiversity and efficiency of PET degrading organisms and enzymes and understand their contribution to environmental plastic degradation.
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spelling pubmed-98882442023-02-01 An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation Dierkes, Robert F. Wypych, Alan Pérez-García, Pablo Danso, Dominik Chow, Jennifer Streit, Wolfgang R. Appl Environ Microbiol Biotechnology Polyethylene terephthalate (PET) is a prevalent synthetic polymer that is known to contaminate marine and terrestrial environments. Currently, only a limited number of PET-active microorganisms and enzymes (PETases) are known. This is in part linked to the lack of highly sensitive function-based screening assays for PET-active enzymes. Here, we report on the construction of a fluorescent biosensor based on Comamonas thiooxidans strain S23. C. thiooxidans S23 transports and metabolizes TPA, one of the main breakdown products of PET, using a specific tripartite tricarboxylate transporter (TTT) and various mono- and dioxygenases encoded in its genome in a conserved operon ranging from tphC-tphA1. TphR, an IclR-type transcriptional regulator is found upstream of the tphC-tphA1 cluster where TPA induces transcription of tphC-tphA1 up to 88-fold in exponentially growing cells. In the present study, we show that the C. thiooxidans S23 wild-type strain, carrying the sfGFP gene fused to the tphC promoter, senses TPA at concentrations as low as 10 μM. Moreover, a deletion mutant lacking the catabolic genes involved in TPA degradation thphA2-A1 (ΔtphA2A3BA1) is up to 10,000-fold more sensitive and detects TPA concentrations in the nanomolar range. This is, to our knowledge, the most sensitive reporter strain for TPA and we demonstrate that it can be used for the detection of enzymatic PET breakdown products. IMPORTANCE Plastics and microplastics accumulate in all ecological niches. The construction of more sensitive biosensors allows to monitor and screen potential PET degradation in natural environments and industrial samples. These strains will also be a valuable tool for functional screenings of novel PETase candidates and variants or monitoring of PET recycling processes using biocatalysts. Thereby they help us to enrich the known biodiversity and efficiency of PET degrading organisms and enzymes and understand their contribution to environmental plastic degradation. American Society for Microbiology 2022-12-12 /pmc/articles/PMC9888244/ /pubmed/36507653 http://dx.doi.org/10.1128/aem.01603-22 Text en Copyright © 2022 Dierkes et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Biotechnology
Dierkes, Robert F.
Wypych, Alan
Pérez-García, Pablo
Danso, Dominik
Chow, Jennifer
Streit, Wolfgang R.
An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation
title An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation
title_full An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation
title_fullStr An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation
title_full_unstemmed An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation
title_short An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation
title_sort ultra-sensitive comamonas thiooxidans biosensor for the rapid detection of enzymatic polyethylene terephthalate (pet) degradation
topic Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9888244/
https://www.ncbi.nlm.nih.gov/pubmed/36507653
http://dx.doi.org/10.1128/aem.01603-22
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