CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain
Abstract Background Oil spills are a major concern due to the economic impact and severe effects on the ecosystem. To mitigate oil spills, hydrocarbon dispersion through emulsification is a promising approach, as it makes oil more susceptible to degradation by microorganisms. Environmental strains o...
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| Format: | Article |
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BMC
2025-07-01
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| Series: | Microbial Cell Factories |
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| Online Access: | https://doi.org/10.1186/s12934-025-02769-y |
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| author | Luis M. Salazar-García Luis C. Damas-Ramos Luisa M. Trejo-Alarcón Daniela Rago Linda Ahonen Pablo Cruz-Morales Patricia Ponce-Noyola Cuauhtémoc Licona-Cassani |
| author_facet | Luis M. Salazar-García Luis C. Damas-Ramos Luisa M. Trejo-Alarcón Daniela Rago Linda Ahonen Pablo Cruz-Morales Patricia Ponce-Noyola Cuauhtémoc Licona-Cassani |
| author_sort | Luis M. Salazar-García |
| collection | DOAJ |
| description | Abstract Background Oil spills are a major concern due to the economic impact and severe effects on the ecosystem. To mitigate oil spills, hydrocarbon dispersion through emulsification is a promising approach, as it makes oil more susceptible to degradation by microorganisms. Environmental strains of Pseudomonas aeruginosa have demonstrated significant potential for producing rhamnolipids (RMLs) and pyocyanin (PYO), secondary metabolites associated to hydrocarbon emulsification. In this study, we isolated and characterized an environmental strain from an oil-contaminated site in the Gulf of Mexico. Upon genome sequencing and taxonomic classification, we developed genetic engineering tools and assessed their capacity to produce PYO and RMLs, molecules relevant for hydrocarbon emulsification. Results Using the CRISPR/Cas9-APOBEC1-UGI system, we generated a targeted cytosine to thymine transition in the rpoS gene to generate a premature STOP codon. The resulting mutant exhibited increased production of PYO and RMLs, along with enhanced gasoline emulsification in cell-free supernatants, demonstrating successful modulation of a key regulatory gene. While the strain IGLPR01 retains certain virulence-associated features, this study contributes to the exploration of environmental isolates as future candidate chassis for biosurfactant production, emphasizing the need for further safety evaluation and rational attenuation strategies. Conclusion This study provides a successful example of implementing CRISPR-based editing in an environmental P. aeruginosa strain. Despite the technical challenges, a genetic editing system was established and validated through a proof of concept to increase production of relevant metabolites. Our work demonstrates the applicability of genetic engineering tools in non-model environmental isolates, facilitating further developments. Importantly, the presence of virulence-associated features highlights the need for in-depth evaluation of pathogenicity and containment strategies before considering any future biotechnological applications. |
| format | Article |
| id | doaj-art-2f82d890f90e4fa784e81ad70bfeee2f |
| institution | Kabale University |
| issn | 1475-2859 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj-art-2f82d890f90e4fa784e81ad70bfeee2f2025-08-20T04:01:43ZengBMCMicrobial Cell Factories1475-28592025-07-0124111710.1186/s12934-025-02769-yCRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strainLuis M. Salazar-García0Luis C. Damas-Ramos1Luisa M. Trejo-Alarcón2Daniela Rago3Linda Ahonen4Pablo Cruz-Morales5Patricia Ponce-Noyola6Cuauhtémoc Licona-Cassani7Centro de Biotecnología FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de MonterreyCentro de Biotecnología FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de MonterreyCentro de Biotecnología FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de MonterreyThe Novo Nordisk Foundation Center for Biosustainability, Technical University of DenmarkThe Novo Nordisk Foundation Center for Biosustainability, Technical University of DenmarkThe Novo Nordisk Foundation Center for Biosustainability, Technical University of DenmarkDepartamento de Biología, División de Ciencias Naturales y Exactas, Universidad de GuanajuatoCentro de Biotecnología FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de MonterreyAbstract Background Oil spills are a major concern due to the economic impact and severe effects on the ecosystem. To mitigate oil spills, hydrocarbon dispersion through emulsification is a promising approach, as it makes oil more susceptible to degradation by microorganisms. Environmental strains of Pseudomonas aeruginosa have demonstrated significant potential for producing rhamnolipids (RMLs) and pyocyanin (PYO), secondary metabolites associated to hydrocarbon emulsification. In this study, we isolated and characterized an environmental strain from an oil-contaminated site in the Gulf of Mexico. Upon genome sequencing and taxonomic classification, we developed genetic engineering tools and assessed their capacity to produce PYO and RMLs, molecules relevant for hydrocarbon emulsification. Results Using the CRISPR/Cas9-APOBEC1-UGI system, we generated a targeted cytosine to thymine transition in the rpoS gene to generate a premature STOP codon. The resulting mutant exhibited increased production of PYO and RMLs, along with enhanced gasoline emulsification in cell-free supernatants, demonstrating successful modulation of a key regulatory gene. While the strain IGLPR01 retains certain virulence-associated features, this study contributes to the exploration of environmental isolates as future candidate chassis for biosurfactant production, emphasizing the need for further safety evaluation and rational attenuation strategies. Conclusion This study provides a successful example of implementing CRISPR-based editing in an environmental P. aeruginosa strain. Despite the technical challenges, a genetic editing system was established and validated through a proof of concept to increase production of relevant metabolites. Our work demonstrates the applicability of genetic engineering tools in non-model environmental isolates, facilitating further developments. Importantly, the presence of virulence-associated features highlights the need for in-depth evaluation of pathogenicity and containment strategies before considering any future biotechnological applications.https://doi.org/10.1186/s12934-025-02769-yPyocyaninRhamnolipidsPseudomonas aeruginosaCRISPR/CasEnvironmental strainBioremediation |
| spellingShingle | Luis M. Salazar-García Luis C. Damas-Ramos Luisa M. Trejo-Alarcón Daniela Rago Linda Ahonen Pablo Cruz-Morales Patricia Ponce-Noyola Cuauhtémoc Licona-Cassani CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain Microbial Cell Factories Pyocyanin Rhamnolipids Pseudomonas aeruginosa CRISPR/Cas Environmental strain Bioremediation |
| title | CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain |
| title_full | CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain |
| title_fullStr | CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain |
| title_full_unstemmed | CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain |
| title_short | CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain |
| title_sort | crispr driven enhanced hydrocarbon emulsification in an environmental pseudomonas aeruginosa strain |
| topic | Pyocyanin Rhamnolipids Pseudomonas aeruginosa CRISPR/Cas Environmental strain Bioremediation |
| url | https://doi.org/10.1186/s12934-025-02769-y |
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