Panayiotis D. Glekas, Ioannis Papageorgopoulos, Stamatios G. Damalas, Víctor de Lorenzo, Diomi Mamma, Esteban Martínez-García, Dimitris G. Hatzinikolaou
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To this end, we explored the combinatorial space of the <i>dsz</i> operon of the naturally occurring strain <i>Rhodococcus qingshengii</i> IGTS8—known to achieve dibenzothiophene degradation—by re-engineering the native regulation of the operon, generating permutations of the order of the cognate genes and their ribosomal-binding sites, testing the effects of multicopy versus monocopy doses and introducing the resulting constructs in the tailor-made host. The combination that emerged as best in terms of catalytic efficacy, moderate physiological burden, and durability was one in which the original <i>dsz</i> operon was refactored by [i] reordering its native gene order to <i>dszBCA</i>, [ii] decompressing their naturally occurring translational coupling with optimised ribosomal-binding sites, [iii] engineering its constitutive expression with a heterologous promoter and [iv] inserting the thereby refactored pathway in the Tn<i>7</i> site of the genome-edited strain <i>P. putida</i> EM384, which is optimised for greater stability and hosting harsh redox reactions. The resulting <i>P. putida</i> DS006 exhibited exceptional DBT desulfurization activity as well as efficiency in model biphasic biodesulfurization systems.</p>","PeriodicalId":209,"journal":{"name":"Microbial Biotechnology","volume":"18 9","pages":""},"PeriodicalIF":5.2000,"publicationDate":"2025-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://enviromicro-journals.onlinelibrary.wiley.com/doi/epdf/10.1111/1751-7915.70148","citationCount":"0","resultStr":"{\"title\":\"Boosting Dibenzothiophene Biodesulfurization Through Implantation of a Refactored DBT Pathway in a Tailored Pseudomonas putida Chassis\",\"authors\":\"Panayiotis D. Glekas, Ioannis Papageorgopoulos, Stamatios G. Damalas, Víctor de Lorenzo, Diomi Mamma, Esteban Martínez-García, Dimitris G. 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To this end, we explored the combinatorial space of the <i>dsz</i> operon of the naturally occurring strain <i>Rhodococcus qingshengii</i> IGTS8—known to achieve dibenzothiophene degradation—by re-engineering the native regulation of the operon, generating permutations of the order of the cognate genes and their ribosomal-binding sites, testing the effects of multicopy versus monocopy doses and introducing the resulting constructs in the tailor-made host. The combination that emerged as best in terms of catalytic efficacy, moderate physiological burden, and durability was one in which the original <i>dsz</i> operon was refactored by [i] reordering its native gene order to <i>dszBCA</i>, [ii] decompressing their naturally occurring translational coupling with optimised ribosomal-binding sites, [iii] engineering its constitutive expression with a heterologous promoter and [iv] inserting the thereby refactored pathway in the Tn<i>7</i> site of the genome-edited strain <i>P. putida</i> EM384, which is optimised for greater stability and hosting harsh redox reactions. 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Boosting Dibenzothiophene Biodesulfurization Through Implantation of a Refactored DBT Pathway in a Tailored Pseudomonas putida Chassis
This study reports the efficacy of a rationally designed Pseudomonas putida strain to bring about the specific removal of S atoms from dibenzothiophene (DBT), the model heterocyclic sulfur-containing component of raw petroleum. The emphasis on DBT as a model compound stems from its prevalence in fossil fuels and its resistance to hydrodesulfurization, which positions it as a critical target for improving biodesulfurization technologies. To this end, we explored the combinatorial space of the dsz operon of the naturally occurring strain Rhodococcus qingshengii IGTS8—known to achieve dibenzothiophene degradation—by re-engineering the native regulation of the operon, generating permutations of the order of the cognate genes and their ribosomal-binding sites, testing the effects of multicopy versus monocopy doses and introducing the resulting constructs in the tailor-made host. The combination that emerged as best in terms of catalytic efficacy, moderate physiological burden, and durability was one in which the original dsz operon was refactored by [i] reordering its native gene order to dszBCA, [ii] decompressing their naturally occurring translational coupling with optimised ribosomal-binding sites, [iii] engineering its constitutive expression with a heterologous promoter and [iv] inserting the thereby refactored pathway in the Tn7 site of the genome-edited strain P. putida EM384, which is optimised for greater stability and hosting harsh redox reactions. The resulting P. putida DS006 exhibited exceptional DBT desulfurization activity as well as efficiency in model biphasic biodesulfurization systems.
期刊介绍:
Microbial Biotechnology publishes papers of original research reporting significant advances in any aspect of microbial applications, including, but not limited to biotechnologies related to: Green chemistry; Primary metabolites; Food, beverages and supplements; Secondary metabolites and natural products; Pharmaceuticals; Diagnostics; Agriculture; Bioenergy; Biomining, including oil recovery and processing; Bioremediation; Biopolymers, biomaterials; Bionanotechnology; Biosurfactants and bioemulsifiers; Compatible solutes and bioprotectants; Biosensors, monitoring systems, quantitative microbial risk assessment; Technology development; Protein engineering; Functional genomics; Metabolic engineering; Metabolic design; Systems analysis, modelling; Process engineering; Biologically-based analytical methods; Microbially-based strategies in public health; Microbially-based strategies to influence global processes