Boosting Dibenzothiophene Biodesulfurization Through Implantation of a Refactored <scp>DBT</scp> Pathway in a Tailored <i>Pseudomonas putida</i> 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.