Advancing Desulfurization in the Model Biocatalyst Rhodococcus qingshengii IGTS8 via an In Locus Combinatorial Approach.

Biodesulfurization poses as an ideal replacement to the high cost hydrodesulfurization of the recalcitrant heterocyclic sulfur compounds, such as dibenzothiophene (DBT) and its derivatives. The increasingly stringent limits on fuel sulfur content intensify the need for improved desulfurization biocatalysts, without sacrificing the calorific value of the fuel. Selective sulfur removal in a wide range of biodesulfurization strains, as well as in the model biocatalyst Rhodococcus qingshengii IGTS8, occurs via the 4S metabolic pathway that involves the dszABC operon, which encodes enzymes that catalyze the generation of 2-hydroxybiphenyl and sulfite from DBT. Here, using a homologous recombination process, we generate two recombinant IGTS8 biocatalysts, harboring native or rearranged, nonrepressible desulfurization operons, within the native dsz locus. The alleviation of sulfate-, methionine-, and cysteine-mediated dsz repression is achieved through the exchange of the native promoter Pdsz, with the nonrepressible Pkap1 promoter. The Dsz-mediated desulfurization from DBT was monitored at three growth phases, through HPLC analysis of end product levels. Notably, an 86-fold enhancement of desulfurization activity was documented in the presence of selected repressive sulfur sources for the recombinant biocatalyst harboring a combination of three targeted genetic modifications, namely, a dsz operon rearrangement, a native promoter exchange, and a dszA-dszB overlap removal. In addition, transcript level comparison highlighted the diverse effects of our genetic engineering approaches on dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. IMPORTANCE Rhodococcus is perhaps the most promising biodesulfurization genus and is able to withstand the harsh process conditions of a biphasic biodesulfurization process. In the present work, we constructed an advanced biocatalyst harboring a combination of three genetic modifications, namely, an operon rearrangement, a promoter exchange, and a gene overlap removal. Our homologous recombination approach generated stable biocatalysts that do not require antibiotic addition, while harboring nonrepressible desulfurization operons that present very high biodesulfurization activities and are produced in simple and low-cost media. In addition, transcript level quantification validated the effects of our genetic engineering approaches on recombinant strains' dsz mRNA ratios and revealed a gene-specific differential increase in mRNA levels. Based on these findings, the present work can pave the way for further strain and process optimization studies that could eventually lead to an economically viable biodesulfurization process.

Deciphering the biodesulfurization potential of two novel Rhodococcus isolates from a unique Greek environment.

Sustainable biodesulfurization (BDS) processes require the use of microbial biocatalysts that display high activity against the recalcitrant heterocyclic sulfur compounds and can simultaneously withstand the harsh conditions of contact with petroleum products, inherent to any industrial biphasic BDS system. In this framework, the functional microbial BDS-related diversity in a naturally oil-exposed ecosystem, was examined through a 4,6-dimethyl-dibenzothiophene based enrichment process. Two new Rhodococcus sp. strains were isolated, which during a medium optimization process revealed a significantly enhanced BDS activity profile when compared to the model strain R. qingshengii IGTS8. In biocatalyst stability studies conducted in biphasic mode using partially hydrodesulfurized diesel under various process conditions, the new strains also presented an enhanced stability phenotype. In these studies, it was also demonstrated for all strains, that the BDS activity losses were decoupled from the overall cells' viability, in addition to the fact that the use of whole-broth biocatalyst positively affected BDS performance.