@article {44698,
	title = {Advancing Desulfurization in the Model Biocatalyst Rhodococcus qingshengii IGTS8 via an In Locus Combinatorial Approach},
	journal = {Appl Environ Microbiol},
	volume = {89},
	number = {2},
	year = {2023},
	note = {Martzoukou, OlgaAmillis, SotirisGlekas, Panayiotis DBreyanni, DimitraAvgeris, MargaritisScorilas, AndreasKekos, DimitrisPachnos, MichalisMavridis, GeorgeMamma, DiomiHatzinikolaou, Dimitris GengResearch Support, Non-U.S. Gov{\textquoteright}t2023/01/24 06:00Appl Environ Microbiol. 2023 Feb 28;89(2):e0197022. doi: 10.1128/aem.01970-22. Epub 2023 Jan 23.},
	month = {Feb 28},
	pages = {e0197022},
	abstract = {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 P(dsz), with the nonrepressible P(kap1) 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{\textquoteright} 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.},
	keywords = {*Rhodococcus/metabolism, *Sulfur Compounds/metabolism, biocatalysis, biodesulfurization, dibenzothiophene, dsz, genetic engineering, metabolic engineering, RNA, Messenger/metabolism, Sulfur/metabolism},
	isbn = {1098-5336 (Electronic)0099-2240 (Print)0099-2240 (Linking)},
	author = {Martzoukou, O and S. Amillis and Glekas, PD and Breyanni, D. and Avgeris, M. and Scorilas, A. and Kekos, D and Pachnos, M. and Mavridis, G. and D. Mamma and D.G. Hatzinikolaou}
}
