Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds

Abstract

The 4S pathway is the most studied bioprocess for the removal of the recalcitrant sulfur of aromatic heterocycles present in fuels. It consists of three sequential functional units, encoded by the dszABCD genes, through which the model compound dibenzothiophene (DBT) is transformed into the sulfur-free 2-hydroxybiphenyl (2HBP) molecule. In this work, a set of synthetic dsz cassettes were implanted in Pseudomonas putida KT2440, a model bacterial "chassis" for metabolic engineering studies. The complete dszB1A1C1-D1 cassette behaved as an attractive alternative -to the previously constructed recombinant dsz cassettes -for the conversion of DBT into 2HBP. Refactoring the 4S pathway by the use of synthetic dsz modules encoding individual 4S pathway reactions revealed unanticipated traits, e.g., the 4S intermediate 2HBP-sulfinate (HBPS) behaves as an inhibitor of the Dsz monooxygenases, and oncesecreted from the cells it cannot be further taken up. That issue should be addressed for the rational design of more efficient biocatalysts for DBT bioconversions. In this sense, the construction of synthetic bacterial consortia to compartmentalize the 4S pathway into different cell factories for individual optimization was shown to enhance the conversion of DBT into 2HBP, overcome the inhibition of the Dsz enzymes by the 4S intermediates, and enable efficient production of unattainable high added value intermediates, e.g., HBPS, that are difficult to obtain using the current monocultures. IntroductionCrude oils contain undesirable contaminant molecules, such as thiophenic aromatics compounds, which have a negative impact on oil processing and pose serious environmental threats (Soleimani et al., 2007). A wide spectrum of desulfurization technologies have been developed to remove sulfur mainly from finished refinery products (Stanislaus et al., 2010). Hydrodesulfurization (HDS) treatment has proved to be the common technology of choice to reduce the level of sulfur in crude oil products.Significant environmental, technical and economic limitations have been reported in applying the HDS process (Babich and Moulijn, 2003).During the past 30 years, research to develop alternative desulfurization technologies resulted in a biotechnological strategy to eliminate sulfur from thiophenic compounds (biodesulfurization (BDS)) via serial reactions known as the 4S pathway (Gray et al., 2003;Gupta et al., 2005;Kilbane, 2006;Monticello, 2000;Nuhu, 2013; Xu et al., 2009). This pathway was firstly reported in the gram-positive bacteriumRhodococcus erythropolis IGTS8 (Gallagher et al., 1993), but the 4S pathway has been also found in other bacteria (Duarte et al., 2001;Kilbane, 2006;Mohebali and Ball, 2008).The 4S pathway provides a nondestructive oxidative process used by the cells to obtain the sulfur required for growth, which involves the transformation of dibenzothiophene (DBT), the model compound for sulfur heterocycles present in oil and refractory to HDS, into 2-hydroxybiphenyl (2HBP) and sulfite ( Fig. 1A) (Gallagher et al...

Desulfurization of Dibenzothiophene by Pseudomonas fluorescens (UCP 1514) Leading to the Production of Biphenyl

“…The inhibitory effects of 2-HBP, the end product of the desulfurization of dibenzothiophene (DBT) by the 4S pathway, were noted early in the study of biodesulfurization [30][31][32]. However, product inhibition has not been fully appreciated as the main impediment to developing improved biocatalysts, and effective means of overcoming the inhibition caused by 2-HBP are just beginning to be developed [33].…”

Cooperation in microbial communities and their biotechnological applications

“…In a recent work, DBT desulphurization was carried out using either an engineered P. putida strain expressing all the dszABCD genes required in the process, or a mixed culture of the same strain expressing only some of the genes. In this experiment, desulfuration of DBT was higher when combining multiple cells ‘specialising’ in one step of the biochemical pathway compared to the case of having all reactions taking place in the same organism (Martínez et al ., ).…”

Molybdenum Dioxide in Carbon Nanoreactors as a Catalytic Nanosponge for the Efficient Desulfurization of Liquid Fuels

“…[6,21,37,39] Consistent with previous studies, we subsequently employed an extraction procedure in which the model fuel was vigorously stirred with acetonitrile (in a 1 to 5 ratio of extractant to fuel) for 30 min. [40] Interestingly, it was noted that 27-30% of DBT can be removed from the fuel simply through extraction alone, reflecting the moderate solubility of DBT in n-hexane. [40] Interestingly, it was noted that 27-30% of DBT can be removed from the fuel simply through extraction alone, reflecting the moderate solubility of DBT in n-hexane.…”