Analysis of Dibenzothiophene Desulfurization in a Recombinant Pseudomonas putida Strain

Abstract

Biodesulfurization was monitored in a recombinant Pseudomonas putida CECT5279 strain. DszB desulfinase activity reached a sharp maximum at the early exponential phase, but it rapidly decreased at later growth phases. A model two-step resting-cell process combining sequentially P. putida cells from the late and early exponential growth phases was designed to significantly increase biodesulfurization.Biodesulfurization (BDS), based on the application of microorganisms that selectively remove sulfur atoms from organosulfur compounds, appears to be a viable technology to complement the traditional hydrodesulfurization of fuels (9,12,18,21,22). Dibenzothiophene (DBT) has been widely employed as the model compound of polycyclic organic sulfur components in fuels. DBT desulfurization in Rhodococcus erythropolis IGTS8, a model bacterium used in BDS, is catalyzed through the 4S pathway composed of two-component, flavin-dependent monooxygenases, i.e., the DszA and DszC oxygenase components and the DszD flavin reductase, and a desulfinase, DszB. DszC catalyzes DBT transformation into dibenzothiophene sulfoxide (DBTO) and dibenzothiophene-sulfone (DBTO 2 ), whereas DszA further oxygenates DBTO 2 to 2-hydroxybiphenyl-2-sulfinic acid (HBPS). In the last step of this pathway, DszB hydrolyzes HBPS into 2-hydroxybiphenyl (HBP) and sulfite (4, 5, 10-13, 20, 21). The 4S pathway has been shown to exist in a wide variety of bacterial genera, such as Paenibacillus, Pseudomonas, Corynebacterium, and Mycobacterium, among others (9,11,12,21,22).Several bottlenecks that limit the commercialization of BDS have been identified, and different strategies based on designing recombinant Dsz pathways and microorganisms have been accomplished to alleviate such limitations (3,7,9,11,12,14,15,19,22). In an attempt to expand our understanding of the BDS process at a molecular level, the activity of the three 4S route enzymes in the recombinant biocatalyst Pseudomonas putida CECT5279, which harbors the R. erythropolis IGTS8 dszABC genes in plasmid pESOX3 and the hpaC gene encoding the Escherichia coli flavin mononucleotide:NADH HpaC reductase (a DszD equivalent) inserted in its chromosome (6), has been determined.Optimal desulfurization capacity of the P. putida recombinant cells. To determine the optimal BDS capacity of P. putida CECT5279, cells were cultured in a bioreactor (2 liter) using basal salt medium, containing 2 mM MgSO 4 as a sulfur source and 20 g liter Ϫ1 glutamic acid as a carbon source, as previously described (17). Samples collected at different growth times were used for resting-cell desulfurization assays using the substrates for all the 4S route enzymes (DBT, DBTO, DBTO 2 , and HBPS). Cells (0.7 g dry cell weight [DCW]/liter) were suspended in 50 mM HEPES buffer (pH 8.0) and 10 M substrate, and desulfurization was carried out at 30°C for 2 h. Samples (0.25 ml) were collected, and the concentrations of DBT, DBTO, DBTO 2 , HBPS, and HBP were determined by high-performance liquid chromatography as previously described (16, 17). The...

Biodesulfurization of dibenzothiophene by resting cells of Pseudomonas putidaCECT5279: influence of the oxygen transfer rate in the scale‐up from shaken flask to stirred tank reactor

“…Resting cells BDS assays were conducted using cells previously collected as commented above. In a previous work it was found that the enzymatic activity depends on the growth time: both monoxygenases (dszC and dszA) have a maximum of activity using cells of 23 h of growth time and the desulfinase (dszB) presents its maximum of activity using cells growth during 5 h. Therefore it was possible to optimise the biocatalyst formulation using mixtures of cells of these two different growth ages; the optimal formulation consists of a mixture of 0.7 g DCW L −1 of 5 h aged cells and 1.4 g DCW L −1 of cells with 23 h of growth time (total biomass concentration: 2.1 g L −1 ) and it is the biocatalyst used in this work.…”

Analysis of the core genome and pangenome of Pseudomonas putida

“…Also, the strain was used as a model system for horizontal and vertical evolution of catabolic pathways in the degradation of alkylaromatic compounds (Ramos et al ., 1986; 1987; Timmis, ; Li et al ., ). The genetic versatility of this bacterium has been proven to be large, making it suitable for metabolic engineering to create superior strains for a variety of biotechnological applications, including the design of new catabolic pathways for pollutants, the biocatalysis of pharmaceutical intermediates and quality improvement of fossil fuels (Calzada et al ., ; Prakash et al ., ; Tao et al ., ; Fernández et al ., ). For P. putida there are 12 complete and 18 in‐progress genomes publicly available on NCBI (http://www.ncbi.nlm.nih.gov/genome/genomes/174, as of June 2014).…”

Analysis of the dibenzothiophene metabolic pathway in a newly isolatedRhodococcusspp.

“…Dibenzothiophene has been widely used as a model compound of the polycyclic organic sulphur components of fossil fuels (Calzada et al , 2009). Organic sulphur is characterized by covalent C–S bonds and can be regarded as an element that is integrated into the macromolecular matrix of coal and in organic sulphur compounds present in oil (Klein et al , 1994).…”