Microbial desulfurization, or biodesulfurization (BDS), of fuels is a promising technology because it can desulfurize compounds that are recalcitrant to the current standard technology in the oil industry. One of the obstacles to the commercialization of BDS is the reduction in biocatalyst activity concomitant with the accumulation of the end product, 2-hydroxybiphenyl (HBP), during the process. BDS experiments were performed by incubating Rhodococcus erythropolis IGTS8 resting-cell suspensions with hexadecane at 0.50 (vol/vol) containing 10 mM dibenzothiophene. The resin Dowex Optipore SD-2 was added to the BDS experiments at resin concentrations of 0, 10, or 50 g resin/liter total volume. The HBP concentration within the cytoplasm was estimated to decrease from 1,100 to 260 M with increasing resin concentration. Despite this finding, productivity did not increase with the resin concentration. This led us to focus on the susceptibility of the desulfurization enzymes toward HBP. Dose-response experiments were performed to identify major inhibitory interactions in the most common BDS pathway, the 4S pathway. HBP was responsible for three of the four major inhibitory interactions identified. The concentrations of HBP that led to a 50% reduction in the enzymes' activities (IC 50 s) for DszA, DszB, and DszC were measured to be 60 ؎ 5 M, 110 ؎ 10 M, and 50 ؎ 5 M, respectively. The fact that the IC 50 s for HBP are all significantly lower than the cytoplasmic HBP concentration suggests that the inhibition of the desulfurization enzymes by HBP is responsible for the observed reduction in biocatalyst activity concomitant with HBP generation. Biodesulfurization (BDS) is a process in which microorganisms, typically, bacteria, are used to reduce the level of sulfur in fuels derived from crude oil, including diesel and gasoline. Over the last 20 years, interest in BDS as an alternative to hydrodesulfurization (HDS), which is the current desulfurization standard in the oil industry, has increased. HDS uses a metal catalyst along with hydrogen gas (H 2 ) at high temperature and pressure to remove sulfur from organic sulfur compounds and generate H 2 S gas (1). Major drawbacks of HDS include steric hindrance of the metal catalysts by certain recalcitrant compounds and large energy consumption due to process operation at high temperature and pressure (1). Recalcitrant compounds include the parent molecule dibenzothiophene (DBT) and some of its alkylated derivatives, such as 4-methyldibenzothiophene (4-DBT) and 4,6-dimethyldibenzothiophene (4,6-DBT). BDS can potentially be used to remove the sulfur that cannot be removed by HDS, though it is likely not a replacement for the current HDS infrastructure. The use of more than one desulfurization technology may be necessary to meet the increasingly stringent sulfur regulations (1).There is a wide range of microorganisms known to have BDS capability (2). Such microorganisms typically desulfurize DBT by one of two pathways: the Kodama pathway or the 4S pathway (1). The Kodama pathway i...

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The anaerobic metabolism of phenylalanine was studied in the denitrifying bacterium Thauera aromatica, a member of the beta-subclass of the Proteobacteria. Phenylalanine was completely oxidized and served as the sole source of cell carbon. Evidence is presented that degradation proceeds via benzoyl-CoA as the central aromatic intermediate; the aromatic ring-reducing enzyme benzoyl-CoA reductase was present in cells grown on phenylalanine. Intermediates in phenylalanine oxidation to benzoyl-CoA were phenylpyruvate, phenylacetaldehyde, phenylacetate, phenylacetyl-CoA, and phenylglyoxylate. The required enzymes were detected in extracts of cells grown with phenylalanine and nitrate. Oxidation of phenylalanine to benzoyl-CoA was catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, phenylacetaldehyde dehydrogenase (NAD+), phenylacetate-CoA ligase (AMP-forming), enzyme(s) oxidizing phenylacetyl-CoA to phenylglyoxylate with nitrate, and phenylglyoxylate:acceptor oxidoreductase. The capacity for phenylalanine oxidation to phenylacetate was induced during growth with phenylalanine. Evidence is provided that alpha-oxidation of phenylacetyl-CoA is catalyzed by a membrane-bound enzyme. This is the first report on the complete anaerobic degradation of an aromatic amino acid and the regulation of this process.

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...

The aerobic metabolism of benzoate in the proteobacterium Azoarcus evansii was reinvestigated. The known pathways leading to catechol or protocatechuate do not operate in this bacterium. The presumed degradation via 3-hydroxybenzoyl-coenzyme A (CoA) and gentisate could not be confirmed. The first committed step is the activation of benzoate to benzoyl-CoA by a specifically induced benzoate-CoA ligase (AMP forming). This enzyme was purified and shown to differ from an isoenzyme catalyzing the same reaction under anaerobic conditions. The second step postulated involves the hydroxylation of benzoyl-CoA to a so far unknown product by a novel benzoyl-CoA oxygenase, presumably a multicomponent enzyme system. An iron-sulfur flavoprotein, which may be a component of this system, was purified and characterized. The homodimeric enzyme had a native molecular mass of 98 kDa as determined by gel filtration and contained 0.