4S Pathway Research Articles

Genomic analysis and biodesulfurization potential of a new carbon-sulfur bond cleaving Tsukamurella sp. 3OW.

Direct combustion of sulfur-enriched liquid fuel oil causes sulfur oxide emission, which is one of the main contributors to air pollution. Biodesulfurization is a promising and eco-friendly method to desulfurize a wide range of thiophenic compounds present in fuel oil. Previously, numerous bacterial strains from genera such as Rhodococcus, Corynebacterium, Gordonia, Nocardia, Mycobacterium, Mycolicibacterium, Paenibacillus, Shewanella, Sphingomonas, Halothiobacillus, and Bacillus have been reported to be capable of desulfurizing model thiophenic compounds or fossil fuels. In the present study, we report a new desulfurizing bacterium, Tsukamurella sp. 3OW, capable of desulfurization of dibenzothiophene through the carbon-sulfur bond cleavage 4S pathway. The bacterium showed a high affinity for the hydrocarbon phase and broad substrate specificity towards various thiophenic compounds. The overall genome-related index analysis revealed that the bacterium is closely related to Tsukamurella paurometabola species. The genomic pool of strain 3OW contains 57 genes related to sulfur metabolism, including the key dszABC genes responsible for dibenzothiophene desulfurization. The DBT-adapted cells of the strain 3OW displayed significant resilience and viability in elevated concentrations of crude oil. The bacterium showed a 19 and 37% reduction in the total sulfur present in crude and diesel oil, respectively. Furthermore, FTIR analysis indicates that the oil's overall chemistry remained unaltered following biodesulfurization. This study implies that Tsukamurella paurometabola species, previously undocumented in the context of biodesulfurization, has good potential for application in the biodesulfurization of petroleum oils.

DszA Catalyzes C-S Bond Cleavage through N5-Hydroperoxyl Formation.

Due to its detrimental impact on human health and the environment, regulations demand ultralow sulfur levels on fossil fuels, in particular in diesel. However, current desulfurization techniques are expensive and cannot efficiently remove heteroaromatic sulfur compounds, which are abundant in crude oil and concentrate in the diesel fraction after distillation. Biodesulfurization via the four enzymes of the metabolic 4S pathway of the bacterium Rhodococcus erythropolis (DszA-D) is a possible solution. However, the 4S pathway needs to operate at least 500 times faster for industrial applicability, a goal currently pursued through enzyme engineering. In this work, we unveil the catalytic mechanism of the flavin monooxygenase DszA. Surprisingly, we found that this enzyme follows a recently proposed atypical mechanism that passes through the formation of an N5OOH intermediate at the re side of the cofactor, aided by a well-defined, predominantly hydrophobic O2 pocket. Besides clarifying the unusual chemical mechanism of the complex DszA enzyme, with obvious implications for understanding the puzzling chemistry of flavin-mediated catalysis, the result is crucial for the rational engineering of DszA, contributing to making biodesulfurization attractive for the oil refining industry.

Biodesulfurization of organosulfur compounds by a trehalose biosurfactant producing Gordonia sp. isolated from crude oil contaminated soil.

Certain factors hinder the commercialization of biodesulfurization process, including low substrate-specificity of the currently reported desulfurizing bacteria and restricted mass transfer of organic-sulfur compounds in biphasic systems. These obstacles must be addressed to clean organic-sulfur rich petro-fuels that pose serious environmental and health challenges. In current study, a dibenzothiophene desulfurizing strain, Gordonia rubripertincta W3S5 (source: oil contaminated soil) was systematically evaluated for its potential to remove sulfur from individual compounds and mixture of organic-sulfur compounds. Metabolic and genetic analyses confirmed that strain W3S5 desulfurized dibenzothiophene to 2-hydroxybiphenyl, suggesting that it follows the sulfur specific 4S pathway. Furthermore, this strain demonstrated the ability to produce trehalose biosurfactants (with an EI24 of 53%) in the presence of dibenzothiophene, as confirmed by TLC and FTIR analyses. Various genome annotation tools, such as ClassicRAST, BlastKOALA, BV-BRC, and NCBI-PGAP, predicted the presence of otsA, otsB, treY, treZ, treP, and Trehalose-monomycolate lipid synthesis genes in the genomic pool of strain W3S5, confirming the existence of the OtsAB, TreYZ, and TreP pathways. Overall, these results underscore the potential of strain W3S5 as a valuable candidate for enhancing desulfurization efficiency and addressing the mass transfer challenges essential for achieving a scaled-up scenario.

Searching for bacteria able to metabolize polycyclic aromatic sulfur compounds in 12-years periodically fed bioreactor.

Biodesulfurization is a promising alternative for removing sulfur molecules from the polycyclic aromatic sulfur compounds (PASC) found in petroleum. PASC consists of recalcitrant molecules that can degrade fuel quality and cause a range of health and environmental problems. Therefore, identifying bacteria capable of degrading PASC is essential for handling these recalcitrant molecules. Microorganisms in environments exposed to petroleum derivatives have evolved specific enzymatic machinery, such as the 4S pathway associated with the dszABC genes, which are directly linked to sulfur removal and utilization as nutrient sources in the biodesulfurization process. In this study, bacteria were isolated from a bioreactor containing landfarm soil that had been periodically fed with petroleum for 12years, using a medium containing dibenzothiophene (DBT), 4.6-dimethylbenzothiophene, 4-methylbenzothiophene, or benzothiophene. This study aimed to identify microorganisms capable of degrading PASC in such environments. Among the 20 colonies isolated from an inoculum containing DBT as the sole sulfur source, only four isolates exhibited amplification of the dszA gene in the dszABC operon. The production of 2-hydroxybiphenyl (HPB) and a decrease in DBT were detected during the growth curve and resting cell assays. The isolates were identified using 16S rRNA sequencing belonging to the genera Stutzerimonas and Pseudomonas. These isolates demonstrated significant potential for biodesulfurization and/or degradation of PASC. All isolates possessed the potential to be utilized in the biotechnological processes of biodesulfurization and degradation of recalcitrant PASC molecules.

Biodesulfurizing Microbes in the Petroleum Refinery Areas of Saudi Arabia

Gordonia sp., Rhodococcus, Paenibaccilus, Mycobacterium and many other desulfurizing strains have shown good potential for dibenzothiophene (DBT), 4, 6-Dimethyldibenzothiophene (4-6-Dimethyl dibenzothiophene) and other organosulfur biodesulfurization. These are microbes which have 4S pathway to remove S from remaining calcitarant organosulfur compounds even after deep desulfurization. Sulfur compounds present in crude oils, diesel and petrol when combust in engines they emerge out in the form of elemental Sulfur, which causes environmental and health problems. Therefore, efforts are going to remove this Sulfur compounds by Hydrodesulfurization (HDS) treatment. Some organosulfur compounds remain there even after HDS, which can only remove by highly evolved microbes residing nearby petroleum-contaminated areas in refineries zone. Nature has such adopted and evolved microbes for the bioremediation of such toxic substances. Here we have isolated and characterized highly evolved and adopted Biodesulfurizing microbes present around oil refineries in Kingdom of Saudi Arabia and prepare the culture collection of such highly evolved and adopted biodesulfurization microorganisms for future application of applied Industrial petroleum refineries, which can reduce the Sulfur load in the petroleum products. The several (10 different types) microbes have been reported in these soils to grow in sulfur compounds. Out of these microbes one microbe desulfurizes by 4S pathway. It was identified to be Rhodococcus erythropolis type named as Rhodococcus erythroplis KAU10. They show good potential for various organosulfur compounds (DBT, 2,4,6-Trimethyl Benzothiophene, Benzothiophene, Dibenzyl sulfide, Benzonaphthothiophene, Dibenzothiophene sulfone, along with crude oil and Petrol and Diesel. Isolated strain Rhodococcus erythroplis KAU10 have good potential for Biodesulfurization.

Deciphering the biodesulfurization pathway employing marine mangrove Bacillus aryabhattai strain NM1-A2 according to whole genome sequencing and transcriptome analyses

In the biogeochemical cycle, sulfur oxidation plays a vital role and is typically referred to as the elemental sulfur or reductive sulfide oxidation process. This study aimed to characterize a subtropical mangrove-isolated bacterial strain using biochemical, whole-genome, and transcriptome sequencing analyses to enhance our understanding of sulfur metabolism and biodegradation from a molecular genetic perspective. Strain NM1-A2 was characterized as Gram-positive and found to have a close molecular phylogenetic relationship with Bacillus aryabhattai. NM1-A2 efficiently converted dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) via a 4S pathway with 95% efficiency, using enzymes encoded by the dsz operon (dszA, dszB, and dszC), which determine monooxygenases (DszA & DszC) and desulfinase (DszB). The whole-genome sequence of NM1-A2 had a length of approximately 5,257,678 bp and included 16 sulfur metabolism-related genes, featuring the ABC transport system, small subunit (ssu) and cysteine (cys) gene families, and adenosine 5′-phosphosulfate (APS) and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis-related genes. Transcriptomic analysis of NM1-A2 using three sulfur groups—magnesium sulfate (MS), sulfur powder (SP), and sodium thiosulfate (ST) resulted in a significant number of differentially expressed genes (1200, 2304, and 2001, respectively). This analysis revealed that intracellular cysteine concentration directly regulated the expression of cys and ssu genes. Sulfate did not directly affect cys gene expression but repressed ssu gene expression. The cys gene expression levels decreased during the conversion of sulfate to sulfide and cysteine. The transcriptomic data was validated by analyzing the expression patterns of NM1-A2 using real-time quantitative PCR validation analysis. The expression levels of cysl, mccB, and nrnA were significantly upregulated, while cysH, metB, and sat were downregulated in the SP, ST, and MS groups, respectively. This research contributes to our understanding of marine mangrove microorganisms' bacterial efficiency through characterization, whole-genome, and transcriptome sequencing-based molecular degradation of organic compounds in the mangrove ecosystem, which may enhance nutrient availability.

Feedback Inhibition of DszC, a Crucial Enzyme for Crude Oil Biodessulfurization

The Rhodococcus erythropolis (strain IGTS8) bacterium has a tremendous industrial interest as it can remove sulfur from crude oil through its four-enzyme (DszA-D) 4S metabolic pathway. DszC is one of the rate-limiting enzymes of the pathway and the one that most suffers from feedback inhibition. We have combined molecular docking and molecular dynamics simulations to identify binding sites through which two products of the 4S pathway, 2-hydroxybiphenyl and 2′-hydroxybiphenyl-2-sulfinate, induce DszC feedback inhibition. We have identified four potential binding sites: two adjacent binding sites close to the 280–295 lid loop proposed to contribute to DszC oligomerization and proper binding of the flavin mononucleotide cofactor, and two other close to the active site of DszC and the substrate binding site. By considering (i) the occupancy of the binding sites and (ii) the similar inhibitor poses, we propose that the mechanism of feedback inhibition of DszC occurs through disturbance of the DszC oligomerization and consequent binding of the flavin mononucleotide due to the weakening of the interactions between the 280–295 lid loop, and both the 131–142 loop and the C-terminal tail. Nevertheless, inhibitor binding close to the active site or the substrate binding sites also compromises critical interactions within the active site of DszC. The disclosed molecular details provide valuable insight for future rational enzyme engineering protocols to develop DszC mutants more resistant against the observed feedback inhibition mechanism.

Bacterial Biological Factories Intended for the Desulfurization of Petroleum Products in Refineries

The removal of sulfur by deep hydrodesulfurization is expensive and environmentally unfriendly. Additionally, sulfur is not separated completely from heterocyclic poly-aromatic compounds. In nature, several microorganisms (Rhodococcus erythropolis IGTS8, Gordonia sp., Bacillus sp., Mycobacterium sp., Paenibacillus sp. A11-2 etc.) have been reported to remove sulfur from petroleum fractions. All these microbes remove sulfur from recalcitrant organosulfur compounds via the 4S pathway, showing potential for some organosulfur compounds only. Activity up to 100 µM/g dry cell weights is needed to meet the current demand for desulfurization. The present review describes the desulfurization capability of various microorganisms acting on several kinds of sulfur sources. Genetic engineering approaches on Gordonia sp. and other species have revealed a variety of good substrate ranges of desulfurization, both for aliphatic and aromatic organosulfur compounds. Whole genome sequence analysis and 4S pathway inhibition by a pTeR group inhibitor have also been discussed. Now, emphasis is being placed on how to commercialize the microbes for industrial-level applications by incorporating biodesulfurization into hydrodesulfurization systems. Thus, this review summarizes the potentialities of microbes for desulfurization of petroleum. The information included in this review could be useful for researchers as well as the economical commercialization of bacteria in petroleum industries.

Sulfamethoxazole degradation by Aeromonas caviae and co-metabolism by the mixed bacteria

Sulfamethoxazole (SMX) is a frequently detected antibiotic in the environment and has attracted much attention. Aeromonas caviae strain GLB-10 was isolated, which could degrade SMX to Aniline and 3-Amino-5-methylisoxazole. Compared to the single bacteria, the mixed bacteria including stain GLB-10, Vibrio diabolicus strain L2-2, Zobellella taiwanensis, Microbacterium testaceum, Methylobacterium, etc, had an ultrahigh degradation efficiency to SMX, with 250 mg/L SMX being degraded in 3 days. In addition to bioproducts of single bacteria, SMX bioproducts by the mixed bacteria also included acetanilide and hydroquinone which were not detected in the single bacteria. The SMX degradation mechanism of the mixed bacteria was more complicated including acetylation, sulfur reduction 4S pathway, and ipso-hydrolysis. The molecular mechanism of the mixed bacteria degrading SMX was also investigated, revealing that the resistance mechanism related to protein outer membrane protein and catalase peroxidase were overexpressed, meanwhile, 6-hydroxynicotinate 3-monooxygenase and ammonia monooxygenase might be the key proteins in SMX degradation. The mixed bacteria could efficiently degrade SMX in different real environments including tap water, river water, artificial lake water, estuary, and, marine water, and have very great research value in bacterial co-metabolism and biodegradation of sulfonamides antibiotics in the environment.

Comparative Genomic Analysis of the Hydrocarbon-Oxidizing Dibenzothiophene-Desulfurizing Gordonia Strains.

A number of actinobacteria of the genus Gordonia are able to use dibenzothiophene (DBT) and its derivatives as the only source of sulfur, which makes them promising agents for the process of oil biodesulfurization. Actinobacteria assimilate sulfur from condensed thiophenes without breaking the carbon-carbon bonds, using the 4S pathway encoded by the dszABC operon-like structure. The genome of the new dibenzothiophene-degrading hydrocarbon-oxidizing bacterial strain Gordonia amicalis 6-1 was completely sequenced and the genes potentially involved in the pathways of DBT desulfurization, oxidation of alkanes and aromatic compounds, as well as in the osmoprotectant metabolism in strain 6-1 and other members of the genus Gordonia, were analyzed. The genome of G. amicalis strain 6-1 consists of a 5,105,798-bp circular chromosome (67.3% GC content) and an 86,621-bp circular plasmid, pCP86 (65.4% GC content). This paper presents a comparative bioinformatic analysis of complete genomes of strain 6-1 and dibenzothiophene-degrading Gordonia strains 1D and 135 that do not have the dsz operon. The assumption is made about the participation in this process of the region containing the sfnB gene. Genomic analysis supported the results of phenomenological studies of Gordonia strains and the possibility of their application in the bioremediation of oil-contaminated environments and in the purification of oil equipment from oil and asphalt-resin-paraffin deposits.

Enhancing biodesulfurization by engineering a synthetic dibenzothiophene mineralization pathway.

A synthetic dibenzothiophene (DBT) mineralization pathway has been engineered in recombinant cells of Pseudomonas azelaica Aramco J strain for its use in biodesulfurization of thiophenic compounds and crude oil. This functional pathway consists of a combination of a recombinant 4S pathway responsible for the conversion of DBT into 2-hydroxybiphenyl (2HBP) and a 2HBP mineralization pathway that is naturally present in the parental P. azelaica Aramco J strain. This novel approach allows overcoming one of the major bottlenecks of the biodesulfurization process, i.e., the feedback inhibitory effect of 2HBP on the 4S pathway enzymes. Resting cells-based biodesulfurization assays using DBT as a sulfur source showed that the 2HBP generated from the 4S pathway is subsequently metabolized by the cell, yielding an increase of 100% in DBT removal with respect to previously optimized Pseudomonas putida biodesulfurizing strains. Moreover, the recombinant P. azelaica Aramco J strain was able to use DBT as a carbon source, representing the best characterized biocatalyst harboring a DBT mineralization pathway and constituting a suitable candidate to develop future bioremediation/bioconversion strategies for oil-contaminated sites.

Improvement in desulfurization of dibenzothiophene and dibenzothiophene sulfone by Paenibacillus strains using immobilization or nanoparticle coating.

Biodesulfurization of fossil fuels is a promising technology for deep desulfurization. Previously, we have shown that Paenibacillus strains 32O-W and 32O-Y can desulfurize dibenzothiophene (DBT) and DBT sulfone (DBTS) effectively. In this work, improvements in DBT and DBTS desulfurization by these strains were investigated through immobilization and nanoparticle coating of cells. Paenibacillus strains 32O-W and 32O-Y immobilized in alginate gel beads or coated with Fe3 O4 magnetite nanoparticles were grown at various concentrations (0.1-2 mmol l-1 ) of DBT or DBTS for 96 h. The production of 2-hydroxybiphenyl (2-HBP) from the 4S pathway biotransformation of DBT or DBTS was measured. The highest amounts of 2-HBP production occurred at concentrations of 0.1 and 0.5 mmol l-1 . Compared to planktonic cultures maximum 2-HBP production increased by 54% for DBT and 90% for DBTS desulfurization with immobilized strains, and 44% for DBT and 66% for DBTS desulfurization by nanoparticle-coated strains. Nanoparticle-coated and immobilized cells may be of use in efforts to increase the efficiency of biodesulfurization. Alginate immobilization or nanoparticle coating of bacterial cells may be useful approaches for the enhancement of biodesulfurization for eventual use on an industrial scale.

Effect of nutrient and physical parameters on dibenzothiophene desulfurization activity of Streptomyces sp. VUR PPR 101 isolated from oil contaminated soils of mechanical workshops

Motor vehicles use petroleum products and release sulfur dioxide gas which causes deleterious effects to environment and humans. The sulfur containing compounds present in petroleum products especially organosulfur compounds serve as major source of sulfur dioxide emission. During refining process, petroleum products are subjected to hydrodesulfurization for the removal of sulfur, which is not an efficient method and most of the organosulfur compounds are not eliminated particularly dibenzothiophene and its derivatives. A process known as biodesulfurization which employs microorganisms was suggested to be an alternative to hydrodesulfurization. The dibenzothiophene was selected as model organosulfur compound to perform biodesulfurization studies due its high recalcitrant nature. The microbes which remove sulfur via 4S pathway from organosulfur compounds (dibenzothiophene) are commercially important. In the present study, the effect of different nutrient sources (carbon, nitrogen and amino acid sources at different concentrations) and physical parameters like temperature and pH on dibenzothiophene desulfurization activity (via 4S pathway) of Streptomyces sp. VUR PPR 101 was studied and optimized. The optimum carbon and nitrogen sources for DBT biodesulfurization activity were found to be 4% glucose and 1% yeast extract, respectively. The best amino acid source reported was 0.3 mg/ml glutamine. At a temperature of 300C and pH 7.0, the organism showed maximum biodedulfurization activity.

Enhancing the biodesulphurization capacity of Rhodococcus sp. FUM94 in a biphasic system through optimization of operational factors

The biodesulfurization activity of bacteria through the 4S pathway in aqueous-oil emulsions is affected by various operational factors. These factors also demonstrate interacting effects that influence the potential for field applications of biodesulfurization technology and can solely be deciphered through multi-variable experiments. In this study, the effects of the influential factors and their interactions on the desulfurizing activity of a newly identified desulfurizing bacterium, Rhodococcus sp, FUM94 were quantitatively investigated. The capacity improvement achieved through optimized values obtained in this study is significant due to its simple implementation to large scale processes. This is the most simple and the most cost-effective way to scale-up a biodesulfurization process.Using response surface methodology (RSM). Optimum values of the factors were identified with the objective of maximizing biodesulfurization activity. Results revealed that the desulfurization activity of the biocatalyst increased from 0.323 ± 0.072 to 46.57 ± 4.556 mmol 2-Hydroxybiphenyl (kg dry cell weight)-1 h-1 at the optimized conditions of 6 h reaction time, 2 g.L-1 biocatalyst concentration, 0.54 mM (100 ppm) dibenzothiophene (DBT) concentration (sulfur source), and 25% oil phase fraction. Desirability analysis proved that the selected conditions are the most desirable combination of factors (desirability value = 0.896) to achieve the highest biodesulfurization activity of the biocatalyst. A comparison between the biodesulfurization capacity achieved in this study and the capacities reported in similar studies published in the past two decades revealed that biodesulfurization under optimized operational conditions outperforms previously proposed techniques.

Experimental and numerical investigation of DBT degradation via Rhodococcus spp. (SL-9) through the use of biological assisted method

Emission of sulfur-based compounds create problem for environment as well as for on human health. To remove sulfur compounds from sulfur-rich fossil fuel, various techniques have been proposed and studied. Based on problem associated with environment as well as for human health, current study focused on DBT removal using novel Rhodococcus spp. (SL-9). Furthermore, study focused on kinetic modeling and simulation of biodesulfurization process for calculating the efficiency of newly isolated bacteria Rhodococcus spp. (SL-9), effect of cell growth, and degradation rate for DBT (dibenzothiophene). Rhodococcus spp. (SL-9) degrade 0.39 mM DBT within 7 days via 4S pathway. The efficiency of Rhodococcus spp. (SL-9) for maximum degradation and conversion of DBT into 2-HBP (2-hydroxybiphenyl) at optimized parameters are 30 °C, 7 pH, and glucose as carbon source. It has been observed through the study that an increase in degradation rate of model DBT compound at 3 mM about 70 hr of incubation period. Rhodococcus sp-SL-9 shows maximum rate of growth about 0.067 h−1 along with biodegradation of DBT rate about 0.89 h−1, which endorses its application in coal desulfurization process.

Combining co-culturing of Paenibacillus strains and Vitreoscilla hemoglobin expression as a strategy to improve biodesulfurization.

Enhancement of the desulfurization activities of Paenibacillus strains 32O-W and 32O-Y were investigated using dibenzothiophene (DBT) and DBT sulfone (DBTS) as sources of sulphur in growth experiments. Strains 32O-W, 32O-Y and their co-culture (32O-W plus 32O-Y), and Vitreoscilla hemoglobin (VHb) expressing recombinant strain 32O-Yvgb and its co-culture with strain 32O-W were grown at varying concentrations (0·1-2mmoll-1 ) of DBT or DBTS for 96h, and desulfurization measured by production of 2-hydroxybiphenyl (2-HBP) and disappearance of DBT or DBTS. Of the four cultures grown with DBT as sulphur source, the best growth occurred for the 32O-Yvgb plus 32O-W co-culture at 0·1 and 0·5mmoll-1 DBT. Although the presence of vgb provided no consistent advantage regarding growth on DBTS, strain 32O-W, as predicted by previous work, was shown to contain a partial 4S desulfurization pathway allowing it to metabolize this 4S pathway intermediate.

Desulphurisation kinetics of thiophenic compound by sulphur oxidizing Klebsiella oxytoca SOB-1.

The major aims of this study are to determine the capability of sulphur oxidizing bacterium (SOB-1) to desulphurize dibenzothiophene (DBT) and crude oil, detection of the reaction kinetics and identify the proposed pathway of DBT desulphurization. The isolate was genetically identified based on 16S rRNA gene sequencing as Klebsiella oxytoca and deposited in the Genebank database under the accession number: MT355440. The HPLC analysis of the remaining DBT concentration revealed that, SOB-1 could desulphurize 90% of DBT (0·25mmoll-1 ) within 96h. The maximum production of sulphate ions from the desulphurization of DBT (0·36mmoll-1 ) and crude oil (0·4mmoll-1 ) could be quantitatively detected after 48h of incubation at 30°C. The high values of correlation coefficient (R2 ) obtained at all studied concentrations; suggested that biodesulfurization kinetics of DBT follows the first-order reaction model. The kinetics studies showed that, DBT may have an inhibitory effect on SOB-1 when the initial concentration exceeded 0·75mmoll-1 . The GC-MS analysis exhibited four main metabolites rather than DBT. The most important ones are 2-hydroxybiphenyl (2-HBP) and methoxybiphenyl n(2-MBP). Klebsiella oxytoca SOB-1 catalyzes the desulphurization of DBT through 4S pathway and forms four main metabolic products. The release of sulphate ion and formation of 2-HBP indicating the elimination of sulphur group without altering the carbon skeleton of DBT. The bacterial strain could also catalyzes desulphurization of crude oil. The desulphurization kinetics follows the first-order reaction model. Klebsiella oxytoca SOB-1 could be used as a promising industrial and environmental biodesulfurizing agent as it is not affecting carbon skeleton of thiophenic compounds and forming less toxic metabolic product (2-MBP).

Phylogenomic Classification and Biosynthetic Potential of the Fossil Fuel-Biodesulfurizing Rhodococcus Strain IGTS8.

Rhodococcus strain IGTS8 is the most extensively studied model bacterium for biodesulfurization of fossil fuels via the non–destructive sulfur–specific 4S pathway. This strain was initially assigned to Rhodococcus rhodochrous and later to Rhodococcus erythropolis thus making its taxonomic status debatable and reflecting the limited resolution of methods available at the time. In this study, phylogenomic analyses of the whole genome sequences of strain IGTS8 and closely related rhodococci showed that R. erythropolis and Rhodococcus qingshengii are very closely related species, that Rhodococcus strain IGTS8 is a R. qingshengii strain and that several strains identified as R. erythropolis should be re-classified as R. qingshengii. The genomes of strains assigned to these species contain potentially novel biosynthetic gene clusters showing that members of these taxa should be given greater importance in the search for new antimicrobials and other industrially important biomolecules. The plasmid-borne dsz operon encoding fossil fuel desulfurization enzymes was present in R. qingshengii IGTS8 and R. erythropolis XP suggesting that it might be transferable between members of these species.

Homology Modeling and In Silico Docking Studies of DszC Enzyme of Streptomyces sp. VUR PPR 101

Sulfur dioxide released during the combustion of fossil fuels causes hazardous effects to living beings. The traditional hydrodesulfurization (HDS) which is employed to remove sulfur components from fossil fuels is not effective in the reduction in aromatic organosulfur compounds like benzothiophene (BT), dibenzothiophene (DBT) and its derivatives. Biodesulfurization process by microorganisms is considered as an effective one over HDS in the removal of sulfur from fuels. DBT, which is a typical recalcitrant organosulfur compound, is considered as a model compound for biodesulfurization studies. Microbes which employ 4S pathway for the removal of sulfur from DBT are commercially so significant. The 4S pathway encompasses four reactions involving three enzymes. The present work deals with the DszC enzyme which catalyzes the first two reaction steps of 4S pathway. The 3D structure of DszC enzyme protein of Streptomyces sp. VUR PPR 101 was modeled and validated. Four mutant DszC enzyme proteins viz., Q119I, H221L, F161A and N129A, were constructed. Wild and mutant DszC proteins were docked against the substrates, DBT and dibenzothiophene sulfoxide (DBTO). Based on the Libdock scores of the docked complexes, the mutant proteins F161A and H221L were found with the highest affinity toward DBT and DBTO, respectively.

Enhancing the substrate tolerance of DszC by a combination of alanine scanning and site-directed saturation mutagenesis.

The biodesulfurization 4S pathway can specifically desulfurize an aromatic S heterocyclic compound (which is difficult to desulfurize by hydrodesulfurization) and maintain the integrity of its combustion value. The four Dsz enzymes in the pathway convert the model compound dibenzothiophene (DBT) into the sulfur-free compound 2-hydroxybiphenyl (HBP). DszC is the first enzyme in the 4S pathway and is subject to feedback inhibition and substrate inhibition. This study is the first attempt to further modify the DszC mutant AKWC to improve its tolerance to DBT. Alanine scanning was performed on the dimeric surface of the DszC mutant AKWC, and the HBP yield of the BAD (AKWCP413A) strain was increased compared to the BAD (AKWC) strain. Site-directed saturation mutagenesis was performed on the 413th amino acid of AKWC, and the substrate inhibition parameter KI value of the mutant AKWCPI was 5.6 times higher than that of AKWC. When the DBT concentration was 0.25mM, the HBP production of the recombinant strain overexpressing AKWCPI was increased by approximately 1.4-fold compared to the BL21(DE3)/BADC*+C* strain. The protein engineering of DszC further improved the substrate tolerance after overcoming the feedback inhibition, which provided a reference for the analysis of the inhibition mechanism of DszC substrate. Overexpression of DszC-beneficial mutants also greatly improved the efficiency of desulfurization.