Desulfurization of alkylated forms of both dibenzothiophene and benzothiophene by a single bacterial strain

Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources. An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes, dszA and dszC. A transversion (G to T) in dszC codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype. By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene. No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity.

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confidence: 99%

Chemostat Approach for the Directed Evolution of Biodesulfurization Gain-of-Function Mutants

Arensdorf¹,

Loomis²,

DiGrazia³

et al. 2002

“…The condensed thiophenes are the most common forms in which sulfur is present (28). Alkyl DBTs and alkyl BTs are highly recalcitrant to chemical catalysts, especially when alkylated at the 4 and 6 positions (9,14,20). Many research efforts have been put into the investigation of biological desulfurization systems using DBTs or alkylated DBTs as model compounds, and the metabolic pathway of desulfurization has been proposed as the so-called "4S" or extended "4SM" pathway (5,7,12,16,18).…”

mentioning

confidence: 99%

Deep Desulfurization of Diesel Oil and Crude Oils by a Newly Isolated Rhodococcus erythropolis Strain

Shi

et al. 2006

131

The soil-isolated strain XP was identified as Rhodococcus erythropolis. R. erythropolis XP could efficiently desulfurize benzonaphthothiophene, a complicated model sulfur compound that exists in crude oil. The desulfurization product of benzonaphthothiophene was identified as ␣-hydroxy-␤-phenyl-naphthalene. Resting cells could desulfurize diesel oil (total organic sulfur, 259 ppm) after hydrodesulfurization. The sulfur content of diesel oil was reduced by 94.5% by using the resting cell biocatalyst for 24 h at 30°C. Biodesulfurization of crude oils was also investigated. After 72 h of treatment at 30°C, 62.3% of the total sulfur content in Fushun crude oil (initial total sulfur content, 3,210 ppm) and 47.2% of that in Sudanese crude oil (initial total sulfur, 1,237 ppm) were removed. Gas chromatography with pulsed-flame photometric detector analysis was used to evaluate the effect of R. erythropolis XP treatment on the sulfur content in Fushun crude oil, and it was shown that most organic sulfur compounds were eliminated after biodesulfurization.

“…strain A11-2 could also desulfurize DBT in addition to BTH, since this bacterium was isolated for its ability to grow in a medium with DBT as the sole source of sulfur. However, it is interesting that the DBTdesulfurizing bacterium R. erythropolis KA2-5-1 could desulfurize alkylated BTH but not BTH (10). On the other hand, for NTH biodesulfurization through the sulfur-specific degradation pathway, there is no report except for our recent publication (3).…”

Section: Discussionmentioning

confidence: 81%

Biodesulfurization of Naphthothiophene and Benzothiophene through Selective Cleavage of Carbon-Sulfur Bonds by Rhodococcus sp. Strain WU-K2R

Kirimura

Furuya

Sato

et al. 2002

Self Cite

Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2-hydroxynaphthylethene, and naphtho Sulfur oxides generated by combustion of fossil fuel lead to acid rain and air pollution. Therefore, today petroleum is treated by hydrodesulfurization (HDS) using metallic catalysts in the presence of hydrogen gas under extremely high temperature and pressure. Although HDS can remove various types of sulfur compounds, some types of heterocyclic sulfur compounds cannot be removed. Dibenzothiophene (DBT) is one such recalcitrant organosulfur compound and is widely recognized as a model target compound for deeper desulfurization, since DBT derivatives can be detected in diesel oil following HDS treatment. Therefore, the application of a biodesulfurization process using a DBT-desulfurizing microorganism following HDS, mainly for diesel oil, has attracted attention for achievement of deeper desulfurization (16,20). Some mesophilic and thermophilic DBT-desulfurizing microorganisms have been isolated, for example, Rhodococcus sp. strain IGTS8 (1, 4, 21, 22), Rhodococcus erythropolis D-1 (8), R. erythropolis H-2 (17, 18), R. erythropolis KA2-5-1 (6), and Paenibacillus sp. strain A11-2, which desulfurizes DBT at 60°C (7, 11). We have also isolated Bacillus subtilis WU-S2B (9) and Mycobacterium phlei WU-F1 (2), which could desulfurize DBT and its derivatives over a wide temperature range of 20 to 50°C and at the highest level at 45 to 50°C. These bacteria desulfurize DBT through the sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur (C-S) bonds without reducing the energy content (4,21,22). Naphtho[2,1-b]thiophene (NTH) (see Fig. 3B), which includes a benzothiophene (BTH) (see Fig. 3A) structure, is an asymmetric structural isomer of DBT. Recently it has become apparent that in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following HDS treatment, although NTH derivatives are minor components in comparison with DBT derivatives (unpublished data). Therefore, NTH may also be a model target compound for deeper desulfurization. Kropp et al. (13) have reported that Pseudomonas sp. strain W1 could degrade NTH. However, this bacterium utilized NTH as the carbon source with reducing the energy content, and the sulfur atom was not remo...