A novel enzyme, 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB), from a dibenzothiophene-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1: gene overexpression and enzyme characterization

Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl (2-HBP), and the gene operon dszABC was required for DBT desulfurization. The final step in the microbial DBT desulfurization is the conversion of 2'-hydroxybiphenyl-2-sulfinate (HBPSi) to 2-HBP catalyzed by DszB. In this study, DszB of a DBT-desulfurizing bacterium Rhodococcus erythropolis KA2-5-1 was overproduced in Escherichia coli by coexpression with chaperonin genes, groEL/groES, at 25 degrees C. The recombinant DszB was purified to homogeneity and characterized. The optimal temperature and pH for DszB activity were 35 degrees C and about 7.5, respectively. The K(m) and k(cat) values for HBPSi were 8.2 microM and 0.123.s(-1), respectively. DszB has only one cysteine residue, and the mutant enzyme completely lost the activity when the cysteine residue was changed to a serine residue. This result together with experiments using inhibitors showed that the cysteine residue contributes to the enzyme activity. DszB was also inhibited by a reaction product, 2-HBP (K(i)=0.25 mM), and its derivatives, but not by the other reaction product, sulfite. The enzyme showed a narrow substrate specificity: only 2-phenylbenzene sulfinate except HBPSi served as a substrate among the aromatic and aliphatic sulfinates or sulfonates tested. DszB was thought to be a novel enzyme (HBPSi desulfinase) in that it could specifically cleave the carbon-sulfur bond of HBPSi to give 2-HBP and sulfite ion without the aid of any other proteinic components and coenzymes.     

红球菌SDUZAWQ对有机硫和无机硫的耐受性研究及脱硫基因的克隆

以筛选得到的红球菌SDUZAWQ为对象,研究其在不同浓度的有机硫化合物二苯并噻吩(DBT)存在下的脱硫能力,以及在0.2mmolLDBT和不同浓度Na2SO4同时存在下的脱硫情况。当DBT浓度高达6mmolL时,菌株仍能生长,而且检测出产物2-羟基联苯(2-HBP)的存在,说明该菌株具有耐受较高浓度DBT的能力。当DBT和Na2SO4同时存在时,DBT为菌株SDUZAWQ所利用,并且也检测出2-HBP,并非如文献所报道的红球菌在无机硫存在下不代谢DBT,表明该菌株能够耐受一定浓度的无机硫酸盐。对相关脱硫基因的克隆和测序结果显示,完整脱硫基因dszABC、其上游调控序列和dszD的序列与模式菌株RhodococcuserythropolisIGTS8的同源性分别是99%、100%和100%。     

Desulfurization of light gas oil by a novel recombinant strain from Pseudomonas aeruginosa

The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa NCIMB 9571 by using a transposon vector. Resting cells of the recombinant strain, PAR41, desulfurized 63 mg sulfur l^–1 of light gas oil (LGO) containing 360 mg S l^–1. The desulfurization activity for LGO by the resting cells of strain PAR41 grown with n-tetradecane (50% v/v) was much higher (1018-fold) than in glucose-grown cells. P. aeruginosa NCIMB 9571 is able to take up water-insoluble compounds from an oil phase which is enhanced by n-alkane.     

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

Thirty-five bacterial strains capable of converting dibenzothiophene into 2-hydroxybiphenyl were isolated. Among them Rhodococcus erythropolis KA2-5-1 was chosen for further characterization because of its ability to retain high desulfurization activity stably. PCR cloning and DNA sequencing of a KA2-5-1 genomic DNA fragment showed that it was practically identical with dszABC genes from Rhodococcus sp. IGTS8, a representative carbon-sulfur-bond-targeted dibenzothiophene-degrading bacterium. KA2-5-1 desulfurized a variety of alkyl dibenzothiophenes through the specific cleavage of their C-S bonds. In addition, unexpectedly, KA2-5-1 also attacked alkyl benzothiophenes in a C-S-bond-targeted fashion. The purified monooxygenase, encoded by dszC of KA2-5-1, converted benzothiophene and dibenzothiophene into benzothiophene sulfone and dibenzothiophene sulfone, respectively, with the aid of an NADH-dependent oxidoreductase. This result raises the possibility that the same enzymatic step may be involved in desulfurization of alkylated forms of both dibenzothiophene and benzothiophene in KA2-5-1 cells.     

Enhanced desulfurization in a transposon-mutant strain of Rhodococcus erythropolis

The dsz desulfurization gene cluster from Rhodococcus erythropolis strain KA2-5-1 was transferred into R. erythropolis strain MC1109, unable to desulfurize light gas oil (LGO), using a transposon-transposase complex. As a result, two recombinant strains, named MC0203 and MC0122, were isolated. Resting cells of strain MC0203 decreased the sulfur concentration of LGO from 120 mg l^–1 to 70 mg l^–1 in 2 h. The LGO-desulfurization activity of strain MC0203 was about twice that of strain MC0122 and KA2-5-1. The 10-methyl fatty acids of strain MC0203 were about 28%–41% that of strain MC1109. It is likely that strain MC0203 had a mutation involving alkylenation or methylation of 9-unsaturated fatty acids caused by the transposon inserted in the chromosome, which increased the fluidity of cell membranes and enhanced the desulfurization activity.     

Desulfurization of Dibenzothiophene and Diesel Oils by a Newly Isolated Gordona Strain, CYKS1

A dibenzothiophene (DBT)-desulfurizing bacterial strain was isolated and identified as Gordona strain CYKS1. Strain CYKS1 was found to transform DBT to 2-hydroxybiphenyl via the 4S pathway and to be able to also use organic sulfur compounds other than DBT as a sole sulfur source. Its desulfurization activity was susceptible to sulfate repression. Active resting cells for desulfurization could be prepared only in the early growth phase. When two types of diesel oils, middle distillate unit feed (MDUF) and light gas oil (LGO) containing various organic sulfur compounds including DBT, were treated with resting cells of strain CYKS1 for 12 h, the total sulfur content significantly decreased, from 0.15% (wt/wt) to 0.06% (wt/wt) for MDUF and from 0.3% (wt/wt) to 0.25% (wt/wt) for LGO. The newly isolated strain CYKS1 is considered to have good potential for application in the biodesulfurization of fossil fuels.     

Operon structure and functional analysis of the genes encoding thermophilic desulfurizing enzymes of Paenibacillus sp. A11-2

Paenibacillus A11-2 can efficiently cleave two carbon&bond;sulfur bonds in dibenzothiophene (DBT) and alkyl DBTs, which are refractory by conventional petroleum hydrodesulfurization, to remove sulfur atom at high temperatures. An 8.7-kb DNA fragment containing the genes for the DBT desulfurizing enzymes of A11-2 was cloned in Escherichia coli and characterized. Heterologous expression analysis of the deletion mutants identified three open reading frames that were required for the desulfurization of DBT to 2-hydroxybiphenyl (2-HBP). The three genes were designated tdsA, tdsB, and tdsC (for thermophilic desulfurization). Both the nucleotide sequences and the deduced amino acid sequences show significant homology to dszABC genes of Rhodococcus sp. IGTS8, but there are several local differences between them. Subclone analysis revealed that the product of tdsC oxidizes DBT to DBT-5,5'-dioxide via DBT-5-oxide, the product of tdsA converts DBT-5,5'-dioxide to 2-(2-hydroxyphenyl) benzene sulfinate, and the product of tdsB converts 2-(2-hydroxyphenyl)benzene sulfinate to 2-HBP. Cell-free extracts of a recombinant E. coli harboring all the three desulfurization genes converted DBT to 2-HBP at both 37 and 50 degrees C. In vivo and in vitro exhibition of desulfurization activity of the recombinant genes derived from a Paenibacillus indicates that an E. coli oxidoreductase can be functionally coupled with the monooxygenases of a gram-positive thermophile.     

Kinetic analysis of microbial desulfurization of model and light gas oils containing multiple alkyl dibenzothiophenes

The reaction mechanism of biodesulfurization was investigated using whole cells of Rhodococcus erythropolis KA2-5-1, which have the ability to convert dibenzothiophene (DBT) into 2-hydroxybiphenyl. The desulfurization patterns of alkyl DBTs were represented by the Michaeis-Menten equation. The values of rate constants, the limiting maximal velocity (Vmax) and Michaelis constant (Km), for desulfurization of alkyl DBTs were calculated. The relative desulfurization activities of various alkyl DBTs were reduced in proportion to the total carbon numbers of alkyl substituent groups. Alkyl DBTs that had a total of six carbons of alkyl substituent groups were not desulfurized. The type or position of alkyl substituent groups had little effect on desulfurization activity. The desulfurization activity of each alkyl DBT, when mixed together, was reduced. This phenomenon was caused by apparent competitive inhibition of substrates. Using the apparent competitive inhibition model, the desulfurization pattern of a multiple components system containing alkyl DBTs was elucidated. This model was also applicable for biodesulfurization of light gas oil.     

Isolation and characterization of a moderate thermophile,Mycobacterium phlei GTIS10, capable of dibenzothiophene desulfurization

An organism, identified as Mycobacterium phlei GTIS10, was isolated based on its ability to use dibenzothiophene (DBT) as a sole source of sulfur for growth at 30-52 degrees C. Similar to other biodesulfurization-competent organisms, M. phlei GTIS10 converts DBT to 2-hydroxybiphenyl (2-HBP), as detected by HPLC. The specific desulfurization activity of the 50 degrees C M. phlei GTIS10 culture was determined to be 1.1+/-0.07 micromol 2-HBP min(-1) (g dry cell)(-1). M. phlei GTIS10 can also utilize benzothiophene and thiophene as sulfur sources for growth. The dszABC operon of M. phlei GTIS10 was cloned and sequenced and was found to be identical to that of Rhodococcus erythropolis IGTS8. The presence of the R. erythropolis IGTS8 120-kb plasmid pSOX, which encodes the dszABC operon, has been demonstrated in M. phlei GTIS10. Even though identical dsz genes are contained in both cultures, the temperature at which resting cells of R. erythropolisIGTS8 reach the highest rate of DBT metabolism is near 30 degrees C whereas the temperature that shows the highest activity in resting cell cultures of M. phlei GTIS10 is near 50 degrees C, and activity is detectable at temperatures as high as 57 degrees C. In M. phlei GTIS10, the rate-limiting step in vivo appears to be the conversion of DBT to dibenzothiophene sulfone catalyzed by the product of the dszC gene, DBT monooxygenase. The thermostability of individual desulfurization enzymes was determined and 2-hydroxybiphenyl-2-sulfinate sulfinolyase, encoded by dszB, was found to be the most thermolabile. These results demonstrate that the thermostability of individual enzymes determined in vitro is not necessarily a good predictor of the functional temperature range of enzymes in vivo.     

Biodesulfurization of dibenzothiophene by recombinant Gordonia alkanivorans RIPI90A

The dszABC genes from newly reported dibenzothiophene biodesulfurizing bacterium, Gordonia alkanivorans RIPI90A were cloned and sequenced. The overall nucleotide sequence similarity between the dszABC genes of G. alkanivorans RIPI90A and those of Rhodococcus erythropolis IGTS8 and Gordonia nitida were 83.1% and 83.2%, respectively. A gene transfer system for G. alkanivorans RIPI90A was established employing the Escherichia coli-Rhodococcus shuttle vector pRSG43 as suitable cloning vector, resulting in transformation efficiencies up to 1.6 x 10(5)CFUs microg(-1) plasmid DNA. This stable vector was applied to cloning and efficient expression of the dsz genes under the control of lac promoter. The recombinant strain was able to desulfurize dibenzothiophene in the presence of inorganic sulfate and sulfur-containing amino acids. The maximum desulfurization activity by recombinant resting cells (131.8 microM2-hydroxybiphenylg(dry cell weight)(-1)h(-1)) was increased 2.67-fold in comparison to the highest desulfurization activity of native resting cells.     

Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain

Rhodococcus sp. KT462, which can grow on either benzothiophene (BT) or dibenzothiophene (DBT) as the sole source of sulfur, was newly isolated and characterized. GC and GC-MS analyses revealed that strain KT462 has the same BT desulfurization pathway as that reported for Paenibacillus sp. A11-2 and Sinorhizobium sp. KT55. The desulfurized product of DBT produced by this strain, as well as other DBT-desulfurizing bacteria such as R. erythropolis KA2-5-1 and R. erythropolis IGTS8, was 2-hydroxybiphenyl. A resting cells study indicated that this strain was also able to degrade various alkyl derivatives of BT and DBT.     

Isolation and characterization of a transposon mutant of Pseudomonas aeruginosa affecting uptake of dibenzothiophene in n-tetradecane

AIMS: Isolation and characterization of a transposon mutant of Pseudomonas aeruginosa affecting the uptake of dibenzothiophene (DBT) in n-tetradecane (n-TD). METHODS AND RESULTS: The dsz desulphurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred to the chromosome of P. aeruginosa NCIMB9571 using a transposon vector. A recombinant (named PARM1) was obtained which was able to desulphurize DBT in water, but not in n-TD. CONCLUSIONS: PARM1 is a mutant deficient in a DBT transport system operational in n-TD. This transport system is independent of rhamnolipids and of the n-alkane transport system. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas aeruginosa NCIMB9571 seems to have a specific system of transporting hydrophobic compounds such as DBT in oil.     

Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8

The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to remove sulfur from certain aromatic compounds without breaking carbon-carbon bonds. In particular, sulfur is removed from dibenzothiophene (DBT) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurization phenotype was imparted to Escherichia coli. Therefore, IGTS8 was mutagenized, and a new strain (UV1) was selected that had lost the ability to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype were isolated, though free plasmid could not be isolated. Instead, vector DNA had integrated into either the chromosome or a large resident plasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This procedure identified individual cosmid clones that, when electroporated into strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which these clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicated that the desulfurization genes reside on a 4.0-kb DNA fragment. Finally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV1, and R. fascians produced 2-hydroxybiphenyl from DBT when they contained appropriate clones, indicating that the genes for the entire pathway have been isolated.     

Genome sequence of Rhodococcus erythropolis XP, a biodesulfurizing bacterium with industrial potential

Rhodococcus erythropolis strains have shown excellent characteristics in petroleum oil biodesulfurization. Here we present the first announcement of the draft genome sequence of an efficient biodesulfurizing bacterium named R. erythropolis XP (7,229,582 bp). The biodesulfurizing genes dszABC are located on a plasmid, while the flavin reductase gene dszD is located on the chromosome.     

Enhanced biodesulfurization by expression of dibenzothiophene uptake genes in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The transfer of dibenzothiophene (DBT) and its derivatives into cells is a critical step for biodesulfurization. The desulfurization reactions of resting cells and cell lysate were studied, which showed that the desulfurization rate of DBT, especially 4, 6-dimethyldibenzothiophene (4, 6-DMDBT) in Rhodococcus erythropolis LSSE8-1 was seriously affected by the transfer into cells. The inhibited effect of NaN₃ on desulfurization reactions was studied, which confirmed that the transfer of DBT into cells was an active transport in R. erythropolis LSSE8-1. The uptake-genes of DBT and its derivatives (HcuABC) of Pseudomonas delafieldii R-8 were introduced into the specific desulfurization bacterium, R. erythropolis LSSE8-1. Compared with the wild type, the strains bearing HcuABC genes showed a higher desulfurization activity. The desulfurization ratio of DBT showed a 19% increase, and 13% increase of 4, 6-DMDBT.     

Effect of high-cell-density fermentation of Candida utilis on kinetic parameters and the shift to respiro-fermentative metabolism

In this study, biodesulfurization (BDS) was carried out using immobilized Rhodococcus erythropolis KA2-5-1 in n-tetradecane containing dibenzothiophene (DBT) as a model oil (n-tetradecane/immobilized cell biphasic system). The cells were immobilized by entrapping them with calcium alginate, agar, photo-crosslinkable resin prepolymers (ENT-4000 and ENTP-4000), and urethane prepolymers (PU-3 and PU-6); and it was found that ENT-4000-immobilized cells had the highest DBT desulfurization activity in the model oil system without leakage of cells from the support. Furthermore, ENT4000-immobilized cells could catalyze BDS repeatedly in this system for more than 900 h with reactivation; and recovery of both the biocatalyst and the desulfurized model oil was easy. This study would give a solution to the problems in BDS, such as the troublesome process of recovering desulfurized oil and the short life of BDS biocatalysts.     

Cloning of a rhodococcal promoter using a transposon for dibenzothiophene biodesulfurization

The expression of biodesulfurization genes (dsz) in Rhodococcus erythropolis strain KA2-5-1 is repressed by sulfate which is the product of biodesulfurization. The application of a sulfate non-repressible promoter could be effective in enhancing biodesulfurization. A promoter-probe transposon was constructed using the promoterless, red-shifted green fluorescence protein gene (rsgfp). A 340 bp putative promoter element, designated kap1, was isolated from a strain KA2-5-1 recombinant that had shown high fluorescence intensity. The activity of kap1 was not affected by 1 mM sulfate. It gave about a 2-fold greater activity than the 16S ribosomal RNA promoter in R. erythropolis strain KA2-5-1 and is therefore useful for expressing desulfurization genes in rhodococcal strains.     

Comparative analysis of phenotypic and genotypic characteristics of two desulfurizing bacterial strains, Mycobacterium phlei SM120-1 and Mycobacterium phlei GTIS10

AIM: To compare few phenotypic and genotypic characteristics of two desulfurizing bacterial strains, Mycobacterium phlei SM120-1 and Mycobacterium phlei GTIS10. METHODS AND RESULTS: In the present study, dibenzothiophene (DBT) desulfurizing activity, composition of fatty acids of cell membranes, DBT sulfone monoxygenase gene (bdsA) and the selection pressure applied during the growth and enrichment of the bacterial strains M. phlei SM120-1 and M. phlei GTIS10 were compared in our laboratory. The DBT desulfurization activity of M. phlei SM120-1 was found to be 0.17 +/- 0.02 micromol 2-HBP min(-1) (gram dry cell weight)(-1) and that of the bacterial strain M. phlei GTIS10 was 1.09 +/- 0.05 micromol 2-HBP min(-1) (gram dry cell weight)(-1). Fatty acid methyl ester analysis of cell membranes of these two bacterial strains in the presence of light gas oil showed that both the strains had different fatty acid profiles in their cell membranes. Comparison of the full gene sequences of the desulfurization gene bdsA in the two bacterial strains showed significant difference in the bdsA gene sequences. There was a significant difference observed in the selection pressure applied during the growth and enrichment of the two bacterial strains. CONCLUSIONS: The results of the comparative study of the bacterial strains, M. phlei SM120-1 and M. phlei GTIS10 showed that there were considerable differences in the phenotypic and genotypic characteristics of these two strains. SIGNIFICANCE AND IMPACT OF STUDY: The present study would broaden the understanding of biodesulfurization trait at intra-species level.     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Batch and Fed-Batch Cultures

The DbtS(sup+) phenotype (which confers the ability to oxidize selectively the sulfur atom of dibenzothiophene [DBT] or dibenzothiophene sulfone [DBTO(inf2)]) of Rhodococcus erythropolis N1-36 was quantitatively characterized in batch and fed-batch cultures. In flask cultures, production of the desulfurization product, monohydroxybiphenyl (OH-BP), was maximal at pH 6.0, while specific productivity (OH-BP cell(sup-1)) was maximal at pH 5.5. Quantitative measurements in fermentors (in both batch and fed-batch modes) demonstrated that DBTO(inf2) as the sole sulfur source yielded a greater amount of product than did DBT. Specifically, 100 (mu)M DBT maximally yielded (apprx=)40 (mu)M OH-BP, while 100 (mu)M DBTO(inf2) yielded (apprx=)60 (mu)M OH-BP. Neither maintaining the pH at 6.0 nor adding an additional carbon source increased the yield of OH-BP. The presence of SO(inf4)(sup2-) in growth media repressed expression of desulfurization activity, but SO(inf4)(sup2-) added to suspensions of cells grown in DBT or DBTO(inf2) did not inhibit desulfurization activity.     

Desulfurization of light gas oil in immobilized-cell systems of Gordona sp. CYKS1 and Nocardia sp. CYKS2

Desulfurizations of a model oil (hexadecane containing dibenzothiophene (DBT)) and a diesel oil by immobilized DBT-desulfurizing bacterial strains, Gordona sp. CYKS1 and Nocardia sp. CYKS2, were carried out. Celite bead was used as a biosupport for cell immobilization. Seven-eight cycles of repeated-batch desulfurization were conducted for each strain. Each batch reaction was carried out for 24 h. In the case of model oil treatment with strain CYKS1, about 4.0 mM of DBT in hexadecane (0.13 g sulfur l(oil)(-1)) was desulfurized during the first batch, while 0.25 g sulfur l(oil)(-1) during the final eighth batch. The mean desulfurization rate increased from 0.24 for the first batch to 0.48 mg sulfur l(dispersion)(-1) h(-1) for the final batch. The sulfur content in the light gas oil was decreased from 3 to 2.1 g l(oil)(-1) by strain CYKS1 in the first batch. The mean desulfurization rate was 1.81 mg sulfur l(dispersion)(-1) h(-1), which decreased slightly when the batch reaction was repeated. No significant changes in desulfurization rate were observed with strain CYKS2 when the batch reaction was repeated. When the immobilized cells were stored at 4 degrees C in 0.1 M phosphate buffer (pH 7.0) for 10 days, the residual desulfurization activity was about 50 approximately 70% of the initial value.