Gordona sp. CYKS1으로부터 탈황 유전자의 클로닝과 탈황을 위한 재조합 대장균의 개발

박시재, 이상엽, 장용근

대전광역시 유성구 구성동 373-1 한국과학기술원 화학공학과 및 생물공정연구센터

Cloning of desulfurization genes from Gordona sp. CYKS1 and development of recombinant Escherichia coli for desulfurization

Si Jae Park, Sang Yup Lee, and Yong Keun Chang

Department of Chemical Engineering and BioProcess Engineering Research Center, KAIST 373-1 Kusong-dong, Yusong-gu, Taejon 305-701

Introduction

As the consumption of fossil fuels increases, pollution of our environment rapidly increases. Sulfur compounds in fossil fuels play a major role in the formation of acid rain when fossil fuels are combusted. Acid rain has lasting deleterious effects on aquatic and forest ecosystems, as well as on agricultural areas located downwind of combustion. (Monticello, D. J. and W. R. Finnerty, 1985). Environmental regulations in every country become more stringent to reduce the emission of sulfur oxides. Because the availability of low sulfur crude oil has decreased constantly, new approach to remove sulfur from the fossil fuels has been investigated to meet this stringent environmental regulations. To meet new stringent environmental regulation, complex heterocyclic organic sulfur compounds including DBT should be desulfurized. BDS has potency to remove sulfur from such a complex organic sulfur compound.

Among the thiophenes, dibenzothiophene (DBT) has been the model compound for biodesulfurization. Two pathways are known to be used for desulfurization of DBT among the aerobes. One is Kodama pathway and the other is 4-S pathway. However, microorganisms that use Kodama pathway gained little practical interest due to the DBT metabolites existing in water phase and emission of CO2 causing the loss of fuel value. Microorganisms removing sulfur specifically from DBT via sulfur specific pathway (4-S pathway) were also screened. 4-S represents metabolic intermediates of this pathway; sulfoxide, sulfone, sulfonate, and sulfate. The dead end product in this pathway is 2-hydroxybiphenyl (2-HBP) and sulfate is metabolized for the cell growth. Rhodococcus sp. IGTS8 (Kilbane and Jackowski, 1992; Denome et al, 1993,1994), Rhodococcus erythropolis D-1 (Izumi et al., 1994), and Corynebacterium sp. SY-1 (Omori et al., 1992) were reported to use this 4-S pathway. Recently, our group screened microorganism able to remove sulfur specifically from DBT (Rhee et al, 1998) and found out that this bacteria use the 4-S pathway.

In this work, the cloning of desulfurization genes from newly isolated strain, Gordona sp. CYKS1 and the development of the recombinant Escherichia coli strains for desulfurization were carried out.

Experimantal

Bacterial strains and plasmids

Nine E. coli strains were tested for desulfurization activity and Plasmid  pBluescript SK(-) was used to clone the desulfurization genes. pTrc99A was used to express the desulfurizing genes under the inducible trc promoter in E. coli.

Cloning of desulfurization genes from Gordona sp. CYKS1

Desulfurization genes were cloned by polymerase chain reaction (PCR). The available dsz DNA sequence from Rhodococcus sp. IGTS8 (Denome et al (1994), Larose et al (1997)) and PCR amplified fragments of Gordona sp CYKS1 were used to design primers. Primers for dszAB were 5-CCCAAGCTTCATGACTCAACAACGACAACTGCATCTGGCC and 5-CCCAAGCTTTCCTATCGGTGACGGTTGAGGCTG. Primers for dszC  were 5-CCCAAGCTTGCATGACTCTGTCCGTTGAAAAGC and 5- TGCTCTAGATCATCATCAGGAGGTGAAGCCGGG. The original 6 base pairs of Gordona sp CYKS1 between dszB and dszC was substituted with HindIII recognition site for easy construction of expression vector. Amplification was 94 oC for 50 s, 52  oC for 50 s, and 72 oC for 2 min for 30 cycles. As a result, the new isolate yielded 2.5 kb fragment for dszAB and 1.3 kb for dszC. PCR amplified fragments were cloned into pBluescript SK(-). and constructed vector was named as pBlueS1ExABC.

Culture media

The complex medium used contains per liter: tryptone, 10 g; yeast extract, 5 g; NaCl, 5 g. BSM medium supplemented with 10 g/L glucose and 2 g/L glycerol, respectively.

BSM medium (pH 6.9) contains per liter: NaH2PO4H2O, 4 g; K2HPO43H2O, 4 g; NH4Cl, 2 g; MgCl26H2O, 0.2 g; CaCl22H2O, 0.001 g; FeCl36H2O, 0.001 g. Casamino acids (0.01%, w/v) and thiamine (5g/mL) were added to BSM medium if necessary. Ampicillin (Ap, 50 mg/L) were added when required.

Test tube cultures

Test tube cultures were carried out in tube containing 10 mL of complex or defined BSM in a shaking incubator at 30 oC and 200 rpm for desulfurization activity.

Desulfurization assays

Cells were grown in LB at 30 oC overnight and induced with IPTG for the protein expression. Cell pellet was separated by centrifugation. Cells washed in BSM were used to inoculate fresh BSM containing 0.2 mM DBT. The cultures in BSM were grown at 30 oC and culture supernatants were analyzed by high-performance liquid chromatography (HPLC).

HPLC analysis

The culture broth was acidified to pH 2.0 with 5 N HCl and extracted with an equal volume of ethyl acetate. After extraction, 10 L of upper ethyl acetate layer was analyzed by using a reverse-phase HPLC with a UV-VIS detector (Model L-6200, Hitachi, Japan) and Nova-Pak C18 column (3.9 x 150 mm). The UV-VIS detector was set at 280 nm. The mobile phase was pure methanol or 70 % acetonitrile and flow rate was 0.6 mL/min.

Results and discussion

Analysis of cloned desulfurization genes

The nucleotide sequence and deduced amino acids of the desulfurization genes sequence were compared with that of previously cloned desulfurization genes from Rhodococcus sp. IGTS8 (Denome et al, 1994). The operon of dszABC was very similar to that of IGTS8 and the orientation of dszABC is the same as that of IGTS8 because PCR using forward primer designed from dszB of Gordona sp. CYKS1 could amplify dszC with partial gene fragment of dszB. Maxium homology of DNA sequence of dszABC is 89 % and the homologies of deduced amino acids are 86 % in DszA, 86 % in DszB, and 90 % in DszC, respectively. dszA consists of 1423 bp and encodes 474 amino acids of Mr of 51,906. dszB consists of 1095 bp and encodes 365 amino acids of Mr of 39,339. dszC consists of 1248 bp and encodes 416 amino acids of Mr of 44,989. dszA and dszB are overlapped about 60 bp, and in dszC there is one amino acid deletion compared with that of IGTS8. There is 13 bp gap between stop codon of dszB and start codon of dszC.

Development of recombinant E. coli for desulfurization

pTrcS1ExABC containing desulfurization genes was constructed by the insertion of a 3.8 kb BspHI and XbaI fragment of pBlueS1ExABC into NcoI and XbaI site of pTrc99A. pTrcS1ExABC was transformed into nine E. coli strains via electroporation. Each recombinant E. coli strains containing pTrcS1ExABC were cultured in LB medium at 30 oC and examined for desulfurization activity in BSM medium with 0.2 mM DBT. For the induction  0.5 mM of IPTG was used and cells were incubated further four hours and transferred to BSM media. The results of desulfurization were summarized in Figure 1. Desulfurization activity differs from E. coli strains and maximum activity was shown in E. coli FMJ123 (pTrcS1ExABC).

Figure 1. Conversion of DBT to 2-HBP in various E. coli strains containing pTrcS1ExABC

Acknowledgement

This work was supported by a grant from R&D management Center for Energy and Resources, as a part of the clean Energy Program.

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