Part:BBa_K1972007
dszBCAD with T7 promoter
We constructed dszBCAD genes with T7 promoter and choose E.coli BL21 as our host cell. When IPTG is added to our system, DszABCD enzymes will be successful expressed and these four enzymes convert dibenzothiophene (DBT) undergoes through three successive oxidation steps and one a hydrolytic step to 2-hydroxybiphenyl (2HBP) by the 4s pathway.
Each single enzyme gene was synthesized by Generay and we constructed them together and suquencing comformed. the sequences can be found in NCBI (GenBank Accession number L37363.1)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1267
Illegal NheI site found at 4441
Illegal NheI site found at 4489 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 489
Illegal BamHI site found at 2071
Illegal XhoI site found at 481
Illegal XhoI site found at 3226
Illegal XhoI site found at 4067 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 196
Illegal NgoMIV site found at 2515
Illegal NgoMIV site found at 2730
Illegal NgoMIV site found at 3034
Illegal NgoMIV site found at 3154
Illegal AgeI site found at 2689
Illegal AgeI site found at 4255 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2221
Illegal SapI.rc site found at 3538
Figure 1. Bio-circuit of BBa K1972007
The native dszABC operon was rearranged and the promoter was replaced in order to avoid overlapping genes, increase the expression of the dsz genes, especially dszB, which encoded the rate-limiting enzyme of the 4S-pathway, and relieve inhibition. Besides, a synthetic dszD cassette which was not linked to the dszABC genes in engineered bacteria IGTS8 was also constructed (Figure 1).
Figure 2. DNA gel electrophoresis of constructed dsz cassette（lane 1~4）
The plasmid that can express T7 RNA polymerase under the induction of IPTG and the plasmid that includes four DSZ genes under T7 promoter were successfully constructed and transformed to BL21. Subsequently, the expression of four DSZ genes was detected by SDS-PAGE. As shown in Figure 3, the four enzymes were expressed in the engineered strain.
Figure 3. SDS-PAGE analysis of DSZ genes expression Control: BL21; 1, 2: Recombinant strain BL21-dszBCAD
The desulfurization activity of the recombinant strain BL21-dszBCAD was further measured by chromogenic reaction.
Figure 4. The desulfurization results of Recombinant strain BL21-dszBCAD tested by HPLC
The result showed the generation of 2-HBP was successful. However, the desulfurization efficiency of the recombinant strain BL21-dszBCAD showed no significant difference compared with that of IGTS8. This might be due to the high promoter activity of T7 promoter. The excessively strong activity of T7 promoter could result in lots of inclusion body, affecting the desulfurization efficiency of the recombinant strain. In order to solve the formation of inclusion body, the T7 promoter was replaced with Lac promoter (as shown in Figure 4). Unfortunately, the desulfurization efficiency was still not significantly improved.
Reference
[1] Li MZ, Squires CH, Monticello DJ, Childs JD. Genetic analysis of the dsz promoter and associated regulatory regions of Rhodococcus erythropolis IGTS8[J].Journal of Bacteriology.1996,178(22):6409-18.
[2] Franchi E RF, Serbolisca L, et al. Vector development, isolation of new promoters and enhancement of the catalytic activity of the Dsz enzyme complex in Rhodococcus sp. strains[J]. Oil & gas science and technology, 2003, 58(4): 515-520.
[3] Abin-Fuentes A, Mohamed Mel S, Wang DI, Prather KL. Exploring the mechanism of biocatalyst inhibition in microbial desulfurization[J].Applied and environmental microbiology.2013,79(24):7807-17.
[4] Li Y, Li W, Gao H, Xing J, Liu H. Integration of flocculation and adsorptive immobilization of Pseudomonas delafieldii R-8 for diesel oil biodesulfurization[J].Journal of Chemical Technology & Biotechnology.2011,86(2):246-50.
[5] Li GQ, Ma T, Li SS, Li H, Liang FL, Liu RL. Improvement of dibenzothiophene desulfurization activity by removing the gene overlap in the dsz operon[J].Biosci Biotechnol Biochem.2007,71(4):849-54.
[6] Li GQ, Li SS, Zhang ML, Wang J, Zhu L, Liang FL, et al. Genetic rearrangement strategy for optimizing the dibenzothiophene biodesulfurization pathway in Rhodococcus erythropolis[J].Applied and environmental microbiology.2008,74(4):971-6.
[7] Hirasawa K, Ishii Y, Kobayashi M, Koizumi K, Maruhashi K. Improvememt of Desulfurization Activity in Rhodococcus erythropolis KA2-5-1 by Genetic Engineering[J].Bioscience, Biotechnology and Biochemistry.2014,65(2):239-46.
[8]Martínez I, Mohamed M E S, Rozas D, et al. Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds[J]. Metabolic engineering, 2016, 35: 46-54.
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