Part:BBa_K1972009
dszBCAD with Lac promoter
We constructed dszBCAD genes with lac promoter(a weaker promoter to aviod the generation of inclusion body) 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 1266
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 488
Illegal BamHI site found at 2070
Illegal XhoI site found at 480
Illegal XhoI site found at 3225
Illegal XhoI site found at 4066 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 195
Illegal NgoMIV site found at 2514
Illegal NgoMIV site found at 2729
Illegal NgoMIV site found at 3033
Illegal NgoMIV site found at 3153
Illegal AgeI site found at 2688
Illegal AgeI site found at 4254 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2220
Illegal SapI.rc site found at 3537
Our Previous work shows 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 1). Unfortunately, the desulfurization efficiency was still not significantly improved.
Figure 1. BBa_K1972009: Bio-circuit after weakening the promoter of BBa_K1972007
Reference
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[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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