Part:BBa_K1972008

dszB with tac promoter induced by IPTG

We constructed dszB with tac promoter. DszB is a a desulfinase which can convert 2-(2-hydroxybiphenyl) sulfinate (HBPS) to 2-hydroxybiphenyl (2HBP) during the 4S pathway, in which dibenzothiophene (DBT) undergoes three successive oxidation steps and one a hydrolytic step leading to the formation of 2-hydroxybiphenyl (2HBP)

This single desulfinase gene was synthesized whith E.coli codon optimized by Generay and the sequence was found in NCBI (GenBank Accession number L37363.1)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 267
  Illegal AgeI site found at 1104
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 864
  

Figure 1. Bio-circuit of BBa_K1972014：Final optimized with dszB copy number increased

Figure 2. Bio-circuit of BBa_K1972008: dszB with tac promoter

Finally, we increased DszB copy number to gain higher efficiency of desulfurization (as shown in Figure 1). We also cultured the recombinant strain BL21-dszB (as shown in Figure 2), and add it (0.5 mgprotein/mL) to the recombinant strain BL21-dszBBACD (optimized)

Figure 3. The desulfurization results of Recombinant strain BL21-dszBBACD (optimized) addition of cell extract from the recombinant strain BL21-dszB tested by HPLC The desulfurization efficiency of the recombinant strain BL21-dszBBACD (optimized) addition of cell extract from the recombinant strain BL21-dszB is further improved (as shown in Figure 3). To know our project working under real industrial condition, we used n-dodecane with DBT (final concentration is 1mM) to simulate oil and the ratio of organic mixture and water is 1:9, which was based on our mathematic modeling. We added induced bacteria into the mixed system at an initial A600 of 2.0. After the reaction of 6 hours, we took 1 ml sample which would be tested by HPLC (as shown in Figure 4). The result shows clearly the generation of 2-HBP which means success of our project. Because it’s only a pre-experiment that we didn’t use equipment such as ultrasonator, the result does not show a high-efficiency desulfurization. We will do 2L-system desulfurization experiment in the next step.

Figure 4. The elements of oil phase and aqueous phase after 6h-reaction The result shows significant conclusions which will guide us in the next step: a.The elements of oil will nearly not be metabolized or absorbed by our engineered bacteria in oil-water phrases. b.Our engineered bacteria can convert DBT into 2-HBP successfully in oil-water phrases. c.Very little organic elements will be left in aqueous phase.

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.