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4S-Pathway
Bio-desulfurization is mainly divided into the initial catalytic reaction center of carbon atoms (Kodama pathway) and the initial catalytic reaction center of sulfur atoms (4S pathway). The Kodama pathway does not destroy the S-C bond, and makes the main sulfur compounds into the water phase, and loses the organic hydrocarbons (reducing the fuel calorific value). It is estimated that oil with sulfur mass fraction of 0.2%, lost about 1.0% after desulfurization, so the current stage is mainly to study the 4S pathway which retains most of the heat value of products
4S-Pathway, the core of our project, was firstly found in Rhodococcus erythropolis (IGTS8 for example), which grows very slowly, and is very demanding on the culture conditions. Through a series of biochemical reactions by Monooxygenase A, Desulfurase B, Monooxygenase C and Flavin reductase D in 4S-pathway, the sulfur-contained hydrophobic complexes in crude oil, that cannot be removed by traditional methods, could be transformed into hydrophilic sulfate, which can be dissolved in water and eventually removed from oil.
Figure 1. The way 4S pathway work, choose DBT as substrate
The representative thiophenes compound DBT was chosen as reaction substrate to study 4S-pathway under laboratory conditions (Figure 1). Firstly DBT is transformed into sulfone and sulfoxide through the catalysis of Monooxygenase C, and then Monooxygenase A breaks one of the C-S bonds and creates a hydroxyl group on the benzene. Finally, Desulfurase B breaks another C-S bond, generating the end product--HBP. Flavin reductase D provides reducing power for the above steps. Through these reactions, sulfur is eventually removed from DBT in the form of sulfur sulfite.
Designs and Results
Alternation of desulfurization host
Gene manipulation for the model organism E. coli is standardized and modularized, which made it appropriate for introducing the desulfurization-system. Based on standard system, the engineered bacteria could be upgraded by introducing relatively parts of the 4S-pathway. It is also convenient for other researchers to use our engineered bacteria or alter it.
Figure 2. Growth curves of E.coli and IGTS8
As shown in the growth curves of E.coli and IGTS8 (Figure 2), the growth rate of BL21 is significantly higher than that of IGTS8. Considering that a large number of bacteria is necessary for the bio-desulfurization process, the BL21 strain was chose as the desulfurization host.
Reprogram expression cassette under the control of inducible promoter
Figure 3. Bio-circuit of 4S pathway in IGTS8
During sequence analysis on IGTS8, we find Enzyme A’s activity is stronger, and it is placed at the front of the expression cassette, Enzyme B’s activity is relatively weak, and it has a position at the backward of the expression cassette. The gene of Enzyme B is even overlapped by genes of A and C (Figure 3).
Figure 4. The desulfurization results of IGTS8 tested by HPLC
The model substrate DBT was mixed with IGTS8 and the concentrations of DBT and HBP were measured. As shown in Figure 4, the DBT consumption is fast, while the HBP generation is relatively slow. The results indicated that the relative ratio of enzyme A, B, and C in IGTS8 is inappropriate and limits the desulfurization efficiency. According to the sequence analysis of DSZ genes in IGTS8 and its desulfurization capacity measurement, the expression cassette of DSZ genes in IGTS8 should be optimized.
Sulfur is removed in the form of sulfur sulfite through 4S-pathway, but sulfur sulfite could inhibit the promoter activity of DSZ genes. Consequently, the native constitutive promoters of DSZ genes were replaced by the inducible T7 promoter to relieve the inhibition and enhance the expression.
Figure 5. Bio-circuit after first optimization
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 5).
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 6, the four enzymes were expressed in the engineered strain.
Figure 6. 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. As shown in Figure 7, Recombinant strain BL21-dszBCAD could make the color of reaction system change to blue when mixed with DBT, indicating that the 4S-pathway works effectively in the recombinant strain.
Figure 7. The desulfurization results of Recombinant strain BL21-dszBCAD tested by HPLC
However, the desulfurization efficiency of the recombinant strain BL21-dszBCAD showed no significant difference compared with that of IGTS8 (as shown in Figure 7). 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 8). Unfortunately, the desulfurization efficiency was still not significantly improved.
Figure 8. Bio-circuit after second optimization
Codon optimization and regulate ratio of DszABCD
The DszB desulfurase catalyzes the rate-limiting step of the 4S-pathway and the Y63F amino acid substitution was previously reported to enhance its activity and stability. Therefore, the Y63F amino acid substitution of the DszB desulfurase was performed.
Furthermore, the desulfurization experiment showed that the activities of enzyme C and A were still stronger than B, which caused undesirable accumulation of intermediate products (DBTO2/HBPS), seriously affecting the activity of enzyme B. The promoters of dsz genes were further adjusted. Gene dszB, dszC and dszA were controlled by the tac promoter, which was strong enough and endogenous for E.coli. At the same time, dszD was under the weak lac promoter as an independent operon, making the expression of enzyme D relatively weak, with the purpose of indirectly attenuating the effect of the enzyme A and C (as shown in Figure 9).
Figure 9. Final bio-circuit
Figure 10. The desulfurization results of Recombinant strain BL21-dszBACD (optimized) tested by HPLC
The desulfurization efficiency of the recombinant strain BL21-dszBACD (optimized) is greatly improved, compared with that of IGTS8 (as shown in Figure 10).
Figure 11. Final bio-circuit with dszB copy number increased
Finally, we increased DszB copy number to gain higher efficiency of desulfurization (as shown in Figure 11). We also cultured the recombinant strain BL21-dszB, and add it (0.5 mgprotein/mL) to the recombinant strain BL21-dszBBACD (optimized)
Figure 12. 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 12).
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South China University of tecnology
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