Difference between revisions of "Team:SCUT-China A/Project"

Desulfurization of oil is an important step during getting production of oil. Sulfur compounds exist in nature as various forms and can be classified into about four main groups: mercaptans, sulfides, disulfides and thiophenes (THs). Traditionally Chemical and Physical methods were used to desulfurize the former three compounds, but the sulfur in thiophenes was hardly removed by these ways. The present project is committed to developing a new desulfurization method. The desulfurization-system of Staphylococcus aureus IGTS8 was transformed into Escherichia coli and the recombinant strains were used to achieve the bio-desulfurization of oil. No one in iGEM has ever done it before.

In order to improve efficiency of desulfurization, we reprogrammed expression cassette under the control of inducible promoter, codon optimized of DszABC and regulate ratio of DszABCD by different promoter.

Fossil fuels are the most widely used sources of energy in the world. Although the percentage of energy obtained from fossil fuels declined over recent years, the consumption of energy from fossil fuels is still over 82% in the world, half of which comes from petroleum. Crude oil, a complex mixture of organic liquids, is the largest source of energy, has been playing a very important role in the world energy mix for centuries. The sulfur content is a property that has a great influence on the value of the crude oil. The sulfur content is expressed as a percentage of sulfur by weight and varies from less than 0.1% to greater than 5% depending on the type and source of crude oils.

Sulfur compounds are undesirable in refining process as they tend to deactivate some catalysts used in crude oil processing and cause corrosion problems in pipeline, pumping, and refining equipment. Naturally occurring sulfur compounds left in fuels lead to the emission of sulfur oxide gases. These gases react with water in atmosphere to form sulfates and acid rain, which damage buildings, destroy automotive paint finishes, acidify soil, and ultimately leads to loss of forests and various other ecosystems. Sulfur emissions also cause respiratory illnesses, aggravate heart disease, trigger asthma, and contribute to formation of atmospheric particulates. Automobiles are also adversely affected by presence of sulfur compounds in liquid fuels. Unfortunately, because of the serious energy crisis, people have to exploit high sulfur crude oil, making the sulfur pollution much more difficult to deal with.

Traditionally, there are three methods of industrial desulfurization.

Chemical method: HDS and ODS (Hydro desulfurization and Oxidative desulfurization). Typically, the HDS process involves catalytic treatment with hydrogen to convert the various sulfur compounds to H2S and sulfur-free organic compounds at high temperature and partial pressure of hydrogen. Conventional catalytic HDS method for reducing sulfur content requires severe conditions of operation. In refineries, the H2S resulting from the HDS reaction is eventually converted to elemental sulfur by a modified version of the Claus process. In ODS, heavy sulfides are oxidized by adding one or two oxygen atoms to the sulfur using appropriate oxidants without breaking any carbon–sulfur bonds, yielding the sulfoxide and sulfone, respectively. These oxidized compounds are then extracted or adsorbed from the light oil due to their increased relative polarity. Thus, the ODS is basically a two stage process; oxidation, followed by liquid extraction. Oxidation of the DBT derivatives to the corresponding sulfones increases their polarity and molecular weight. This facilitates their separation by extraction, distillation, or adsorption. Any of these separation methods could be used for separation of sulfur from the organic phase.

Physical method: ADS (Adsorptive desulfurization). Adsorption has been applied variously for removal of sulfur compounds from liquid hydrocarbon fuels. Removal of DBT and other sulfur compounds has been studied over zeolites, alum inosilicates, activated carbon (AC), alumina, zinc oxide, etc.

However, all of these traditional ways are environmentally harmful and energy-intensive. Especially they cannot remove the sulfur contained thiophenes (THs) compound in crude oil.

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 make the main sulfur compounds into the water phase, and loss the organic hydrocarbons (reducing the fuel calorific value). It is estimated that the oil whose sulfur mass fraction was 0.2%, lost about 1.0% after desulfurization, so the current stage is mainly to study the 4S pathway which retain the most of the heat value of products

4S-Pathway, the core of our project, was firstly found in Staphylococcus aureus (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, which cannot be removed by traditional methods, could be transformed into hydrophilic sulfate, which can be dissolved in water and eventually removed from oil.

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.

Gene manipulation for the model organism E. coli is standardized and modular, 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 researcher to use our engineered bacteria or alter it.

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.

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).

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.

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.

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.

However, the desulfurization efficiency of the recombinant strain BL21-dszBCAD shown no significant difference with that of IGTS8 (as shown in Figure 8). The results may due to the high promoter activity of T7 promoter. The excessively strong activity of T7 promoter could result to 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 9). Unfortunately, the desulfurization efficiency was still not significantly improved.

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 too many intermediate products (DBTO2/HBPS) accumulate, 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, in order to indirectly attenuate the effect of the enzyme A and C(as shown in Figure 10).

After all of these work, our engineered bacteria already have the ability to desulfurization. We sincerely hope that our project will be able to be applied to the processing of crude oil in refineries. The original intention of our project is to solve a problem that needs to be solved urgently. Although our project is still not mature enough, our project has begun to take shape. In the future, we are confident that we will continue to improve the project, and finally apply it to industrial production. We will use our killer sulfur to contribute our strength to the solution of environmental problems.

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The desulfurization efficiency of the recombinant strain BL21-dszBACD (optimized) is greatly improved, compared with that of IGTS8 (as shown in Figure 11).

Finally, we increased DszB copy number to gain higher efficiency of desulfurization (as shown in Figure 12). We also cultured the recombinant strain BL21-dszB, and add it (0.5 mgprotein/mL) to the recombinant strain BL21-dszBBACD (optimized)

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 13).

During our experiments, we find that there are some unanticipated limitations should be taken into consideration when designing efficient BDS system. The high activity of Dsz A, C will cause intermediate products accumulate, which seriously inhibit the activity of Dsz B. The high activity of Dsz also accelerated the rate of oxidation reaction catalyzed by Dsz C and A. The strong activity of promoter may cause inclusion body. Too much rare codons in the expression cassette reduced the translation efficiency. Finally we solved these problems, and achieved a pretty high level of desulfurization.

A good biological response system is really essential. In our project, we have not done enough on the optimization of the reaction conditions. For now we have just completed the optimal ratio of oil and water in mixed system, which severely limits the efficiency of the BDS. In the future, we will further optimize the temperature, PH, oxygen content and other conditions. We believe that after further optimization of the reaction conditions, the desulfurization efficiency will be greatly improved.