Team:Tokyo-NoKoGen/Project/solubilization

Bitumen is hydrocarbon which adhered to oil sand. Bitumen is consisted of poisonous organic compounds such as naphthalene, phenanthrene, benzopyrene and so on. It is difficult to recycle bitumen as fuels because it is hard to extract target compound from bitumen.
Surfactants enable to solubilize organic compound, but it is difficult to biodegradate artificial surfactants which we usually use. On the other hand, biosurfactants which microbes produce by themselves are able to biodegradate. That is why, biosurfactants are very ecology.

We try to extract organic compound such as naphthalene from bitumen using solubilization device. Pseudomonas aeruginosa is known to produce biosurfactants called as rhamnolipids. Rhamnolipids form micelle with organic compound and micelle were able to solubilize. Then, we cloned RhlA and RhlB genes which are necessary to produce rhamnolopids from Pseudomonas aeruginosa. E. coli was transformed with plasmid involved RhlA or RhlB gene. We tried to construct solubilization device which E. coli encoded rhamnolopids genes.

For the first of all, we have to make the target be easily taken up from an mixture. Oil sand is a good example. It is a naturally occurring mixture of sand, clay and several minerals, and bitumen. This bitumen is a material for a usable fuels such as gasoline and diesel, though it is too viscous to produce fuels. One of the solutions is to solubilize a object from a mixture. This is because solubilization system is the first necessary step in Eco Tanker.

This device can produce two enzymes. One is transacylase, the other is rhamnosyltransferase. Each protein is expressed by the gene, RhlA and RhlB respectively.

Fig.1 shows Rhamnolipids Biosynthetic Pathway, and you can see where two genes work for.

Transacylase:

It catalyzes the transfer of β-hydroxydecanoyl moieties from acyl carrier protein (ACP) to coenzyme A (CoA). With this reaction, 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), which Fig.2 shows, are produced.

Rhamnosyltransferase:

It catalyzes the reaction, which uses dTDP-L-rhamnose and an HAA as precursors, yielding mono-rhamnolipids(rhamnolipids contains both mono-rhamnolipids and di-rhamnolipid naturally(Fig.3).).