Team:NCTU Formosa/BP design

In traditional genetic engineering method, we use highly expressing promoter to initiate our gene, so E.coli will over express the protein we need in synthetic pathway. However, this overexpression will let E.coli waste its limited growth resources and too much intermediates will be accumulated. These exceeded intermediates are poisonous to organisms, which then slow down the growing of E.coli and cannot achieve optimum productivity.

In the new method we design, we control the pathway by stopping the mechanism when it reach the non-toxic intermediate production step which we want to accumulate, then under specific thermal control, the mechanism would continue to express. The advantage of our new method is that the precursor is much less toxic for E.coli than our target product be. Applying this new method to our project, we first accumulate lots of the non-toxic intermediate as the precursor, which is 2-Ketoisovalerate, to a certain amount, and then convert all the non-toxic precursor into the product, isobutanol, all at once.

To optimize the performance of the butanol pathway, we use the intermediates of E. coli’s amino acid metabolic system as raw material to produce butanol through the butanol biosynthesis pathway by adjusting the performace of gene we put into to the host cell.

We clone the genes which will be translated into enzymes such as alsS, ilvC, ilvD ,kivD and assemble the genes into two circuits as following. Those enzymes are crucial for producing butanol.

A. ( BBa_K539691)

In circuit A ( BBa_K539691), we used the lacI regulated promoter to initiate the circuit to produce alss, ilvC and ilvD. This circuit also inhibit circuit B (BBa_K539742)by producing tetR(regulated by 37℃ RBS)

B. (BBa_K539742)

In circuit B (BBa_K539742), we use Ptet to initiate the expression of kivD. This circuit is regulated by circuit A ( BBa_K539691)as the temperature rise up to 37℃ or higher.

By viewing our two circuits we could observe one circumstance that when the temperature reaches 37℃, tetR will be expressed and inhibit Ptet. In this way, circuit B(BBa_K539742) will not be expressed, and if we keep the temperature over 37℃, we can accumulate the intermediate, 2-Ketoisovalerate, which is non-toxic for E.Coli.

When we lower the temperature under 37℃, the circuit B(BBa_K539742) will not be inhibited by tetR. At the same time, kivD will be produced and start to catalyze 2-Ketoisovalerate into isobutanol.

In order to achieve the goal of our experimental design, we apply fermentation of carbohydrate to gain our product. First, we choose glucose as the resource to get through biosynthetic pathway, and we can harvest isobutanol which is the derivative of butanol.Glucose can be catalyzed into isobutanol through consequent different enzymes.We can also stop any step to accumulate the intermediate we want.