Team:NCTU Formosa/introduction

Introduction

Our program is to design “Pathway Commander”, just like its name, which is able to direct a pathway’s going. The goal is to control a pathway to stop at any intermediate step. In traditional pathway design, one circuit will express itself all to the end, unable to stop at any step involved. However, with our “RNA Thermometer”, which is temperature-controlled, the pathway can be easily controlled at specific temperature. The circuit we design can stop at any intermediate state we want and accumulate intermediate products, and then continue to express if the condition is changed. Even branched circuit can also be created.

Our team put the new idea in use to improve the production of butanol which is catalyzed by alpha-ketoisovalerate decarboxylase encoded by kivd gene. We use low-temperature-induced pathway because our product will easily vaporize in high temperature, and the specific enzyme involved has better activity at 30℃. We know that butanol is fatal to E.coli if the amount is above 2%. Now, we can exploit our “Pathway Commander” idea to the fullest in this situation. First, intermediate products accumulate until enough quantity to be bio-fuel and then we lower the temperature to induce the enzyme which catalyzes precursors into butanol. (Figure 1.)Of course, the E.coli dies due to butanol of high concentration, and there won’t be environmental issue and get enough butanol to produce bio-fuel!

We also apply “Pathway Commander” to other pigment metabolic pathway in order to test the idea of temperature-induced method. We design two examples to demonstrate our idea, which are Carotenoid pathway and Volacein pathway.

In Carotenoid pathway, we can stop the pathway at three states. In every state, it can turn to the next state after accumulate enough intermediates. For instance, when the temperature is below 37℃, E.coli synthesizes enzyme CrtE,CrtB and CrtI, which catalyze the colorless farnesyl pyrophosphate into pigment-Lycopen, and the red color is displayed. Later, when the temperature rises to 37℃, E.coli expresses crtY which catalyzes accumulative Lycopen into β-carotene, which shows obvious orange color. Finally, we raise the temperature up to 42℃ and E.coli expresses crtZ which catalyzes β-carotene into Zeaxanthin, and the yellow color is displayed consequently.(Figure 2.)

In Violacein pathway, we bear the same idea in mind controlling the pathway with branches. Intermediate PVA can turn into Deoxychromoviridans or Protoviolaceinic acid regulated by different temperatures. The Protoviolaceinic acid will turn into violacein eventually and then two branches are created.(Figure 3.)