Product Synthesis

The CO₂ fixation of module II ends with 3-Phosphogylcerat generated by the Calvin cycle. This 3-Phosphogylcerat is transformed to pyruvate by the glycolysis of the cell. The pyruvate is now used as the initial point for the product synthesis. Pyruvate is the starting point of the producing pathways of a variety of high value products like isobutanol, isoprene, putrescine or even antibiotics. We decided to introduce an isobutanol production pathway which starts with pyruvate and is called 2-keto-acid, or Ehrlich, pathway. (Peralta-Yahya et al., 2012)
For this we use and improve existing BioBricks from iGEM Team NCTU Formosa 2011/2012. We use the coding sequences of the genes of four out of five required proteins for the isobutanol production:

• AlsS (α-acetolactate synthase)
• IlvC (Ketol-acid reductoisomerase)
• IlvD (Dihydroxyacid dehydratase)
• KivD (α-ketoisovalerate decarboxylase)

Figure 1: Schematic illustration of module III - the production of isobutanol

Isobutanol

Isobutanol is an amino-acid-based alcohol which is an organic substance.

Figure 2: Chemical structure of isobutanol.

It can be produced by the 2-keto-acid, or Ehrlich, pathway. In this pathway 2-ketoisovalerate is first decarboxylated into isobutyraldehyde by the ketoacid decarboxylase and then reduced to alcohols. Keto acids are immediate amino-acid precursors. By using this pathway amino-acid-based alcohols can be produced in E. Coli. These include n-butanol from norvaline, n-propanol from isoleucine and isobutanol from valine. Although the energy contents of isobutanol and n-butanol are similar, isobutanol is the closest to industrial use. Isobutanol has a better octane number. (Peralta-Yahya et al., 2012)
This indicates that isobutanol could be a possible alternative to ethanol as a fuel additive which is presented in one of our application scenarios.
In the following table 1 you can find some general information about isobutanol.