Product Synthesis

The CO₂ fixation of module II generates 3-phosphoglycerate generated by the Calvin cycle. The 3-phosphoglycerate is transformed to pyruvate by glycolysis. 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 industrially relevant products like isobutanol, isoprene, putrescine or even antibiotics. We decided to introduce an isobutanol production pathway which starts with pyruvate (Figure 1). (Atsumi et al., 2008) This pathway is explained in more detail in the section Genetical Approach.
For this we used and improved some of the BioBricks from iGEM Team NCTU Formosa 2011/2012, which were available at the parts registry. We used the gene coding sequences of four out of five required proteins for the isobutanol production. For further information about our cloning strategic, please check our Genetical Approach section.
We used coding sequences for the following proteins:

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

Figure 1: Schematic illustration of the isobutanol pathway (Atsumi et al., 2008)

Figure 2: Schematic illustration of module III - the production of isobutanol. 3-phosphoglycerate from the CO2 fixation is converted to pyruvate in the glycolysis. In the implemented pathway pyruvate is then converted to isobutanol (Atsumi et al., 2008)

Isobutanol

Isobutanol is an amino-acid-based alcohol and consequently an organic substance.

Figure 3: 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 keto-acid decarboxylase and then reduced to an alcohol by an alcoholdehydrogenase. Keto-acids are immediate amino-acid precursors. By using this enzymatic conversion amino-acid-based alcohols can be produced in E. coli. These alcohols include n-butanol, n-propanol and isobutanol. Although the energy contents of isobutanol and n-butanol are similar, isobutanol is the closest to industrial use. Isobutanol has a better octane number because it is a branched-chain alcohol in comparison to straight-chain alcohols like ethanol. This indicates that isobutanol could be a possible alternative to ethanol as a fuel additive. In contrast to ethanol, the traditional biofuel respectively biofuel supplement, isobutanol as a higher alcohol has a lower hygroscopicity. Implementation of these characteristics in one of our application scenarios. (Atsumi et al., 2008; Peralta-Yahya et al., 2012)
In the following table 1 you can find some general information about isobutanol.

References

• Atsumi S, Hanai T, Liao JC., 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. In: Nature 451, 86–89.
• Bizzari, S.N., R. Gubler, and A. Kishi., 2002. CEH Marketing Research Report for Plasticizer Alcohols. In: IHS Chemical
• Dow Chemical (DOW): Isobutyl Acetate, version: 10/2014
• INCHEM: SIDS Initial Assessment Report For SIAM 19, version: 10/2014
• Pamela P. Peralta-Yahya, Fuzhong Zhang, Stephen B. del Cardayre & Jay D. Keasling, 2012. Microbial engineering for the production of advanced biofuels. In: Nature 488, 320–328