Module III - Isobutanol production

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)
Adh (alcohol dehydrogenase), the fifth required protein, was not available as a BioBrick but because of Escherichia coli's own AdhE the pathway works. (Atsumi et al., 2008)

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)
The product synthesis can be changed through the modularity of BioBricks, so that variable high value products derived from pyruvate can be implemented in the future.


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 alcohol dehydrogenase. 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.

Table 1: General information about isobutanol (INCHEM, 2004)
CAS Number78-83-1
IUPAC Name2-methyl-propan-1-ol
Synonymsisobutyl alcohol
fermentation butyl alcohol
2-methylpropyl alcohol
Molecular FormulaC4H10O
Structural Formula(CH3)2-CH-CH2OH
Molecular Weight74.12 g/mol
density802.00 kg/m3
Physical stateLiquid
Melting point-108°C
Boiling point108°C
Water solubility85.0 g/l at 25°C


In 1998 the U.S. EPA Inventory Update Report (IUR) listed 16 manufacturing facilities in the United States. Altogether these facilities produced between 100 and 500 million pounds of isobutanol per year, which are 45.4 – 227.3 thousand metric tons. Manufacturing facilities of other regions or countries in 2002 including their manufacturing capacities are listed in the following table 2. (INCHEM, 2004)

Table 2: Information about the producing facilities in various regions and countries including their manufacturing capacities in 2002 (INCHEM, 2004; Bizzari, 2002)
Region or countryNumber of producersManufacturing capacities
[metric tons]
Western Europe4160,000
Eastern Europe369,000 (including some n-butyl alcohol)
Indian.a.8,000 (including some n-butyl alcohol)


Isobutanol has many applications. In the following table 3 you can find a list of the span of applications and how much isobutanol is applied for the various uses in the United States.

Table 3: Industrial applications of isobutanol and their required amounts (INCHEM, 2004; Bizzari, 2002)
ApplicationAmount [metric tons]
lube oil additives (in which isobutyl alcohol is an intermediate to produce the lube oil additive ZDDP)19,000
conversion to isobutyl acetate10,000
direct solvent9,000
conversion to amino resins7,000
conversion to isobutylamines1,000
conversion to acrylate and methacrylate esters1,000
other uses1,000

As the table shows there are three big markets for isobutanol in the United States. The largest one is the production of zinc dialkyldithiophosphates (ZDDP). ZDDP is an additive for lube oils, greases and hydraulic fluids, which work as anti-wear and corrosion inhibitors.
The conversion of isobutanol to isobutyl acetate is the second largest market. Applications of isobutyl acetate are for example coating, adhesives, or cosmetic/personal care solve (DOW).
The use of isobutanol as a solvent is the third largest market. It is mainly used for surface coatings and adhesives. Hence it is used as a latent solvent in surface coatings or even as a processing solvent in the production of e.g. pesticides and pharmaceuticals.(INCHEM, 2004) These tables show the importance of isobutanol for industrial use and large amounts are needed all over the world.
We thought about different additional applications of isobutanol during our lively policy and practices discussion. You can find our suggestions here

  • 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