Short summary

Within the third module of our project the products of the carbon dioxide fixation will be used by the cell to produce the key metabolite pyruvate. Pyruvate can be converted in different products. We decided to introduce an isobutanol production pathway. For this we want to use and improve existing BioBricks (iGEM Team Formosa 2011/2012).
The modularity of BioBricks enables the exchange of a variety of producing systems. The producing pathway of variable high value products derived from pyruvate can be implemented. Hence, products like isoprene, putrescine or even antibiotics are possible candidates with industrial application.

Here you will find our results of the production of Isobutanol.
You can find more information about the underlying theory, our genetical approach and an outlook on our wiki

Product Synthesis

The third module benefits from the two modules before. E. coli has gained energy equivalents and reduction equivalents to generate pyruvate by binding carbon dioxide. By using the Calvin cycle the product of our CO2 fixation is pyruvate. This substance is now used as the initial point for the product synthesis. Pyruvate is a starting point of the producing pathways of a variety of high value products like isobutanol, isoprene, putrescine or even antibiotics. Our project implements the producing pathway of isobutanol by using and improving existing BioBricks (iGEM Team Formosa 2011/2012).

Figure 1: Schematic illustration of module III

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

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

It can be produced by the 2-keto-acid, or Ehrlich, pathway. Keto acids, the immediate amino-acid precursors, are decarboxylated into aldehydes and reduced to alcohols. By using this pathway amino-acid-based alcohols can be produced. These include n-butanol from norvaline, n-propanol from isoleucineand isobutanol from valine. Although the energy contents of isobutanol and n-butanol are similar, isobutanol is the closest to industrial use. Because of its branching it has improved properties, like a better octane number. This number is nessesarry to measure a fuel's resistance to knocking in spark ignition engines. (Pamela P. Peralta-Yahya et al., 2012)
In the following table you can find some general information about isobutanol.

CAS Number	78-83-1
IUPAC Name	2-methyl-propan-1-ol
Synonyms	isobutyl alcohol
	IBA, IBOH
	fermentation butyl alcohol
	1-hydroxymethylpropane
	isobutanol
	isopropylcarbinol
	2-methylpropanol
	2-methyl-1-propanol
	2-methylpropan-1-ol
	2-methylpropyl alcohol
Molecular Formula	C4H10O
Structural Formula	(CH3)2-CH-CH2OH
Molecular Weight	74.12 g/mol
Physical state	Liquid
Melting point	-108°C
Boiling point	108°C
Water solubility	85.0 g/l at 25°C

Production

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

Region or country	Number of producers	Manufacturing capacities [metric tons]
Western Europe	4	160,000
Eastern Europe	3	69,000 (including some n-butyl alcohol)
Russia	3	48,000
Iran	1	6,000
Japan	3	43,000
China	2	14,000
India	n.a.	8,000 (including some n-butyl alcohol)
Indonesia	1	10,000
Korea	2	25,000
Brazil	1	19,000

Use

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

Application	Amount
lube oil additives	19 thousand metric tons
(in which isobutyl alcohol is an intermediate
to produce the lube oil additive ZDDP)
conversion to isobutyl acetate	10 thousand metric tons
direct solvent	9 thousand metric tons
conversion to amino resins	7 thousand metric tons
conversion to isobutylamines	1 thousand metric tons
conversion to acrylate and methacrylate esters	1 thousand metric tons
other uses	1 thousand metric tons
All in all 47 thousand metric tons produced in the United States

As the table shows there are 3 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.
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 for as a latent solvent in surface coatings or even as a processing solvent in the production of e.g. pesticides and pharmaceuticals (INCHEM, 2004).
All in all isobutanol is an important substance for industrial use and large amounts are needed all over the world.