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)

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

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

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.

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)
In the following table you can find some general information about isobutanol.

Table 1: General information about isobutanol (INCHEM, 2004)

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.

References

• 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
• INCHEM: SIDS Initial Assessment Report For SIAM 19, version: 15/10/2014
• Atsumi S, Hanai T, Liao JC., 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. In: Nature 451, 86–89.