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Thaddeus Chukwuemeka Ezeji1’*, Nasib Qureshi2, Victor Ujor1

1The Ohio State University, Department of Animal Sciences and Ohio State Agricultural Research and Development Center (OARDC), Wooster, OH, USA, 2United States Department of Agriculture, a National Center for Agricultural Utilization Research, ARS, Bioenergy Research, Peoria, IL, USA

Corresponding author email: ezeji.1@osu. edu

OUTLINE

BACKGROUND/INTRODUCTION

Isobutanol (Inte’mational Union of Pure and Applied Chemistry nomenclature: 2-methylpropan-1-ol) is a branched four-carbon alcohol [(CH3^CHCH2OH], with a boiling point of 108 °C, a melting point of —108 °C, and a relative density of 0.806 at 15 °C (Budavari,

1996) . It is also known as isobutyl alcohol or 2-methyl-

1- propanol. Isobutanol has a vapor pressure of 10.43 mm Hg or 13.9 hPa at 25 °C (Daubert and Danner, 1985) and a water solubility of 85.0 g/l at 25 °C (Valvani et al., 1981). These properties reveal that isobutanol is lighter than, and also soluble in water. While isobutanol is produced industrially via carbonylation (incorpora­tion of carbon monoxide into organic/inorganic com­pounds) of propylene, isobutanol can be produced biologically via fermentation of glucose with a potential to use lignocellulosic biomass. Isobutanol is naturally
produced in low amounts by Saccharomyces cerevisiae as a degradation product of valine. The first report of biolog­ical production of isobutanol was by Dickinson et al. (1998) who demonstrated that S. cerevisiae was able to produce isobutanol using 13C-labeled valine as substrate. It was hypothesized that the product of valine transami­nation, a-ketoisovalerate, had four potential routes to isobutanol, which include (1) catalysis of a-ketoisovalerate by branched-chain a-keto acid dehy­drogenase to produce isobutyryl-CoA and subsequently isobutanol; (2) catalysis of a-ketoisovalerate to isobutanol by pyruvate decarboxylase (PDC); (3) reduction of a-ketoisovalerate to a-hydroxyisovalerate by a-ketoiso — valerate reductase; and (4) use of the PDC-like enzyme encoded by YDL080c to produce isobutanol. Given the fact that riddance of branched-chain a-keto acid dehydro­genase activity in an lpd1 disruption mutant did not — prevent the formation of isobutanol, S. cerevisiae cell

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Bioenergy Research: Advances and Applications http://dx. doi. org/10.1016/B978-0-444-59561-4.00007-3

homogenates could not convert a-hydroxyisovalerate to isobutanol, and a strain with a disrupted PDC-like gene, YDL080c, produced wild-type levels of isobutanol, hence, routes 1, 3, and 4 were eliminated in S. cerevisiae. Notably, elimination of PDC activity in a pdc1 pdc5 pdc6 triple mutant abolished isobutanol production thus, buttressing the notion that this route is the right channel to isobutanol biosynthesis.

As a feedstock chemical, isobutanol is used for the production of isobutyl acetate, which is subsequently used for the production of lacquers. It also finds use as a direct solvent, production of amino resins, isobutyl amines, and acrylate and methyl acrylate esters. The largest use for isobutanol is for the production of zinc dialkyldithiophosphates, an additive for lube oils, greases and hydraulic fluids, in which it functions as an antiwear and antioxidant additive. The second most significant applications of isobutanol are in the produc­tion of isobutyl acetate and as a solvent, primarily for surface coatings and adhesives (Bizziari et al., 2002).

Recent advances in liquid biofuel technology (Mariano et al., 2011,2012; Atsumi et al., 2008), depletion of petroleum reserves, global population growth, envi­ronmental and energy security concerns, have revived research efforts aimed at producing environmentally friendly liquid fuel chemicals. Indeed, global population is projected to reach 8.92 American billion by 2050 and world energy use may increase 53% by 2035. Conse­quently, there is an exigent need to source for or develop new fuels to fill potential shortfalls and, possibly replace our fast depleting petroleum reserves. Between 1980 and 2010, efforts have been focused on engineering microor­ganisms to make production of ethanol from biomass more efficient for use as a biofuel. Compared to ethanol, longer chain alcohols (e. g. n-propanol, n-butanol and isobutanol) have greater energy content, lower vapor pressure, and lower hygroscopicity, which make them superior alternatives to ethanol as a biofuel (Ladisch, 1991; Ezeji et al., 2005).

Isobutanol has the potential to substitute gasoline or serve as a gasoline supplement and can be produced from domestically abundant biomass sources including lignocellulosic biomass. Lignocellulosic biomass, which may contain xylan, arabinan, galactan, glucuronic, ace­tic, ferulic, and coumaric acids, is the most abundant renewable resource on the planet (Koukiekolo et al.,

2005) and has great potential as a substrate for isobuta­nol production (Higashide et al., 2011). Substrate cost has long been recognized to have significant influence on biofuel price and has been identified as a major factor affecting economic viability of n-butanol production by fermentation (Qureshi and Blaschek, 2000). Production of isobutanol from low-cost lignocellulosic biomass which does not compete with food crops may be critical for cost-effective fermentative production of isobutanol.

Whereas majority of producing microorganisms including S. cerevisiae use glucose as preferred substrate for growth and alcohol production, recent advances in genetic engineering have made it possible to metaboli — cally engineer these microorganisms to expand their substrate range to include pentose sugar components of lignocellulosic biomass hydrolysates such as xylose and arabinose, as in the case of ethanologenic microor­ganisms such as Escherichia coli (Dien et al., 1999, 2000), Zymomonas mobilis (Zhang et al., 1995; Deanda et al., 1996), and S. cerevisiae (Jin et al., 2005; Wisselink et al., 2007; Garcia Sanchez et al., 2010). This chapter describes the biochemistry of isobutanol production from biomass, latest developments in isobutanol production technology, and efforts directed toward development of more efficient and cost-effective processes for isobuta­nol production from biomass.