Christopher J. Brigham, Claudia S. Gai, Jingnan Lu, Daan R. Speth, R. Mark Worden, and Anthony J. Sinskey

Abstract Isobutanol (IBT) can be used as a 100% replacement for gasoline in existing automobile engines, has >90% of the energy density of gasoline and is compatible with established fuel distribution infrastructure. The facultatively auto­trophic bacterium Ralstonia eutropha can utilize H2 for energy and CO2 for carbon and is also employed in industrial processes that produce biodegradable plastics. Using a carefully designed production pathway, R. eutropha, a genetically tractable organism, can be modified to produce biofuels from autotrophic growth. Microbial

C. J. Brigham • C. S. Gai

Department of Biology, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

J. Lu

Department of Chemistry, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

D. R. Speth

Department of Biology, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

Department of Microbiology, IWWR, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands

R. M. Worden

Department of Chemical Engineering and Materials Science,

Michigan State University, East Lansing, MI 48824, USA

A. J. Sinskey (H)

Department of Biology, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

Engineering Systems Division, Massachusetts Institute of Technology,

77 Massachusetts Avenue, Cambridge, MA 02139, USA

Health Sciences Technology Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA e-mail: asinskey@mit. edu

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_39, 1065 © Springer Science+Business Media New York 2013

production of IBT can be achieved by directing the flow of carbon through a synthetic production pathway involving the branched-chain amino acid biosynthe­sis pathway, a heterologously expressed ketoisovalerate decarboxylase, and a broad substrate specificity alcohol dehydrogenase. We discuss the motivations and the methods used to engineer R. eutropha to produce the liquid transportation fuel IBT from CO2, H2, and O2.

1 Introduction

Increased demand for fossil fuels along with dwindling reserve supplies reveals an immediate need for alternative fuel sources. Bio-based fuels, or biofuels, are pro­duced from many sources of biomass. Microbially produced biofuels offer a sus­tainable approach to fuel production using inexpensive carbon sources, such as agricultural waste or CO2 [1] . The availability of H2 derived from solar-powered electrolysis will eventually increase dramatically, creating demand for microbes that use this energy source to convert CO2 into value-added chemical compounds, including liquid transportation fuels. Ethanol has been long discussed as a biofuel since Beall et al. [2, 3] developed a method for producing ethanol from sugars using a recombinant Escherichia coli strain. However, ethanol is not the most suitable alcohol for biofuel use as its hygroscopicity is higher and its energy density is lower than for longer chain alcohols [4]. Isobutanol (IBT), on the other hand, can be used without gasoline-blending in existing internal combustion engines and is compati­ble with the existing fuel infrastructure [5] . Recently, an automobile competed in the American Le Mans racing Series running on 100% IBT [6]. Although the source of IBT was not disclosed, the American Le Mans racing Series had recently approved IBT from corn, sugarcane, and cellulosic feedstocks for use as a fuel [7].