894 1997, 2000; Hazelwood et al. 2008) about how these alcohols are produced in fermentations. The Ehrlich pathway is the shortest path to higher alcohols through the direct degradation of amino acids (Fig. 1). The amino acids first undergo transamination by an amino acid aminotransferase enzyme to form 2-ketoacids, the reverse direction of the biosynthetic reaction. The next step is a decarboxylation reaction by a 2-ketoacid decarboxylase (KDC), of which the most well known is pyruvate decarboxylase for ethanol production found in a diverse array of yeast and bacteria. Decarboxylation of a 2-ketoacid yields an aldehyde and CO2, and the final reduction from aldehyde to alcohol via an alcohol dehydrogenase (ADH) is a standard fermentation reaction. For example, isoleucine added to a yeast fermentation can be converted to its 2-ketoacid, 2-keto-3-methylvalerate (KMV), which can then be decarboxylated to form 2-methyl-1-butyraldehyde, which is in turn reduced to 2-methyl-1-butanol (active amyl alcohol). Likewise, leucine is converted to 3-methyl-1-butanol, and several other amino acids can also use this pathway. However, it is not necessary to add amino acids to a fermentation to produce these higher alcohols. The yeast and bacteria themselves synthesize all of the amino acids as well as their 2-ketoacid precursors. Yeast can produce higher alcohols from their native amino acid production pathways (Eden et al. 2001; Schoondermark-Stolk et al. 2005). In fact, the first push toward engineering the production of pentanols in microorganisms came about by exploiting this biosynthetic pathway. By using mutagenesis and selection based on the biosynthetic amino acid path- ways, several groups have been able to increase higher alcohol formation in yeast (Kielland-Brandt et al. 1979; Fukuda et al. 1993; Suzzi et al. 1998). By using the toxicity of chemical analogues of the amino acids isoleucine and leucine as selection pressure, they were able to augment production of 2-methyl-1-butanol and 3-methyl-1-butanol, respectively, during fermentation. Because these studies were attempting to improve flavor rather than produce either alcohol in particular, the overall titers were still fairly Fig. 1 Ehrlich degradation pathway from amino acid to alcohol. AAT amino acid amino- transferase, KDC 2-ketoacid decarboxylase, ADH alcohol dehydrogenase, 2KG 2-ketoglutarate, Glu glutamate Appl Microbiol Biotechnol (2010) 85:893–899 low. This endogenous production and improvement in yeast can serve as a model from which to engineer the production of pentanols in other organisms. Engineered production of 2-methyl-1-butanol in Escherichia coli In E. coli, the KDC enzyme catalyzing the decarboxylation of higher 2-ketoacids is missing. Combined with an ADH, these two enzymes are all that is needed for conversion of 2-ketoacids in E. coli into higher alcohols. Atsumi et al. (2008b) have shown that the broad substrate range KDC from Lactococcus lactis encoded by the kivd gene is functional in E. coli and enables the conversion of many 2-ketoacids into alcohols, including KMV to 2-methyl-1- butanol and 2-ketoisocaproate (KIC) to 3-methyl-1-butanol. The synthesis of 2-methyl-1-butanol is dependent on the 2-ketoacid precursor of the amino acid isoleucine whose biosynthetic pathway is naturally occurring in bacteria such as E. coli (Fig. 2). Thus, the isoleucine pathway, up to the common precursor KMV, is the primary target for the engineering of 2-methyl-1-butanol production. The first committed reaction in the biosynthesis of 2-methyl-1-butanol is the condensation of pyruvate and 2-ketobutyrate (2KB), catalyzed by the enzyme aceto- hydroxyacid synthase (AHAS). This enzyme also catalyzes the condensation of two pyruvate molecules in the first step of valine production, though most organisms have only one isozyme to carry out this reaction. In the enterobacteria, however, there are often three isozymes present, one of which is better suited for reacting with 2KB (Barak et al. 1987; Gollop et al. 1990) and thus better for the production of 2-methyl-1-butanol. Cann and Liao (2008) confirmed that overexpression of this isozyme, AHAS II, is in fact a good choice for 2-methyl-1-butanol production, especially AHAS II from Salmonella typhimurium. For most microorganisms, the source of 2KB for the AHAS reaction is the amino acid threonine. Threonine is Amino acid 2KG Glu 2-Ketoacid CO2 Aldehyde Alcohol NADH Isoleucine 2KG 2-Keto-3- Glu methyl-valerate 2-Methyl-1- CO2 butyraldehyde NADH 2-Methyl-1- butanol AAT KDC ADH