Appl Microbiol Biotechnol (2010) 85:893–899 895 Isoleucine Threonine 2KB KMV 2-Methyl- 1-butanol Glucose PEP Pyruvate AceCoA KIV KIC 3-Methyl- 1-butanol iBuOH Valine Leucine Fig. 2 Production of 2-methyl-1-butanol and 3-methyl-1-butanol from the amino acid biosynthetic pathways of isoleucine and leucine, respectively. PEP phosphoenol pyruvate, 2KB 2-ketobutyrate, KIV 2-ketoisovalerate, KMV 2-keto-3-methylvalerate, KIC 2-ketoisocaproate, iBuOH isobutanol converted to 2KB in one reaction carried out by the enzyme threonine deaminase. Several studies have given insights to increasing threonine production in E. coli and other organisms (Shimizu et al. 1995; Lee et al. 2007), and much of this knowledge is transferrable to 2-methyl-1-butanol biosynthesis. In the case of Corynebacterium glutamicum, it was shown that a lysine-producing strain can be converted into an isoleucine-producing strain by over- expression and removal of feedback inhibition of the threonine biosynthetic pathway and threonine deaminase (Morbach et al. 1996). Recent work has validated this approach for the production of higher alcohols in E. coli, including 2-methyl-1-butanol, where the efficacy of over- expressing a feedback-resistant threonine deaminase and threonine biosynthetic operon (thrABC) with artificial transcription regulation has been clearly demonstrated (Cann and Liao 2008; Shen and Liao 2008). Further improvement in 2-methyl-1-butanol production was achieved through gene knockouts. Many knockout strains were cultured and tested for improved 2-methyl-1- butanol production, mostly genes in competing pathways and genes that lead to side product formation. However, only knockout strains leading to increased flux to threonine and 2KB (metA and tdh) were significantly improved (Cann and Liao 2008). The best strain tested achieved 1.25 g/L of 2-methyl-1-butanol with a yield of 0.17 g/g glucose. Additional work in strain design should lead to even better 2-methyl-1-butanol production strains. Engineered production of 3-methyl-1-butanol in E. coli The leucine biosynthetic pathway in E. coli produces the 2-ketoacid precursor of 3-methyl-1-butanol, KIC (Fig. 2). Analogous to 2-methyl-1-butanol production, the expres- sion of kivd from L. lactis facilitates the conversion of KIC to 3-methyl-1-butanol. Leucine biosynthesis also builds from the valine pathway in E. coli, consuming the precursor of valine, 2-ketoisovalerate (KIV). Previous work in isobutanol production has demonstrated the ability to produce enough KIV to enable 22 g/L of isobutanol in E. coli cultures (Atsumi et al. 2008b). A key factor in this level of production was the expression of alsS from Bacillus subtilis coding for aceto-lactate synthase for very efficient condensation of two pyruvate molecules in the first committed step in KIV production. Another important aspect of high KIV production for isobutanol is a strain in which competing fermentation pathways are deleted, leading to an increased availability of both NADH and pyruvate. The strategies shown here to increase KIV are useful for further conversion of KIV to 3-methyl-1-butanol. The leucine biosynthetic pathway combines KIV with acetyl coenzyme-A (acetyl-CoA) to produce KIC for 3- methyl-1-butanol production. However, simply overex- pressing the native leucine operon from E. coli (leuABCD) does little to divert KIV from isobutanol production (Connor and Liao 2008). It was previously demonstrated that removal of feedback inhibition of the leucine operon is effective for increasing the production of leucine in micro- organisms (Gusyatiner et al. 2002). Connor and Liao (2008) showed that in addition to the expression of a feedback- resistant leucine operon, a synthetic ribosomal binding site is important for the efficient expression of the operon. These additions led to a dramatic increase in 3-methyl-1- butanol production in E. coli. Further improvement in the production of 3-methyl- 1-butanol can come from the deletion of competing pathways. As mentioned previously, gene knockouts of fermentation pathways such as lactate (ldhA), ethanol (adhE), acetate (pta), and succinate (frdBC), as well as knockouts of pflB and fnr to increase efficiency in low oxygen conditions (Atsumi et al. 2008a), contributed to an increase in the pools of NADH, pyruvate, and acetyl- CoA for increased production of 3-methyl-1-butanol. Two other vital precursors, KIV and KIC, are also precursors for the transamination reaction to form the amino acids valine and leucine, respectively. Deletion of the two genes responsible for the transamination of most of the KIV and KIC, ilvE and tyrB, further increased 3- methyl-1-butanol production in E. coli to a final titer of nearly 1.3 g/L with a yield of 0.11 g/g glucose (Connor and Liao 2008).