896 Appl Microbiol Biotechnol (2010) 85:893–899 Isopentenol production from the terpenoid pathway 1-Pentanol production using novel pathway functions An oxidized extension of the pentanol family, isopentenol can be found in trace amounts in the resin of certain tree species (Dewick 2002). Isopentenol is derived from the five-carbon building blocks of terpenoid synthesis, isopen- tenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP; Fig. 3). There are at least two biosynthetic pathways to IPP and DMAPP common in microorganisms, one dependent on the intermediate mevalonate, made from acetyl-CoA, and found in Saccharomyces cerevisiae, and another dependent on methylerythritol phosphate, from pyruvate and glyceraldehyde-3-phosphate, and found in E. coli. There have been many studies concerning the engineering of these pathways for the production of higher terpenoids (Farmer and Liao 2000; Martin et al. 2003), but very little work has been done to engineer the production of isopentenol. Isopentenol was detected at very low levels in E. coli cultures as a by-product of the overexpression of most of the native methylerythritol phosphate pathway (Rohdich et al. 2002). More recently, Withers et al. (2007) actually discovered some enzymes responsible for the direct production of isopentenol from IPP and DMAPP from B. subtilis. They accomplished this by screening a genomic library for the enzymatic relief of toxic levels of farnesyl diphosphate, a product of IPP and DMAPP polymerization, in E. coli cultures. Expression of the nudF gene from B. subtilis allowed the production of about 110 mg/L of isopentenol. Glucose G3P Pyruvate AceCoA MEP DMAPP Mevalonate IPP Terpenoids Isopentenol Fig. 3 Pentenol production via parallel pathways involving either the intermediate mevalonate or methylerythritol phosphate. G3P glyceraldehyde-3-phosphate, AceCoA acetyl coenzyme-A, MEP meth- ylerythritol phosphate, DMAPP dimethylallyl diphosphate, IPP isopentenyl diphosphate Normal pentanol, the straight chain isomer, is a semi- natural biological compound. Its presence has been detected in trace amounts in yeast fermentation (Williams et al. 1981; Mauricio et al. 1997), but the biosynthetic pathway is not well understood. Recently, however, it has been shown how 1-pentanol can be produced in microorganisms by expanding the range of substrates for known biosynthetic pathways (Zhang et al. 2008). It was previously established that the KDC coded by kivd from L. lactis has a wide substrate range for converting 2-ketoacids into aldehydes (de la Plaza et al. 2004; Atsumi et al. 2008b), and we have already seen how well this enzyme helps in the production of two other pentanols, 2-methyl-1- butanol and 3-methyl-1-butanol. However, the 2-ketoacid that would be converted to 1-pentanol, 2-ketocaproate, is not normally found in microorganisms. Interestingly, it turns out that the leucine biosynthetic pathway in E. coli (leuABCD) can also be engineered to accommodate a broad substrate range (Fig. 4). Previous work has already shown that the leucine pathway, which normally converts KIV to KIC, can convert 2KB to 2-ketovalerate, the precursor in 1-butanol produc- tion (Atsumi et al. 2008b; Shen and Liao 2008). The natural substrate for the leucine pathway is very similar to 2- ketovalerate; however, overexpression in E. coli of wild- type leuABCD along with kivd produces only a small amount of 1-butanol, and no 1-pentanol is detected (Zhang et al. 2008). Protein engineering of the two promiscuous enzymes LeuA and Kivd proved to be instrumental in the production of 1-pentanol. Removal of leucine-induced feedback inhibition of LeuA first allowed 1-pentanol to be detected, and an enlargement of the Kivd substrate-binding pocket allowed a production level of 750 mg/L of 1- pentanol in E. coli cultures (Zhang et al. 2008). Considering that 1-pentanol was not the final goal of this protein engineering, it seems likely that more focused work could yield even greater production of 1-pentanol in the future. Applications of pentanols The current push for alternative fuels has brought about our recent discussion of pentanols and other higher alcohols as potential biofuels. As discussed, pentanols have good physical properties for use as liquid transportation fuels, and they are currently used as fuel additives in gasoline blends. This section will discuss the many other applica- tions of pentanol isomers, both current and potential. There are a couple of ways to diversify the type of fuel that pentanols can become. For example, current biodiesel production relies mostly on methanol to esterify fatty acid