Keto acids are organic acids with ketone functional group on the second carbon, typically known as a-carbon, and are present in microorganisms as intermediate pro­ducts of amino acids production pathways, and degra­dation of biosynthesized amino acids to alcohols is what is commonly known as Ehrlich pathway (Figure 7.1). Different strains of S. cerevisiae and yeasts belonging to genera such as Endomycopsis, Candida, and Hansenula are known to produce higher alcohols via keto acid pathways (Singh and Kunkee, 1976; Cronk et al., 1979).

In addition to ethanol and CO2 production, S. cerevisiae produces a variety of relatively low — molecular weight flavor compounds such as alcohols, diacetyl, esters, organic acids, organic sulfides, and carbonyl compounds during fermentation (Ter Schure

Amino acid

‘ <

tt-Keto acid (e. g. L-glutamic acid)

<‘

Aldehyde (e. g. isobutyraldehyde)

У

Alcohol (e. g. isobutanol)

FIGURE 7.1 Simplified biochemistry of branched chain higher alcohol production from amino acids.

et al., 1998; Hazelwood et al., 2008). These compounds are formed via the Ehrlich pathway involving branched — chain amino acids such as isoleucine, leucine, methio­nine, phenylalanine, tryptophan, tyrosine, and valine, and biocatalysts such as transaminase, decarboxylase, and alcohol dehydrogenase (ADH) (Figure 7.1, Table 7.1 Ryan and Kohlhaw, 1974). This pathway is prevalent in yeast and is especially active when yeast is cultivated in growth medium whose carbon source is solely amino acids. Notably, catabolism of isoleucine, leucine, methi­onine, phenylalanine, tryptophan, tyrosine, and valine by S. cerevisiae via Ehrlich pathway generates

2- methylbutanol (active amyl alcohol), 3-methylbutanol (isoamyl alcohol), methionol, 2-phenylethanol, trypto — phol, p-hydroxyphenyl ethanol, and isobutanol, respec­tively (Figure 7.1; Table 7.1). These relatively long-chain alcohols are often referred to as fusel oils or fusel alcohols. During ethanolic fermentation by S. cerevisiae, small quan­tities of these alcohols (fusel oil) are produced (Singh and Kunkee, 1976). Whereas this mixture of alcohols may contribute flavor and body to wines, it can produce an off-flavor in wines when the acceptable concentration threshold is exceeded. Indeed, the catabolism of amino acids in S. cerevisiae and its regulation has been studied extensively (Ter Schure et al., 1998; Hazelwood et al., 2008; Dickinson et al., 1998,2000).

By transamination of amino acids to a-keto acids followed by decarboxylation of a-keto acids to aldehydes, these aldehydes can undergo reduction reaction to produce alcohols (Figure 7.1; Table 7.1). Scientists are
exploiting this biosynthetic pathway to take advantage of the amino acid biosynthesis capability of producing microorganisms such as E. coli and S. cerevisiae to pro­duce fusel alcohol, of which isobutanol appears to be the most attractive. In particular, the specificity of decar­boxylases has been suggested to be an important factor influencing the composition of fusel alcohols (Harrison and Collins, 1968; Suomalainen and Keranen, 1967). Furthermore, the amount of fusel alcohols produced by different yeasts and specific ADH activities with the cor­responding alcohols as substrates was found to be related as well (Singh and Kunkee, 1976). In recent years, metabolic engineering strategy using heterologous hosts such as E. coli and Clostridium cellulolyticum to produce higher alcohols from glucose and cellulose, respectively, is under investigation (Atsumi et al., 2008; Higashide et al., 2011). Depending on the source, 2-ketoacid decar­boxylase (KDC, encoded by the kivd gene) and ADH (encoded by the adh2 gene), which play critical roles in fusel oil production, may have broad substrate specific­ities toward the catalysis of 2-ketoacids and generation of isobutanol (Figures 7.1 and 7.2). When these two genes, kivd from Lactococcus lactis and adh2 from S. cere — visiae, were cloned and overexpressed in E. coli, approx­imately six long-chain alcohols including 1-propanol,

1- butanol, isobutanol, 2-methyl-1-butanol, 3-methyl — 1-butanol, and 2-phenylethanol were produced (Atsumi et al., 2008). This strategy exploits the presence of a highly active amino acid biosynthetic pathway in the host microorganism, keto acid pathway, and the ability

FIGURE 7.2 Schematic diagram depicting pathways leading to valine and isobutanol biosynthesis in S. cerevisiae. Genes encoding enzymes that catalyze each step are indi­cated and are as follows: ADH2 (alcohol dehydrogenase), Batl and Bat2 (branched chain amino acid aminotransfer­ases), ILV2 (acetolactate synthase), ILV3 (dihydroxyacid dehydratase), ILV5 (acetohydroxyacid reductoisomerase), Kdc/kivd/Pdc, 2-ketoacid decarboxylase (pyruvate decar­boxylase); and PDA (pyruvate dehydrogenase complex). (For color version of this figure, the reader is referred to the online version of this book.)

Cytosol

of the host to reroute its 2-ketoacid intermediates for alcohol synthesis (Atsumi et al., 2008). The amount of individual alcohol produced is compared with the level of its corresponding ketoacid. For example, when alsS from Bacillus subtilis and ilvCD from E. coli were overex­pressed in E. coli, the resulting strain accumulated remarkable amounts of 2-ketoisovalerate (KIV) in the fermentation broth. Furthermore, when kivd and adh2 were co-expressed in this recombinant strain, approxi­mately 22 g/l isobutanol was produced over the course of 112 h of fermentation (Atsumi et al., 2008). Notably, AlsS of B. subtilis, kivd from L. lactis, adh2 from S. cerevi­siae, and YqhD (nicotinamide adenine dinucleotide phos­phate (NADPH)-dependent ADH) from E. coli have high affinity for pyruvate and 2-ketoacids, 2-ketoacids, isobu — tyraldehyde, and isobutyraldehyde, respectively.