Acetolactate Synthase from Bacillus subtilis Serves as a 2-Ketoisovalerate Decarboxylase for Isobutanol Biosynthesis in Escherichia coli

A pathway toward isobutanol production previously constructed in Escherichia coli involves 2-ketoacid decarboxylase (Kdc) from Lactococcus lactis that decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde. Here, we showed that a strain lacking Kdc is still capable of producing isobutanol. We found that acetolactate synthase from Bacillus subtilis (AlsS), which originally catalyzes the condensation of two molecules of pyruvate to form 2-acetolactate, is able to catalyze the decarboxylation of KIV like Kdc both in vivo and in vitro. Mutational studies revealed that the replacement of Q487 with amino acids with small side chains (Ala, Ser, and Gly) diminished only the decarboxylase activity but maintained the synthase activity.We have previously shown that 2-keto acids generated from amino acid biosynthesis can serve as precursors for the Ehrlich degradation pathway (15) to higher alcohols (3). In order to produce isobutanol, the valine biosynthesis pathway was used to generate 2-ketoisovalerate (KIV), the precursor to valine, which was then converted to isobutanol via a decarboxylation and reduction step (Fig. ​(Fig.1A).1A). The entire pathway to isobutanol from glucose is shown in Fig. ​Fig.1A.1A. To produce isobutanol, we overexpressed five genes, alsS (Bacillus subtilis), ilvC (Escherichia coli), ilvD (E. coli), kdc (Lactococcus lactis), and ADH2 (Saccharomyces cerevisiae) (Fig. ​(Fig.1A).1A). This E. coli strain produced 6.8 g/liter isobutanol in 24 h (Fig. ​(Fig.1B)1B) and more than 20 g/liter in 112 h (3). More recently, we have found that an alcohol dehydrogenase (Adh) encoded by yqhD on the E. coli genome can convert isobutyraldehyde to isobutanol efficiently (5) (Fig. ​(Fig.1B1B).Open in a separate windowFIG. 1.Schematic representation of the pathway for isobutanol production. (A) The Kdc-dependent synthetic pathway for isobutanol production. (B) Isobutanol production with the Kdc-dependent and -independent synthetic pathways. IlvC, acetohydroxy acid isomeroreductase; IlvD, dihydroxy acid dehydratase. (C) Enzymatic reaction of Als, Ahbs, and Kdc activities.One key reaction in the production of isobutanol is the conversion of KIV to isobutyraldehyde catalyzed by 2-ketoacid decarboxylase (Kdc) (Fig. ​(Fig.1C).1C). Since E. coli does not have Kdc, kdc from L. lactis was overexpressed. Kdc is a nonoxidative thiamine PP_i (TPP)-dependent enzyme and is relatively rare in bacteria, being more frequently found in plants, yeasts, and fungi (8, 19). Several enzymes with Kdc activity have been found, including pyruvate decarboxylase, phenylpyruvate decarboxylase (18), branched-chain Kdc (8, 19), 2-ketoglutarate decarboxylase (10, 17, 20), and indole-3-pyruvate decarboxylase (13).In this work, unexpectedly, we find that Kdc is nonessential for E. coli to produce isobutanol (Fig. ​(Fig.1).1). An E. coli strain overexpressing only alsS (from B. subtilis), ilvC, and ilvD (both from E. coli) is still able to produce isobutanol. Since E. coli is not a natural producer of isobutanol, it cannot be detected from the culture media in any unmodified strain. We identify that AlsS from B. subtilis, which was introduced in E. coli for acetolactate synthesis (Als), catalyzes the decarboxylation of 2-ketoisovalerate like Kdc both in vivo and in vitro. AlsS is part of the acetoin synthesis pathway and catalyzes the aldo condensation of two molecules of pyruvate to 2-acetolactate (Als activity) (Fig. ​(Fig.1C)1C) (11). The overall reaction catalyzed by AlsS is irreversible because of CO₂ evolution. The first step in catalysis is the ionized thiazolium ring of TPP reacting with the first pyruvate, followed by decarboxylation. This intermediate then reacts with the second pyruvate. Deprotonation followed by C-C bond breakage produces 2-acetolactate. In this work, mutational approaches were used to assess the importance of Q487 in the Kdc activity of AlsS.