Acetolactate synthase from Bacillus subtilis serves as a 2-ketoisovalerate decarboxylase for isobutanol biosynthesis in Escherichia coli

Abstract

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.

FIG. 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.

FIG. 2.

Summary of results for isobutanol production without Kdc in E. coli. The cells were grown in M9 medium containing 5 g/liter yeast extract and 36 g/liter glucose in shake flasks at 30°C with 0.1 mM IPTG for 24 h. Overexpressed and deleted genes and KIV supplementation are indicated below the graphs. (A) Isobutanol production using various enzymes. (B and C) Isobutanol production with the supply of KIV. ND, not detectable.

FIG. 3.

Effect of Q487 on the decarboxylase activity of AlsS. (A) Isobutanol production with the AlsS variants. The cells were grown in M9 medium containing 5 g/liter yeast extract and 36 g/liter glucose in shake flasks at 30°C with 0.1 mM IPTG for 24 h. (B and C) Specific growth rate of E. coli strain KS145 (ΔilvI ΔilvB) with the AlsS variants in M9 medium containing 1% glucose and 39.5 μg·ml⁻¹ l-isoleucine (B) and 35 μg·ml⁻¹ l-valine and 39.5 μg·ml⁻¹ l-leucine (C). The ilvC and ilvD genes were overexpressed with the alsS gene.