Metabolic engineering of Clostridium cellulolyticum for production of isobutanol from cellulose

Abstract

Producing biofuels directly from cellulose, known as consolidated bioprocessing, is believed to reduce costs substantially compared to a process in which cellulose degradation and fermentation to fuel are accomplished in separate steps. Here we present a metabolic engineering example for the development of a Clostridium cellulolyticum strain for isobutanol synthesis directly from cellulose. This strategy exploits the host's natural cellulolytic activity and the amino acid biosynthesis pathway and diverts its 2-keto acid intermediates toward alcohol synthesis. Specifically, we have demonstrated the first production of isobutanol to approximately 660 mg/liter from crystalline cellulose by using this microorganism.

Fig. 1.

The pathway for isobutanol production in C. cellulolyticum (A) and the ferredoxin promoter (black arrow)-driven operons used in this study (B). The asterisk indicates the adenine insertion in the alsS gene sequence.

Fig. 2.

The first 120 bp of the alsS sequence with the adenine insertion mutation. The adenine insertion (solid box), the putative start GTG, which restores the alsS reading frame (underline), the premature stop codon (*), and the putative Shine-Dalgarno sequence (dashed box) are indicated.

Fig. 3.

Activity assays of isobutanol pathway enzymes for C. cellulolyticum strains expressing the empty vector, *alsS ilvCD kivd adhA, and kivd yqhD alsS ilvCD, determining the activity for AlsS (one specific unit of Als activity corresponds to the formation of 1 nmol of acetoin per min per mg of soluble protein at 34°C) (A), IlvC (one specific unit of IlvC activity corresponds to the oxidation of 1 nmol of NADPH per min per mg of soluble protein at 34°C) (B), IlvD (one specific unit of IlvD activity corresponds to the formation of 1 nmol of 2-ketoisovalerate per min per mg of soluble protein at 34°C) (C), Kivd (one specific unit of Kivd activity corresponds to the oxidation of 1 nmol of NADPH per min per mg of soluble protein at 34°C) (D), and AdhA and YqhD activity [one specific unit of ADH activity corresponds to the oxidation of 1 nmol of NAD(P)H per min per mg of soluble protein at 34°C] (E).

Fig. 4.

AlsS activity of E. coli and C. cellulolyticum expressing the vector, the *alsS ilvCD construct, or the alsS ilvCD construct. One specific unit of AlsS activity corresponds to the formation of 1 nmol of acetoin per min per mg of soluble protein at 34°C.

Fig. 5.

Growth of C. cellulolyticum strains on cellobiose (A) and cellulose (B) and the isobutanol production (mg/liter) on cellobiose (C) and cellulose (D). The figure shows one data set representative of three independent experiments, with all three showing comparable results.