Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose

Abstract

Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose. We tested 12 different enzyme combinations to identify an n -butanol pathway with high titer and thermostability in C. thermocellum . The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum , the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n -butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum . The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold. Here we report engineering C. thermocellum to produce n- butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n- butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n- butanol titer by 2.2-fold. We further increased n -butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n -butanol from cellulose within 120 h.