Metabolic engineering of for -butanol production from cellulose.

Background: Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose.

Results: We tested 12 different enzyme combinations to identify an -butanol pathway with high titer and thermostability in . The best producing strain contained the thiolase-hydroxybutyryl-CoA dehydrogenase-crotonase (Thl-Hbd-Crt) module from , the trans-enoyl-CoA reductase (Ter) enzyme from and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from sp. X514 and was able to produce 88 mg/L -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 . 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.

Conclusions: Here we report engineering to produce butanol. Our initial pathway designs resulted in low levels (88 mg/L) of butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the butanol titer by 2.2-fold. We further increased -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 -butanol from cellulose within 120 h.