Abstract
BackgroundBiofuel 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.ResultsWe 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.ConclusionsHere 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.
Highlights
Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products
Pathway combinations To find the best combination of pathway enzymes for thermophilic n-butanol production in C. thermocellum, we tested pathway genes from several different species and engineered strains
12 different combinations were constructed on plasmids (Fig. 2) and the native C. thermocellum promoter from gene Clo1313_2638 [32] was used to drive expression
Summary
Biofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. A common feature of these approaches is that they eliminate ferredoxin-linked enzymes, such as butyryl-CoA dehydrogenase/electron transfer protein (Bcd/EtfAB) and ferredoxin: NAD(P)+ oxidoreductase (Fnor), and use the Ter enzyme instead (Fig. 1b). This CoA-dependent pathway has allowed high levels (up to 30 g/L) of n-butanol production in E. coli [14, 15]. A third option for n-butanol production involves using the threonine biosynthesis pathway and/or the citramalate pathway to produce alpha-ketobutyrate, followed by decarboxylation and reduction to n-butanol [17] (Fig. 1c) Introducing this pathway into S. cerevisiae has allowed production of 835 mg/L n-butanol. In addition to these three pathways, which allow conversion of sugar to n-butanol, some organisms have a native ability to convert butyrate to n-butanol, and these organisms may be a source of enzymes for n-butanol production [18, 19]
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