Retooling A Bacterial Biofuel Factory

The native Clostridium pathway isn't optimized to make the most butanol possible, so it can be tweaked to increase output, says Chang, who also presented her team's work in the Division of Biological Chemistry. The pathway includes an enzyme that catalyzes a reversible double-bond reduction. To boost butanol output in E. coli, her team replaced the gene encoding this Clostridium enzyme with another gene, called ter, encoding trans-enoyl-coenzyme A reductase, which makes the reduction irreversible. This irreversibility prevents chemical intermediates from being diverted backward, away from butanol.

Different enzymes change reactions' activation energies—that is, the amount of energy input required to make a reaction go—by different amounts, Chang explained. Using the Ter enzyme makes it nearly impossible for the double-bond reduction to go in reverse because it eliminates an intermediate state. Energetically, the new setup means that going backward would require a far higher energy input than before, Chang explained. As a result, Chang's flasks of E. coli produced nearly 5 g of n-butanol per liter—about 10 times more than previous engineered E. coli but not yet comparable to levels from Clostridium.

Chang's work "serves as a reminder that there is a lot more to engineering a functional and well-performing pathway than just stitching the pathway genes together," comments Massachusetts Institute of Technology metabolic engineer Gregory N. Stephanopoulos.

At UCLA, Shen, Liao, and colleagues coupled the irreversible Ter reaction to two driving forces based on the enzyme cofactor NADH and acetyl coenzyme A. Clostridium's butanol biosynthesis requires a pool of acetyl coenzyme A as a starting material and a pool of NADH to drive various transformations, Liao explained. Bacteria produce both of these compounds, but other reactions consume them immediately, Liao said. So his team knocked out enzymes that divert the two feedstocks elsewhere and overexpressed a gene that produces NADH, thereby boosting E. coli's butanol production. The result was a yield of 15 g/L in flasks and 30 g/L in a fermentor, comparable to Clostridium's output.

"Everybody knows how to push equilibrium" in principle, Liao said. "But in this case it is surprisingly effective."

Liao's team used a 1-L bioreactor for their fermentation, a much smaller scale than would be used industrially. Economic factors play a role in whether butanol will become a viable fuel, Liao said. "But certainly our next step is to scale up."

Until now, butanol production levels from engineered microbes haven't been encouraging for industrial use, said MIT chemical engineer Kristala Jones Prather, who attended the meeting. The new work presented at the ACS meeting, she said, suggests that the approach has promise after all.