Production of advanced biofuels in engineered E. coli.

Commercial fermentation processes have long taken advantage of the synthetic power of living systems to rapidly and efficiently transform simple carbon sources into complex molecules. In this regard, the ability of yeasts to produce ethanol from glucose at exceptionally high yields has served as a key feature in its use as a fuel, but is also limited by the poor molecular properties of ethanol as a fuel such as high water miscibility and low energy density. Advances in metabolic engineering and synthetic biology allow us to begin constructing new high-flux pathways for production of next generation biofuels that are key to building a sustainable pipeline for liquid transportation fuels.

Biochemical and structural characterization of the trans-enoyl-CoA reductase from Treponema denticola.

The production of fatty acids is an important cellular pathway for both cellular function and the development of engineered pathways for the synthesis of advanced biofuels. Despite the conserved reaction chemistry of various fatty acid synthase systems, the individual isozymes that catalyze these steps are quite diverse in their structural and biochemical features and are important for controlling differences at the cellular level. One of the key steps in the fatty acid elongation cycle is the enoyl-ACP (CoA) reductase function that drives the equilibrium forward toward chain extension. In this work, we report the structural and biochemical characterization of the trans-enoyl-CoA reductase from Treponema denticola (tdTer), which has been utilized for the engineering of synthetic biofuel pathways with an order of magnitude increase in product titers compared to those of pathways constructed with other enoyl-CoA reductase components. The crystal structure of tdTer was determined to 2.00 Å resolution and shows that the Ter enzymes are distinct from members of the FabI, FabK, and FabL families but are highly similar to members of the FabV family. Further biochemical studies show that tdTer uses an ordered bi-bi mechanism initiated by binding of the NADH redox cofactor, which is consistent with the behavior of other enoyl-ACP (CoA) reductases. Mutagenesis of the substrate binding loop, characterization of enzyme activity with respect to crotonyl-CoA, hexenoyl-CoA, and dodecenoyl-CoA substrates, and product inhibition by lauroyl-CoA suggest that this region is important for controlling chain length specificity, with the major portal playing a more important role for longer chain length substrates.

Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways.

Living systems have evolved remarkable molecular functions that can be redesigned for in vivo chemical synthesis as we gain a deeper understanding of the underlying biochemical principles for de novo construction of synthetic pathways. We have focused on developing pathways for next-generation biofuels as they require carbon to be channeled to product at quantitative yields. However, these fatty acid-inspired pathways must manage the highly reversible nature of the enzyme components. For targets in the biodiesel range, the equilibrium can be driven to completion by physical sequestration of an insoluble product, which is a mechanism unavailable to soluble gasoline-sized products. In this work, we report the construction of a chimeric pathway assembled from three different organisms for the high-level production of n-butanol (4,650 ± 720 mg l⁻¹) that uses an enzymatic chemical reaction mechanism in place of a physical step as a kinetic control element to achieve high yields from glucose (28%).