Rice University engineers reverse ubiquitous metabolic pathway to enable speedy microbial production of fuels, chemicals
|Comparison of n-alcohols synthesis via the fatty acid biosynthesis pathway (top) and the engineered reversal of the β-oxidation cycle (bottom). Dellomonaco et al. Click to enlarge.|
Rice University engineering researchers have developed a new method for rapidly converting glucose into biofuels and renewable petrochemical substitutes by reversing the ubiquitous β-oxidation metabolic pathway in bacteria. The work is reported in the journal Nature.
Species ranging from single-celled bacteria to humans use β-oxidation to break down fatty acids and generate energy. Ramon Gonzalez, associate professor of chemical and biomolecular engineering at Rice and lead co-author of the Nature study, and his team reversed the β-oxidation cycle by selectively manipulating about a dozen genes in the bacteria Escherichia coli—i.e., essentially reversing the process cells use to break apart fatty acids. On a cell-per-cell basis, the bacteria produced butanol about 10 times faster than any previously reported organism.
Advanced (long-chain) fuels and chemicals are generated from short-chain metabolic intermediates through pathways that require carbon-chain elongation. The condensation reactions mediating this carbon–carbon bond formation can be catalysed by enzymes from the thiolase superfamily, including β-ketoacyl-acyl-carrier protein (ACP) synthases, polyketide synthases, 3-hydroxy-3-methylglutaryl-CoA synthases, and biosynthetic thiolases. Pathways involving these enzymes have been exploited for fuel and chemical production, with fatty-acid biosynthesis (β-ketoacyl-ACP synthases) attracting the most attention in recent years.
Degradative thiolases, which are part of the thiolase superfamily and naturally function in the β-oxidation of fatty acids, can also operate in the synthetic direction and thus enable carbon-chain elongation. Here we demonstrate that a functional reversal of the β-oxidation cycle can be used as a metabolic platform for the synthesis of alcohols and carboxylic acids with various chain lengths and functionalities.
This pathway operates with coenzyme A (CoA) thioester intermediates and directly uses acetyl-CoA for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA), characteristics that enable product synthesis at maximum carbon and energy efficiency. The reversal of the β-oxidation cycle was engineered in Escherichia coli and used in combination with endogenous dehydrogenases and thioesterases to synthesize n-alcohols, fatty acids and 3-hydroxy-, 3-keto- and trans-Δ2-carboxylic acids.
The superior nature of the engineered pathway was demonstrated by producing higher-chain linear n-alcohols (C≥4) and extracellular long-chain fatty acids (C>10) at higher efficiency than previously reported. The ubiquitous nature of β-oxidation, aldehyde/alcohol dehydrogenase and thioesterase enzymes has the potential to enable the efficient synthesis of these products in other industrial organisms.—Dellomonaco et al.
The team also showed that selective manipulations of particular genes could be used to produce fatty acids of particular lengths, including long-chain molecules like stearic acid and palmitic acid, which have chains of more than a dozen carbon atoms.
This is not a one-trick pony. We can make many kinds of specialized molecules for many different markets. We can also do this in any organism. Some producers prefer to use industrial organisms other than E. coli, like algae or yeast. That’s another advantage of using reverse-beta oxidation, because the pathway is present in almost every organism.—Ramon Gonzalez
Clementina Dellomonaco, James M. Clomburg, Elliot N. Miller & Ramon Gonzalez (2011)Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals. Nature doi: 10.1038/nature10333