Fatty Acid Decarboxylase Engineering for Continuous Hydrocarbon Fuel Production

Fatty acids are a primary candidate for the production of third generation hydrocarbon biofuels. However, major issues associated with fatty acid production will severely limit their technical viability as a targeted intermediate, including the need for a high temperature hydrotreating step for deoxygenation to fuel blendstocks coupled to the inability of most organisms to secrete fatty acids during fermentation. A potential means to simultaneously overcome both of these problems involves the application of in vivo decarboxylase enzymes, which directly convert fatty acids to alkenes. Given their hydrophobic nature, these alkenes are then able to be secreted by most organisms through passive transport mechanisms and are directly separable from fermentation broths through phase separation. Upgrading of alkenes to alkanes through hydrotreating can be conducted near room temperature. Despite this promise, the newly discovered class of decarboxylase enzymes does not yet exhibit sufficient catalytic activity for viable industrial application.

We improved the activity of these decarboxylase enzymes via a three pronged approach. We investigated the mechanism of decarboxylase enzymes using cutting edge quantum mechanical calculations with the aim to understand the rate limiting steps in decarboxylation. In parallel, we developed and implemented a high throughput evolutionary strategy for experimentally improving enzyme activity. Future work will be directed at employing structural biology and biophysical assays on the improved variants produced via an evolutionary approach to understand the mechanistic basis for the enzyme activity improvements.