Identification of Mechanistic Targets for Optimization of a Cyanobacterial Hydrocarbon Production Bioprocess

Abstract

Cyanobacteria utilize sunlight to fix carbon dioxide to form biomolecules such as fatty acids. These microbes can convert fatty acids into diesel fuel hydrocarbons via a two‐enzyme pathway, which has been targeted for application in renewable fuel bioprocesses. The first enzyme, acyl‐acyl carrier protein reductase (AAR), catalyzes a two‐electron, NADPH‐dependent reduction of its C n ‐fatty acyl‐ACP substrate to yield a C n ‐fatty aldehyde. This reaction provides the substrate for the second enzyme, aldehyde‐deformylating oxygenase (ADO), which catalyzes the oxygenative conversion of the fatty aldehyde into the corresponding C n‐1 ‐alkane and formate. To gain insight into how to improve product turnover of the ADA enzyme, kinetic and spectroscopic techniques were used. This revealed an O 2 ‐based unproductive pathway that compromises alkane yields. Time dependent traces showed that under high O 2 conditions a delay of only 30s was necessary for maximum intermediate formation, while for the low O 2 conditions it took the intermediate 50s to generate. Mix Freeze‐Quench experiments were performed under analogous conditions for the stopped‐flow experiment, but once the intermediate was formed, it was reacted with reduced ferredoxin and frozen in a cryosolvent. Electron Paramagnetic Resonance spectra showed formation of the peroxyl radical under high and low O 2 conditions. Under high O 2 conditions more peroxyl radical was formed and less alkane product, but the alkane product can be recovered under low O 2 conditions. This suggests that ADO requires O 2 , however under conditions of excessive O 2 alkane formation is inhibited. Future efforts in bioprocesses should focus on avoiding this unproductive pathway to maximize alkane production. Through investigation of ADO catalysis, opportunities for optimization of this pathway have been identified to make it a viable option for the production of renewable biofuels.