Biocatalytic conversion of fatty acids to olefins through a solvent-free chimeric decarboxylase biofluid system

• Grant number: 22/13956-4
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: October 01, 2023
• End date: April 30, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Leticia Maria Zanphorlin
• Grantee: Mayara Chagas de Ávila
• Supervisor: Alex Brogan
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Institution abroad: King's College London, England
• Associated to the scholarship: 19/12599-0 - Elucidation of the mechanisms of non-conventional peroxygenases with application in the production of advanced biofuels, BP.DD

Abstract

Since the industrial era, a large number of greenhouse gas (GHG) emissions into the atmosphere have been a concerning reality because of fossil fuels usage, the most used energy source worldwide. The development of renewable energy technologies from different sources is growing, meanwhile, they are part of the solution to reduce GHG emissions. Production of advanced biofuels, also called drop-in biofuels, such as butanol, alkanes, and alkenes, can be an excellent alternative to meet this demand. Drop-in biofuels are a mixture of hydrophobic bio hydrocarbons with high energy potential, which makes them chemical and physically similar to petroleum-based fuels. Nowadays, drop-in biofuels are produced by chemical processes such as hydrogenation, which requires very high temperatures and metal catalysts, and is not environment-friendly. Biological routes, such as enzymes, are applied in processes with mild conditions resulting in a more ecologically and friendly option. Considering the importance of finding a way to synthesize bio hydrocarbons through an enzymatic route from low-cost substrates, as this alternative would result in a drop-in biofuels production from a specific, efficient, and sustainable process, our research group has dedicated the last three years to studying the enzymes from the P450 superfamily, which have an efficient activity to produce alkenes from fatty acids. More specifically, we are concentrated in the enzymes from the 152 family (CYP152), which evolved into a reaction mechanism named peroxide shunt, able to use hydrogen peroxide (H2O2) as the source of oxygen and an oxidizing agent (hence called peroxygenases). Recent studies of peroxygenases showed that excess H2O2 can either prejudice the biocatalysis or completely denatured the enzyme. Therefore, it is important to control the H2O2 supply in the reaction. A breakthrough in the field seeking to mediate H2O2 addition in the reaction has been proposed in the literature, combining alditol oxidases (AldO) with decarboxylases. AldO enzymes have the capability of oxidazing glycerol and produce H2O2 as a co-product. Thus, the fusion of those enzymes allows the controlled H2O2 supply in the decarboxylation reaction of free fatty acids (FFA), avoiding oxidative inactivation and improving alkene production. Our research group has identified a new cytochrome P450-based mechanism from Rothia nassimurium (OleTRN) that preferably decarboxylates a range of saturated and unsaturated substrates. Because of the successful combination of oxidases with decarboxylases, the chimeric OleTRN-AldO was constructed, heterologously produced, and proved to produce alkenes in the presence of glycerol. Although enzymes from the 152 family can be a sustainable solution for drop-in production, the need to operate in an aqueous environment can be a restraint for industrial application. Solvent-free liquid proteins are a recent biomaterial technology developed by Dr. Brogan's group. Those materials have presented excellent enzyme activities in anhydrous conditions and allow reactions in ionic liquids. Ionic liquids present many advantages, such as non-volatility, non-flammability, the high solubility of recalcitrant substrates, and excellent chemical and thermal stability, which makes them a more sustainable option when compared with organic solvents. Dr. Alex Brogan coordinates his research group at King's College London (London, England, UK) and has been working in this field for more than 10 years. Considering all the exceptional results that Dr. Brogan obtained in developing solvent-free proteins to expand their activity significantly into ionic liquids, this current project intends to apply these techniques with the chimeric enzyme OleTRN-AldO. We pursue to provide a robust and sustainable alternative, applicable to industry conditions, to produce drop-in biofuels from fatty acids. (AU)