Elucidation of the mechanisms of non-conventional peroxygenases with application in the production of advanced biofuels

• Grant number: 19/12599-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Effective date (Start): August 01, 2020
• Effective date (End): April 08, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Leticia Maria Zanphorlin
• Grantee: Mayara Chagas de Ávila
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovações (Brasil). Campinas , SP, Brazil

Abstract

Biological strategies, through the discovery of new enzymes, added to the Microbial Engineering to promote the biomass use in advanced biofuel production and/or other bioproducts is certainly a more sustainable and promisor way to complement the technologies based in petroleum. The biological approach, in addition to having a lower environmental impact, because allows the use of mild process conditions as temperature and pressure, and less toxic chemical amounts, it also allows the use of enzymes that present higher specificity and high conversion index in the bioprocess. In this sense, the discovery of the enzyme peroxygenase/decarboxylase CYP152 OleTJE made the scientific and technologic interesting in this enzyme family to increase significantly, because of the huge industry and economic appeal of the generated product, 1- alkene, which presents chemical composition and physical characteristic similar to the conventional petroleum fuels. The decarboxylation activity of the long chain fatty acids (C12 - C20) promoted by this enzymatic family could contribute to solving one of the biggest hindrances of the drop-in biofuel production since it promotes the removal of the oxygen molecule of the chain. However, very little is known about peroxygenases from family 152, and one of the big challenges is to obtain enzymes that have preferential decarboxylation activity instead of hydroxylation, considering that CYP152 enzymes can catalyze both reactions. Recently, its notorious importance encouraged Wang and colleagues (2017) publish the article "Peroxygenases en route to becoming dream catalysts. What are the opportunities and challenges?", which levers the use of peroxygenases in much biotechnology applications. Our research group, through bioinformatics strategies, identified an innovative peroxygenases from Rothia nasimurium and Nosocomiicoccus sp bacteria (32 and 73% of identity with OleTJE, respectively), which were predicted with decarboxylation activity to a deep investigation of its molecular, functional, and structural mechanisms. Thus, this doctoral project aims to answer questions that together will be able to support the use of peroxygenases to produce biofuels, as (i) the identified peroxygenases are hydroxylases or decarboxylases?; (ii) which are the molecular components that drive to the formation of alkenes or not, to be applied in the industry?; (iii) which is the specificity in terms of fatty acid size?; (iv) if they are not decarboxylases, which mutations could we propose to transform them? To answer these questions, a combination of structural, spectroscopic, and biochemical methods will be used, which are already widely applied in our laboratory. By the end of this doctoral project, we plan, beyond increase the arsenal of peroxygenases with the activity of decarboxylation of long chain fatty acid, to propose a biological alternative to produce hydrocarbons, which are precursors to the biodegradable synthesis, for example, the green diesel and the aviation biokerosene. (AU)