Enzyme Prospecting and Evolutionary Engineering to Overcome the Guaiacol Demethylation Bottleneck in Pseudomonas putida KT2440

• Grant number: 25/04392-8
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: July 01, 2025
• End date: June 30, 2029
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Priscila Oliveira de Giuseppe
• Grantee: Gabriel Gonçalves Dias
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Associated research grant: 23/11786-7 - Investigation and rational engineering of molecular mechanisms for the bioconversion of aromatic compounds, AP.R

Abstract

Lignin is the second most abundant biopolymer in the world and an important source of renewable aromatic compounds, with potential to generate energy and various types of materials. After the fragmentation process of this heteropolymer, a large heterogeneity of molecules is observed, which hinders its full utilization. To facilitate lignin valorization, a promising strategy is biological funneling, which involves the use of microorganisms to convert a wide variety of molecules into a central compound, thus making it easier to purify and transform it into industrially relevant products. Guaiacol and its chemical derivatives are aromatic compounds abundant in liquors resulting from the fragmentation of lignins rich in Guaiacyl units (G unit), and O-demethylation of these molecules is a key strategy to convert them into platform chemicals. Although an important component of industrial lignins, guaiacol is still underexplored as a substrate in biological conversion processes, mainly due to the lack of efficient O-demethylase enzymes for its in vivo demethylation in the main microbial platforms for converting lignin-derived compounds, such as Pseudomonas putida KT2440. In this sense, this project aims to apply a multidisciplinary approach, including the prospecting of enzymes from the Brazilian biodiversity, laboratory adaptive evolution, and reverse engineering, to obtain new P. putida KT2440 strains efficient for guaiacol demethylation. It is expected to deepen the understanding of the molecular mechanisms that favor guaiacol demethylation in vivo, thus contributing to the advancement of new biotechnologies that promote lignin valorization as a sustainable raw material.