Strategic construction of a gene library for enzyme-directed evolution/semi-rational design: towards substrate recognition comprehension and enhanced enzyme specificity

• Grant number: 24/03956-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: July 20, 2024
• End date: July 19, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Priscila Oliveira de Giuseppe
• Grantee: Anna Julyana Viana Chianca Brilhante
• Supervisor: Peter Kast
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Institution abroad: Swiss Federal Institute of Technology Zurich, Switzerland
• Associated to the scholarship: 22/01070-1 - Structure-function relationships investigation of active dehydrogenases on aromatics: unraveling and optimizing enzymatic mechanisms for vanillin production from renewable sources, BP.DR

Abstract

Enzyme engineering is a highly valuable tool for optimizing processes in the field of biocatalysis. Among the current enzyme engineering strategies, directed evolution and semi-rational design emerge as powerful approaches, compared to rational design, when there is limited information on the structure-function relationships of the target enzyme. Moreover, these approaches enable the faster evaluation of comprehensive libraries, making them highly favorable for addressing complex challenges such as modulating the specificity of an enzyme aiming to reduce its substrate promiscuity. In this project, we propose to apply directed evolution and semi-rational design to optimize the enzyme CalB, a coniferaldehyde dehydrogenase from Pseudomonas sp. HR199 that can be utilized in the eugenol-to-vanillin bioconversion pathway. Despite this potential application, CalB - that acts in the intermediary step from coniferaldehyde to ferulic acid - also recognizes the target product, vanillin, as a substrate. While this characteristic currently limits CalB application, trying to modulate its substrate specificity through enzyme engineering could enable its use in sustainable and efficient vanillin production. Nevertheless, directing CalB's specificity towards coniferaldehyde and also mitigate its activity against vanillin is a challenging task to be optimally achieved through rational design alone, given the structural similarity of vanillin and coniferaldehyde. Facing these challenges, as well as the absence of high-resolution structures for dehydrogenases that are active on coniferaldehyde/vanillin, and the difficulty in obtaining the crystallographic structure of CalB, the establishment of a gene library for directed evolution and semi-rational enzyme design emerges as a pivotal strategy. We expect that screening a significantly larger number of variants will greatly enhance the prospects of acquiring a promising variant with heightened activity towards coniferaldehyde while rejecting vanillin as a substrate. At the same time, such experiments should yield a better understanding of structure-function relationships that govern substrate selection by enzymes. Additionally, this BEPE proposal encompasses both a deliverable that will contribute to the doctoral project and an opportunity for a comprehensive learning experience, providing the necessary skills to enhance our group's expertise by incorporating knowledge of the directed enzyme evolution technique.