Structure-function relationships investigation of active dehydrogenases on aromatics: unraveling and optimizing enzymatic mechanisms for vanillin production from renewable sources

• Grant number: 22/01070-1
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: October 01, 2022
• Status: Discontinued
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Priscila Oliveira de Giuseppe
• Grantee: Anna Julyana Viana Chianca Brilhante
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Associated scholarship(s): 24/03956-2 - Strategic construction of a gene library for enzyme-directed evolution/semi-rational design: towards substrate recognition comprehension and enhanced enzyme specificity, BE.EP.DR

Abstract

Vanillin is a molecule that ranks as the most used flavoring agent in the world. Currently, petroleum is vanillin's primary raw material. However, the growing demand for natural vanillin, the appeal for renewable sources, and the disadvantages associated with current vanillin production processes have driven the search for alternative production routes. The production of vanillin from renewable molecules, such as eugenol, is one of the most promising alternatives for the biosynthesis of vanillin on an industrial scale. However, the knowledge and development of enzymatic technologies for this application are still incipient. In a previous project, we characterized two dehydrogenases with potential application in the bioconversion pathway of eugenol to vanillin. XarA is an aryl alcohol dehydrogenase with high specificity for coniferyl alcohol, converting it to coniferaldehyde (step 2 of the eugenol-to-vanillin pathway) and XarB is an aldehyde dehydrogenase that converts coniferaldehyde to ferulic acid (step 3 of the pathway). Although XarA has high specificity for coniferyl alcohol (~100 times greater than other substrates tested), XarB is less specific, showing specificity constants in the same order of magnitude for the substrates coniferaldehyde and vanillin. If, on the one hand, the low selectivity of XarB makes its application in the eugenol-to-vanillin pathway unfeasible, since it is also active on the product of interest, on the other hand, this makes it a great model for rational engineering studies to increase its specificity to the desired substrate. Considering that coniferyl alcohol and coniferaldehyde retain the same structure, except for the difference of a hydrogen in the aliphatic tail, our working hypothesis is that knowing the mechanism that confers high specificity for XarA in the use of coniferyl alcohol, this can guide us to optimize the specificity of XarB over coniferaldehyde. However, there are still large gaps about the molecular mechanisms that determine the specificity of enzymes with this type of activity. Coniferyl alcohol dehydrogenase or coniferaldehyde dehydrogenase structures are not yet available in the Protein Data Bank. Biochemical data on the specificity of homologous enzymes are also very scarce. Thus, in this project, we aim to unravel the molecular basis of the specificity of coniferyl alcohol dehydrogenases and coniferaldehyde dehydrogenases by combining enzymology, X-ray crystallography, site-directed mutation and computational biology approaches (such as molecular dynamics and docking). As study models we will use the enzymes XarA and XarB, as well as the enzymes commonly used in the engineering of microorganisms for the production of vanillin from eugenol (CalA and CalB from Pseudomonas sp. HR199). As a proof of concept for the application of this knowledge in rational engineering strategies, we will use XarB as an enzymatic framework and try to optimize its specificity to coniferaldehyde. In this way, we will fill, with this project, knowledge gaps about the mechanisms that govern the specificity of aryl-alcohol and aryl-aldehyde dehydrogenases and we will investigate the applicability of this knowledge in the modulation of the specificity of aryl-aldehyde dehydrogenases for industrial applications, using in this case, the route of production of vanillin from eugenol as an example. (AU)