Application of CRISPR/Cas9-based tools to develop a Saccharomyces cerevisiae strain capable of producing 4-vinylguaiacol from lignocellulose hydrolysates

• Grant number: 20/10593-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: December 01, 2020
• End date: November 30, 2021
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Fábio Márcio Squina
• Grantee: Nathália Vilela
• Supervisor: Kevin Verstrepen
• Host Institution: Pró-Reitoria de Pós-Graduação, Pesquisa, Extensão e Inovação. Universidade de Sorocaba (UNISO). Sorocaba , SP, Brazil
• Institution abroad: University of Leuven, Leuven (KU Leuven), Belgium
• Associated to the scholarship: 17/08166-6 - Genetic engineering of yeast for conversion lignocellulosic material to ferulic acid, BP.DR

Abstract

Aroma compounds represent a substantial fraction of the world's market for food additives and are mainly produced by petroleum-derived chemical synthesis. There is an increasing demand to replace compounds derived from fossil fuels; thus, renewable lignocellulosic biomass can be an attractive alternative for aroma production. In this sense, the physicochemical pretreatment is an important step to reduce biomass recalcitrance and facilitate further processing of plant lignocellulose into bioproducts. The alkaline deacetylation of lignocellulose is a mild pre-treatment that can remove most lignocellulose phenolic groups, including ferulic acid. This hydroxycinnamic acid, besides its industrial relevance, plays a role in the formation of the aroma compound 4-vinylguaiacol (4-VG), which has broad applicability in medicine, food, perfumery, and cosmetic industries. Several microorganisms are capable of decarboxylating ferulic acid into 4-VG using a cofactor-free enzyme, the phenolic acid decarboxylase. Differently, in Saccharomyces cerevisiae the ferulic acid decarboxylase (Fdc1) requires a cofactor, a modified flavin mononucleotide (FMN), that is produced by the phenylacrylic acid decarboxylases (Pad1). Two independent strategies have been described to improve the 4-VG production by S. cerevisiae: i) the overexpression of the endogenous PAD1 gene, which improves the cofactor recycling; ii) the heterologous expression of the cofactor free enzyme, a phenolic acid decarboxylase. In this project, we intend to apply CRISPR/Cas9-based tools to develop an industrial S. cerevisiae strain capable of converting ferulic acid (from lignocellulosic hydrolysates) into 4-VG at high yields. Thereby, using CRISPR/Cas9 tools, the gene coding the cofactor free phenolic acid decarboxylase from the lignolytic yeast Rhodosporidium fluviale, will be integrated in an industrial S. cerevisiae strain. Furthermore, the ABC transporters from R. fluviale will be expressed in the mutant S. cerevisiae strain, aiming to improve its robustness related to the conversion of ferulic acid (derived from lignocellulosic biomass hydrolysate) into 4-VG. Kevin Verstrepen's group has expertise in the genetic modification in S. cerevisiae, along with a large collection of yeasts used in the industry. His group has recently developed a synthetic biology tool, using CRISPR/Cas9 strategy, for rapidly shuffling yeast promoters in front of desired pathway genes to balance the genetic expression, that will be applied in this project. (AU)