Genome-wide functional mapping of genes underlying 3HP tolerance in yeast

• Grant number: 23/00824-5
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: March 01, 2023
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Nádia Maria Vieira Sampaio
• Grantee: Thaís Tereza Aguiar dos Reis
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Associated research grant: 20/16759-0 - Synthetic biology tools for scalable production of biorenewable 3-hydroxypropionic acid, AP.JP
• Associated scholarship(s): 25/10188-4 - Improving 3-HP Production from Lignocellulosic Biomass Through User Defined Accelerated Evolution, BE.EP.DD

Abstract

The ability to engineer microbes for the production of bio-based chemicals is critical for achieving a sustainable economy. The platform chemical 3-hydroxypropionic acid (3HP) can be converted to a variety of valuable derivatives, such as 1,3-propanediol, acrylic acid, malonic acid and building blocks for biodegradable polymers. Accordingly, production of 3HP through fermentation of renewable carbon sources would represent a significant step towards achieving a more sustainable production route for a variety of products, such as paints, super absorbent polymers, coatings, adhesives and others. However, bio-based production of 3HP has not yet reached commercial scale. In order to achieve efficient bio-based production of 3HP it is fundamental to engineer robust microbes that display both high productivity and high tolerance to this cytotoxic organic acid. Laboratory adaptive evolution experiments have linked mutations in the gene SFA1 to 3HP resistance in yeast. Evolved strains were capable to tolerate up to 50 g/L 3HP (pH 3.5), however, titers in the range of 50-100 g/L are necessary to support economical fermentative production of most building block acids. Thus, additional strategies need to be devised in order to engineer strains with higher tolerance levels. The laboratory evolution approach works so that when a cell with a highly beneficial mutation emerges it rapidly enriches in the population masking the potentially important effect of additional genes to the phenotype of interest. This research project will apply the modern synthetic biology tool SATAY that enables the interrogation of the functional role of every gene in the yeast genome. First, we will screen a collection of >20 yeast strains to identify those that naturally display high levels of tolerance. We will then apply the SATAY technique to create libraries of strains where every non-essential gene is disrupted. The libraries will be exposed to toxic concentrations of 3HP and samples before and after the treatment will be sequenced. A gene enrichment analysis pipeline will then be performed to identify a complete set of genes involved in 3HP resistance. The function of a subset of the top hits identified will be confirmed experimentally and their expression will be manipulated in order to generate highly tolerant strains amenable to scale-up. (AU)