Synthetic biology tools for the construction of a highproducing 3-hydroxypropionic acid yeast strain

• Grant number: 23/00498-0
• 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: Alex Johan Mendes Comodaro
• 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

Abstract

The construction of microorganisms capable of producing renewable biochemicals is fundamental for the development of a sustainable bioeconomy. However, several factors still challenge the achievement of an efficient and competitive biosynthesis with processes derived from the petrochemical industry. The integration of heterologous pathways in microorganisms for the synthesis of non-native molecules often causes a significant metabolic burden, negatively impacting the growth and productivity of genetically modified strains. This obstacle has prevented the production of 3-hydroxypropionic acid (3HP) from abundant and renewable substrates such as sugar. Efforts carried out so far demonstrate that the integration of a heterologous pathway for 3HP production in Saccharomyces cerevisiae results in a final 3HP concentration of approximately 9 g/L, insufficient for large-scale production. This low production is accompanied by a reduction of about 50% in the growth rate of the engineered strains. This negative impact on growth is important for two main reasons. First, it indicates that the balance in expression of enzymes in the 3HP biosynthesis pathway is not optimized. Second, the reduction in the growth of producer cells results in a selective advantage for eventual mutants that lost the heterologous pathway. Mutant enrichment can cause a significant loss of productivity during the scale-up process for large-scale fermentation. This project will address these two challenges. In Objective 1, a combinatorial expression library will be performed to optimize the expression balance of 3HP pathway enzymes. It is expected that this strategy will result in a strain with higher productivity than reported in the literature. In Objectives 2 and 3, the production stability of the best strains generated will be evaluated in simulated large-scale fermentations. The main routes of mutations that lead to loss of productivity will be determined by sequencing the genome of mutant isolates. This information will be used for new rounds of engineering with the aim of increasing the genetic stability of the lineages. The end result will be obtaining a strain capable of producing 3HP at high concentrations and stable for large-scale production. (AU)