Evaluation of TesB Enzymes improved by directed evolution to drive metabolic intermediates of Pseudomonas sp. LFM046 towards the production of 3-hydroxyacids

• Grant number: 21/11068-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2022
• End date: February 28, 2023
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: José Gregório Cabrera Gomez
• Grantee: Vital Pegorin Oliveira
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

3-hydroxyacids (3HA) are intermediary compounds produced by many organisms, among them are the bacteria from the Pseudomonas genus, which serve as monomers in the metabolic pathway to produce polyhydroxyalkanoates (PHA), a biopolymer with potential use as bioplastic, as well as rhamonolipids (RL), a biosurfactant. On these pathways, 3HA is found to bound to coenzyme A (CoA) or acyl carrier protein (ACP) by a thioester bond, preventing this compound to be secreted from the cell; in order to obtain a high concentration of this molecule in the medium, it is necessary to break this bond, enabling it to leave the cell. This project proposes to use Pseudomonas mutants, unable to polymerize PHA or synthesize RL, testing thioesterase enzymes (TesB) from two different bacterial strains (Escherichia coli and Pseudomonas putida). One of these genes will be selected and submitted to direct evolution experiments searching for evolved enzymes presenting higher efficiency or peculiar specificities (ACP or CoA, different 3HA). The 3HAs present in PHA or RL is always in the "R" configuration, being considered important precursor molecules for the biosynthesis of biologically active compounds. In addition, they have also been considered important components of the ketogenic feed.(AU)