Genetic tool development for Clostridium thermocellum

• Grant number: 24/00350-6
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: March 01, 2024
• End date: February 28, 2026
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Daniel Groban Olson
• Grantee: Fernanda Souza Lopes
• Host Institution: Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 18/25682-0 - Advanced second generation biofuels laboratory, AP.BIOEN.SPEC

Abstract

This postdoctoral project focuses on improving genetic tools for Clostridium thermocellum, a bacterium renowned for its superior lignocellulose degradation capabilities compared to commercial cellulase preparations. The goal is to merge this native deconstruction ability with ethanol production at commercially viable levels, necessitating the development of enhanced genetic tools, a profound understanding of C. thermocellum's metabolic features, ethanol pathways in anaerobes, and the application of these tools to engineer improved strains.The primary focus will be on the development of genetic tools, including inducible systems to control gene expression, an effective CRISPR/Cas system for genomic editing, and a conditional plasmid system to facilitate plasmid loss. The methodology is based on analogous tools developed at SBRC Nottingham, particularly in Parageobacillus thermoglucosidasius.For inducible systems, theophylline-responsive riboswitches, previously utilized in Clostridium, will be evaluated. The short length (84 nt) of the riboswitch allows inducible control when added to any promoter. Different promoters of C. thermocellum will be tested, with nucleotide adjustments to modulate stem: loop structure and enhance the dynamic range.Developed promoter-riboswitches will be linked to the expression of nuclease genes suitable for genomic editing. Cas9 from S. thermophilus, a thermophile, will be tested alongside other thermophilic enzymes. Inducible systems will also be utilized to develop conditionally replicating plasmids through modulation or interference in the replication process, or to control a toxin/antitoxin (TA/AT) system demonstrated in P. thermoglucosidasius, inducing plasmid loss as necessary selection.