Development of inducible promoters for thermophiles

• Grant number: 24/06970-6
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: September 02, 2024
• End date: July 01, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Daniel Groban Olson
• Grantee: Edson Yu Sin Kim
• Supervisor: Adam Guss
• Host Institution: Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Institution abroad: Oak Ridge National Laboratory (ORNL), United States
• Associated to the scholarship: 22/16264-6 - Integrative Metabolic Engineering to Improve Ethanol Production by Thermophilic Bacteria: Technique Development, Cumulative Integration of Mutants, and Strain Characterization, BP.PD

Abstract

Inducible promoter systems are pivotal for precise control of gene expression, facilitating various biotechnological applications. However, the functionality of existing systems at high temperatures (>50°C) remains limited, posing challenges for thermophilic industrial processes. This project aims to address this gap by developing thermophilic inducible promoters with high dynamic range and low leakage. This research focuses primarily on Clostridium thermocellum, a lignocellulose-degrading anaerobe, and Thermoanaerobacterium saccharolyticum, both significant candidates for industrial biotechnology. We employ a systematic approach, including the development of a dual-selection system for promoter characterization, characterization of existing inducible promoters, and library-based selection to optimize the best performing systems. A key aspect of this project involves collaboration with the Guss lab at Oak Ridge National Laboratory (ORNL), responsible for developing many techniques related to thermophiles and non-model organisms, and also has consolidated much of the protocols and infrastructure needed for handling thermophilic anaerobes. The BEPE component is essential for acquiring methodologies like high temperature anaerobic enzymatic assay design and CRISPR genome editing protocols tailored for thermophilic bacteria. The work plan encompasses screening assays, promoter library generation, transformation, and high throughput screening, alongside learning to handle T. saccharolyticum and implementing the optimized promoters in proof-of-concept experiments. We will also attempt to implement SAGE techniques in the mentioned organisms Methodologies involve plasmid cloning, promoter library generation, strain selection, and gene expression analysis through RNA extraction, qPCR, and enzymatic assays. The project's success will advance industrial biotechnology by providing tailored tools for precise gene expression control in thermophilic organisms.