Effects of alcohol dehydrogenase enzyme cofactor specificity on ethanol tolerance and production in ethanologenic strains of the thermophilic bacteria T. saccharolyticum

• Grant number: 25/00691-0
• Support Opportunities: Scholarships abroad - Research
• Start date: September 01, 2025
• End date: August 31, 2026
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Luana Walravens Bergamo
• Grantee: Luana Walravens Bergamo
• Host Investigator: Lee Howard Lynd
• Host Institution: Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Institution abroad: Dartmouth College, United States
• Associated research grant: 18/25682-0 - Advanced second generation biofuels laboratory, AP.BIOEN.SPEC

Abstract

Clostridium thermocellum is a promising candidate organism for producing biofuels from biomass due to its strong native ability to consume cellulose but requires improved ethanol production to be commercially useful. Ethanol production can be improved by metabolic engineering, knocking out genes and/or heterologous expression of genes and pathways from thermophilic bacteria with high-functioning ethanol pathways such as engineered strains of Thermoanaerobacterium saccharolyticum. For that reason, deeply understanding the ethanol production pathway of T. saccharolyticum is extremely important. Although many advances have been achieved in the last years, some gaps still persist, such as the functioning genes responsible for acetaldehyde to ethanol production, known as alcohol dehydrogenases. Different strains of T. saccharolyticum use different nicotinamide cofactors (i.e. NAD+ and NADP+), and the impact of cofactor use on ethanol tolerance and ethanol production is not completely understood. The present proposal aims to address this question, focusing on one strain that has NADPH-linked ADH activity (i.e. LL1049) and other that has NADH-linked ADH activity (i.e. LL1145). Fermentation, ethanol tolerance, Adaptive Laboratory Evolution (ALE), enzymatic assay and genetic engineering experiments will be conducted. The expertise and infrastructure of the Lynd Lab, where the "Bolsa de Pesquisa no Exterior" (BPE) will be held, will allow these experiments to be carried out under the supervision of the leading experts in the field, allowing subsequent technology transfer to Brazil. It is anticipated that this project will complement other studies under development in our research group at the A2G lab and will generate publications in the near future. Considering both energy and food demand, this proposal is aligned with multiple UN Sustainable Development Goals (SDGs), notably including poverty alleviation, affordable and clean energy, and climate action.