Metabolic Engineering of C. thermocellum to improve ethanol production

• Grant number: 24/01728-2
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: April 01, 2024
• End date: March 31, 2025
• Field of knowledge: Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
• Principal Investigator: Daniel Groban Olson
• Grantee: Maria Augusta Crivelente Horta
• 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

The lignocellulose-deconstruction capability of C. thermocellum has been shown to be decisively better than commercial cellulase preparations (Lynd et al., 2022). Combining this native deconstruction capability with ethanol production at commercially viable titers and yields requires development of genetic tools for thesenon-model microbes, better understanding of metabolic features of both C. thermocellum and higher-functioning ethanol pathways in other anaerobes, and using these tools and understanding to engineer C.thermocellum strains with improved ethanol production. Key metabolic engineering objectives will be pursued by this post doc in collaboration with other members of the A2G Biotechnology group. These include:1) Engineer C. thermocellum to get a thermodynamic push for ethanol production. This can be doneby finishing the implementation a "regular glycolytic pathway" and coupling it to the evolved T.saccharolyticum ethanol pathway consuming 1NADPH and 1 NADH. In a hydrogenase minus background, this would also require to have functional i) ferredoxin NADP+ reductase (potentiallyNfnB Wild Type or evolved) and ii) glycerol pathway.2) Make the strain more rigid in its metabolism. For example L-valine is normally not producedextracellularly by Clostridia because they do not possess an exporter for this amino acid. It might then be easy to avoid L-alanine and L-valine production by just deleting the genes encoding the exporters used by C. thermocellum. Lactate could be eliminated through the deletion of the lactate dehydrogenase and the methylglyoxal synthase encoding genes3) During the hydrolysis of unpretreated lignocellulosic biomass, there is a liberation of acetate. This acetate could be potentially converted to acetyl-CoA using the acetate producing pathway and then reduced to ethanol. This would require to work in a hydrogenase minus strain and have a functional oxidative pentose-phosphate pathway (to produce NADPH and CO2 from the oxidation of carbohydrates) and express a gluconeogenic-NADPH dependent glycerol-3-P dehydrogenese encoding gene to produce both the NADH and NADPH needed to reduce acetyl-CoA to ethanol.