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Pathway improvement for ethanol production by thermophilic bacteria involving pyruvate decarboxylase: library development and screening

Grant number: 22/05802-7
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): June 01, 2022
Effective date (End): September 14, 2022
Field of knowledge:Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
Principal researcher:Daniel Groban Olson
Grantee:Pamela Magalí Bermejo
Home 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.SPEC

Abstract

Clostridium thermocellum is a thermophilic candidate for consolidated bioprocessing by carrying out both cellulose solubilization and fermentation. However, despite significant efforts, the maximum ethanol titer achieved to date remains below industrially required targets (90% of theoretical yield and 40 g/L titer). In microorganisms, fermentation of pyruvate to ethanol can proceed either with or without acetyl coenzyme A (acetyl-CoA) as an intermediate. In yeasts and Zymomonas mobilis, pyruvate is decarboxylated directly to acetaldehyde, which is then reduced to ethanol. In many other organisms, including C. thermocellum, pyruvate is oxidatively decarboxylated to acetyl-CoA, which is reduced to acetaldehyde and further reduced to ethanol. Several lines of evidence suggest that conversion of pyruvate to acetyl-CoA limits ethanol titer in C. thermocellum. The pathway comprising direct conversion of pyruvate to acetaldehyde by the enzyme pyruvate decarboxylase (PDC) is simpler than the native one because it requires fewer enzymatic reactions, and it is more thermodynamically favorable at standard conditions. Although the PDC enzyme has been successfully transferred to several mesophilic organisms, it does not seem to function well above 45 °C in vivo. There have been several attempts to introduce pdc genes into C. thermocellum, however the maximum titer we achieved was 21 g/L, and the Acetobacter pasteurianus Pdc activity was 100-fold lower than what we observed in Escherichia coli (0.3 U/mg vs. 31 U/mg). We think this problem is due to folding and therefore the challenge in this project will be to increase the thermostability of the Pdc protein. To this end, we propose to build a single-site saturation mutagenesis library of the A. pasteurianus PDC protein (10,621 members). We will then screen this library in C. thermocellum for increased ethanol production. Promising mutations will also be used to develop computational models of protein stability. (AU)

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