Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production

• Published in: Biotechnology for biofuels Vol. 11; no. 1; p. 242
• Main Authors: Hon, Shuen, Holwerda, Evert K., Worthen, Robert S., Maloney, Marybeth I., Tian, Liang, Cui, Jingxuan, Lin, Paul P., Lynd, Lee R., Olson, Daniel G.
• Format: Journal Article
• Language: English
• Published: London BioMed Central 06.09.2018; BioMed Central Ltd; Springer Science + Business Media; BMC
• Subjects: acetyl coenzyme A; Alcohol; Bacteria; Bacterial proteins; Binding sites; Biodiesel fuels; Biotechnology; Biotechnology & Applied Microbiology; Cellobiose; Cellulose; Chemistry; Chemistry and Materials Science; Clonal deletion; Clostridium; Clostridium thermocellum; Consolidated bioprocessing; Dehydrogenases; E coli; Energy & Fuels; Engineering; Environmental Engineering/Biotechnology; Enzymes; Ethanol; ethanol production; Fermentation; Ferredoxin; Gene expression; Genes; Genetic aspects; Isobutanol; Metabolic engineering; Metabolism; Methods; Microbiology; Oxidoreductase; Plant Breeding/Biotechnology; Production processes; Properties; Pyruvate ferredoxin oxidoreductase; pyruvate synthase; Pyruvic acid; Renewable and Green Energy; Substrates; Thermoanaerobacterium saccharolyticum; Thermophiles; Pyruvate ferredoxin oxidoreductase; Ethanol; Consolidated bioprocessing; Isobutanol; Thermoanaerobacterium saccharolyticum; Clostridium thermocellum
• ISSN: 1754-6834; 1754-6834; 2731-3654
• DOI: 10.1186/s13068-018-1245-2

Background Clostridium thermocellum has been the subject of multiple metabolic engineering strategies to improve its ability to ferment cellulose to ethanol, with varying degrees of success. For ethanol production in C. thermocellum , the conversion of pyruvate to acetyl-CoA is catalyzed primarily by the pyruvate ferredoxin oxidoreductase (PFOR) pathway. Thermoanaerobacterium saccharolyticum , which was previously engineered to produce ethanol of high yield (> 80%) and titer (70 g/L), also uses a pyruvate ferredoxin oxidoreductase, pforA , for ethanol production. Results Here, we introduced the T. saccharolyticum pforA and ferredoxin into C. thermocellum . The introduction of pforA resulted in significant improvements to ethanol yield and titer in C. thermocellum grown on 50 g/L of cellobiose, but only when four other T. saccharolyticum genes ( adhA , nfnA , nfnB , and adhE G544D ) were also present. T. saccharolyticum ferredoxin did not have any observable impact on ethanol production. The improvement to ethanol production was sustained even when all annotated native C. thermocellum pfor genes were deleted. On high cellulose concentrations, the maximum ethanol titer achieved by this engineered C. thermocellum strain from 100 g/L Avicel was 25 g/L, compared to 22 g/L for the reference strain, LL1319 ( adhA ( Tsc )- nfnAB ( Tsc )- adhE G544D ( Tsc )) under similar conditions. In addition, we also observed that deletion of the C. thermocellum pfor4 results in a significant decrease in isobutanol production. Conclusions Here, we demonstrate that the pforA gene can improve ethanol production in C. thermocellum as part of the T. saccharolyticum pyruvate-to-ethanol pathway. In our previous strain, high-yield (~ 75% of theoretical) ethanol production could be achieved with at most 20 g/L substrate. In this strain, high-yield ethanol production can be achieved up to 50 g/L substrate. Furthermore, the introduction of pforA increased the maximum titer by 14%.