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

Hon, Shuen; Holwerda, Evert K.; Worthen, Robert S.; Maloney, Marybeth I.; Tian, Liang; Cui, Jingxuan; Lin, Paul P.; Lynd, Lee R.; Olson, Daniel G.

doi:10.1186/s13068-018-1245-2

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

Journal Article · Thu Sep 06 00:00:00 EDT 2018 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/s13068-018-1245-2· OSTI ID:1618728

Hon, Shuen; Holwerda, Evert K.; Worthen, Robert S.; Maloney, Marybeth I.; Tian, Liang; Cui, Jingxuan; Lin, Paul P.; Lynd, Lee R.; Olson, Daniel G.

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 adhEG544D) 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 ((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%.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Biological and Environmental Research (BER), DOE BioEnergy Science Center; DOE Center for Bioenergy Innovation; DOE Joint Genome Institute

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1618728

Alternate ID(s):: OSTI ID: 1626996

Journal Information:: Biotechnology for Biofuels, Journal Name: Biotechnology for Biofuels Vol. 11 Journal Issue: 1; ISSN 1754-6834

Publisher:: Springer Science + Business MediaCopyright Statement

Country of Publication:: Netherlands

Language:: English

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Cited by: 22 works

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MOESM2 of Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production Hon, Shuen; Holwerda, Evert; Worthen, Robert https://doi.org/10.6084/m9.figshare.7056812 The current record is supplemented by this text	text	January 2018
MOESM2 of Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production Hon, Shuen; Holwerda, Evert; Worthen, Robert https://doi.org/10.6084/m9.figshare.7056812.v1 The current record is supplemented by this text	text	January 2018
MOESM1 of Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production Hon, Shuen; Holwerda, Evert; Worthen, Robert https://doi.org/10.6084/m9.figshare.7056824 The current record is supplemented by this dataset	dataset	January 2018
MOESM1 of Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production Hon, Shuen; Holwerda, Evert; Worthen, Robert https://doi.org/10.6084/m9.figshare.7056824.v1 The current record is supplemented by this dataset	dataset	January 2018
MOESM3 of Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production Hon, Shuen; Holwerda, Evert; Worthen, Robert https://doi.org/10.6084/m9.figshare.7056836 The current record is supplemented by this dataset	dataset	January 2018
MOESM3 of Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production Hon, Shuen; Holwerda, Evert; Worthen, Robert https://doi.org/10.6084/m9.figshare.7056836.v1 The current record is supplemented by this dataset	dataset	January 2018
Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production Hon, Shuen; Holwerda, Evert; Worthen, Robert https://doi.org/10.6084/m9.figshare.c.4224053 The current record is supplemented by this collection	collection	January 2018
Expressing the Thermoanaerobacterium saccharolyticum pforA in engineered Clostridium thermocellum improves ethanol production Hon, Shuen; Holwerda, Evert; Worthen, Robert https://doi.org/10.6084/m9.figshare.c.4224053.v1 The current record is supplemented by this collection	collection	January 2018