Characterization of the Clostridium thermocellum AdhE, NfnAB, ferredoxin and Pfor proteins for their ability to support high titer ethanol production in Thermoanaerobacterium saccharolyticum

Metabolic Engineering
Jingxuan CuiLee R Lynd

Abstract

The thermophilic anaerobes Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are good candidates for lignocellulosic ethanol production. T. saccharolyticum has been successfully engineered to produce ethanol at high titer (70 g/L). The maximum ethanol titer of engineered strains of C. thermocellum is only 25 g/L. We hypothesize that one or more of the enzymes in the ethanol production pathway in C. thermocellum is not adequate for ethanol production at high titer. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from pyruvate to ethanol. In T. saccharolyticum, we replaced all of the genes encoding proteins in this pathway with their homologs from C. thermocellum and examined what combination of gene replacements restricted ethanol titer. We found that a pathway consisting of Ct_nfnAB, Ct_fd, Ct_adhE and Ts_pforA was sufficient to support ethanol titer greater than 50 g/L, however replacement of Ts_pforA by Ct_pfor1 dramatically decreased the maximum ethanol titer to 14 g/L. We then demonstrated that the reason for reduced ethanol production is that the Ct_pfor1 is inhibited by accumulation of ethanol and NADH, while Ts_pforA is not.

References

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Oct 4, 2016·Applied and Environmental Microbiology·Liang TianLee R Lynd
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Dec 5, 2016·Metabolic Engineering·Daniel G OlsonLee R Lynd
Mar 28, 2018·Proceedings of the National Academy of Sciences of the United States of America·Percival Yang-Ting ChenCatherine L Drennan

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Citations

Feb 7, 2020·Metabolic Engineering Communications·Jingxuan CuiLee R Lynd
May 10, 2021·Trends in Biotechnology·Li ZhuSheng Yang

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