Abstract
BackgroundClostridium 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.ResultsHere, 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 (adhA(Tsc)-nfnAB(Tsc)-adhEG544D (Tsc)) under similar conditions. In addition, we also observed that deletion of the C. thermocellum pfor4 results in a significant decrease in isobutanol production.ConclusionsHere, 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%.
Highlights
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
Four proteins from a strain of Thermoanaerobacterium saccharolyticum engineered for high levels of ethanol production—namely, an NADPH-dependent alcohol dehydrogenase (AdhA), the NADH-dependent reduced ferredoxin:NADP+ oxidoreductase complex (NfnAB), and a mutant bifunctional alcohol dehydrogenase (AdhEG544D)—were introduced into wild-type C. thermocellum, to improve ethanol yield, titer, and production rate [4]
The maximum ethanol titer achieved by this engineered C. thermocellum strain (LL1319) was only 15 g/L, which is far short of the 70 g/L ethanol titer that engineered T. saccharolyticum is capable of producing [5]
Summary
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. The maximum ethanol titer achieved by this engineered C. thermocellum strain (LL1319) was only 15 g/L, which is far short of the 70 g/L ethanol titer that engineered T. saccharolyticum (strain M1442) is capable of producing [5] In both C. thermocellum and T. saccharolyticum, the oxidative decarboxylation of pyruvate to acetylCoA is primarily catalyzed by a pyruvate ferredoxin oxidoreductase (PFOR) enzyme or enzyme complex [6,7,8,9]. In C. thermocellum, there are five candidates (Table 1) [10]; of these, the pfors encoded by the genes Clo1313_0020-0023 and Clo1313_1353-1356 were reported to be cumulatively responsible for approximately 80% of the PFOR activity [10] It is not known which of these five pfors is important for ethanol production. Prior to this work, all strains of C. thermocellum that had been engineered to produce ethanol with the pfor pathway have relied on the native pfors; among these strains, the maximum ethanol titers
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