초록
<P><B>Background</B></P><P><I>Clostridium thermocellum</I> 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 <I>C. thermocellum</I>, the conversion of pyruvate to acetyl-CoA is catalyzed primarily by the pyruvate ferredoxin oxidoreductase (PFOR) pathway. <I>Thermoanaerobacterium saccharolyticum</I>, which was previously engineered to produce ethanol of high yield (> 80%) and titer (70 g/L), also uses a pyruvate ferredoxin oxidoreductase, <I>pforA</I>, for ethanol production.</P><P><B>Results</B></P><P>Here, we introduced the <I>T. saccharolyticum pforA</I> and ferredoxin into <I>C. thermocellum</I>. The introduction of <I>pforA</I> resulted in significant improvements to ethanol yield and titer in <I>C. thermocellum</I> grown on 50 g/L of cellobiose, but only when four other <I>T. saccharolyticum</I> genes (<I>adhA</I>, <I>nfnA</I>, <I>nfnB</I>, and <I>adhE</I><SUP><I>G544D</I></SUP>) were also present. <I>T. saccharolyticum</I> ferredoxin did not have any observable impact on ethanol production. The improvement to ethanol production was sustained even when all annotated native <I>C. thermocellum pfor</I> genes were deleted. On high cellulose concentrations, the maximum ethanol titer achieved by this engineered <I>C. thermocellum</I> strain from 100 g/L Avicel was 25 g/L, compared to 22 g/L for the reference strain, LL1319 (<I>adhA</I>(<I>Tsc</I>)-<I>nfnAB</I>(<I>Tsc</I>)-<I>adhE</I><SUP>G544D</SUP> (<I>Tsc</I>)) under similar conditions. In addition, we also observed that deletion of the <I>C. thermocellum pfor4</I> results in a significant decrease in isobutanol production.</P><P><B>Conclusions</B></P><P>Here, we demonstrate that the <I>pforA</I> gene can improve ethanol production in <I>C. thermocellum</I> as part of the <I>T. saccharolyticum</I> 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 <I>pforA</I> increased the maximum titer by 14%.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s13068-018-1245-2) contains supplementary material, which is available to authorized users.</P>