초록
<P><B>Abstract</B></P> <P>Succinic acid is a platform chemical of recognized industrial value and accordingly faces a continuous challenge to enable manufacturing from most attractive raw materials. It is mainly produced from glucose, using microbial fermentation. Here, we explore and optimize succinate production from sucrose, a globally applied substrate in biotechnology, using the rumen bacterium <I>Basfia succiniciproducens</I> DD1. As basis of the strain optimization, the yet unknown sucrose metabolism of the microbe was studied, using <SUP>13</SUP>C metabolic flux analyses. When grown in batch culture on sucrose, the bacterium exhibited a high succinate yield of 1molmol<SUP>−1</SUP> and a by-product spectrum, which did not match the expected PTS-mediated sucrose catabolism. This led to the discovery of a fructokinase, involved in sucrose catabolism. The flux approach unraveled that the fructokinase and the fructose PTS both contribute to phosphorylation of the fructose part of sucrose. The contribution of the fructokinase reduces the undesired loss of the succinate precursor PEP into pyruvate and into pyruvate-derived by-products and enables increased succinate production, exclusively via the reductive TCA cycle branch. These findings were used to design superior producers. Mutants, which (i) overexpress the beneficial fructokinase, (II) lack the competing fructose PTS, and (iii) combine both traits, produce significantly more succinate. In a fed-batch process, <I>B. succiniciproducens</I> Δ<I>fruA</I> achieved a titer of 71gL<SUP>−1</SUP> succinate and a yield of 2.5molmol<SUP>−1</SUP> from sucrose.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Metabolic flux analysis unravels sucrose metabolism in <I>Basfia succiniciproducens</I>. </LI> <LI> The sucrose PTS, the fructose PTS and a fructokinase are involved in sucrose assimilation. </LI> <LI> Fructokinase and the fructose PTS are key targets to improve succinate production. </LI> <LI> The best strain produces 71 g L<SUP>-1</SUP> succinate from sucrose with a yield of 2.5 mol mol<SUP>-1</SUP>. </LI> </UL> </P>