초록
<P><B>Abstract</B></P> <P>Succinate is a versatile petrochemical compound that can be produced by microorganisms, often from carbohydrate based carbon sources. Phototrophic cyanobacteria including <I>Synechocystis</I> sp. PCC 6803 can more efficiently produce organic acids such as succinate without sugar supplementation, via photosynthetic production of glycogen followed by glycogen utilization, typically under dark conditions. In this study, <I>Synechocystis</I> 6803 bioproduction of organic acids under dark anoxic conditions was found to increase with elevation of temperature from 30 °C to 37 °C. The further enhancement of succinate bioproduction by overexpression of the rate limiting enzyme phosphoenolpyruvate carboxylase resulted in improved glycogen utilization. To gain more insight into the mechanisms underlying the increased organic acid output, a novel temperature dependent metabolomics analysis was performed. Adenylate energy charge was found to decrease along with elevating temperature, while central metabolites glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, glycerol 3-phosphate, malate, fumarate and succinate increased. Temperature dependent <SUP>13</SUP>C-labeling metabolomics analysis further revealed a glycolysis to TCA bottleneck, which could be overcome by addition of CO<SUB>2</SUB>, leading to even higher organic acid production. Optimization of initial cell concentration to 25 g-dry cell weight/L, in combination with 100 mM NaHCO<SUB>3</SUB> supplementation, afforded a succinate titer of over 1.8 g/L, the highest reported autotrophic succinate titer. Succinate titers remained high after additional knockout of <I>ackA</I>, resulting in the highest reported autotrophic D-lactate titer as well. The optimization of <I>Synechocystis</I> 6803 organic acid production therefore holds significant promise for CO<SUB>2</SUB> capture and utilization.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Autotrophic production of organic acid by <I>Synechocystis</I> was enhanced by elevating temperature. </LI> <LI> Temperature dependent <SUP>13</SUP>C labeling metabolomics elucidated a glycolysis to TCA bottleneck. </LI> <LI> Overexpression of the PEPC gene alleviated the bottleneck and improved glycogen utilization. </LI> <LI> CO<SUB>2</SUB> utilization enhanced succinate production to the highest autotrophic titer of 1.8 g/L. </LI> <LI> Additional knockout of <I>ackA</I> increased D-lactate production, providing clues for succinate optimization. </LI> </UL> </P>