Bio-based succinic acid holds promise as a sustainable platform chemical. Its production through microbial fermentation concurs with the fixation of CO2, through the carboxylation of phosphoenolpyruvate. Here, we studied the effect of the available CO2 on the metabolism of Pseudoclostridium thermosuccinogenes, the only known succinate producing thermophile. Batch cultivations in bioreactors sparged with 1 and 20% CO2 were conducted that allowed us to carefully study the effect of CO2 limitation. Formate yield was greatly reduced at low CO2 concentrations, signifying a switch from pyruvate formate lyase (PFL) to pyruvate:ferredoxin oxidoreductase (PFOR) for acetyl-CoA formation. The corresponding increase in endogenous CO2 production (by PFOR) enabled succinic acid production to be largely maintained as its yield was reduced by only 26%, thus also maintaining the concomitant NADH re-oxidation, essential for regenerating NAD+ for glycolysis. Acetate yield was slightly reduced as well, while that of lactate was slightly increased. CO2 limitation also prompted the formation of significant amounts of ethanol, which is only marginally produced during CO2 excess. Altogether, the changes in fermentation product yields result in increased ferredoxin and NAD+ reduction, and increased NADPH oxidation during CO2 limitation, which must be linked to reshuffled (trans) hydrogenation mechanisms of those cofactors, in order to keep them balanced. RNA sequencing, to investigate transcriptional effects of CO2 limitation, yielded only ambiguous results regarding the known (trans) hydrogenation mechanisms. The results hinted at a decreased NAD+/NADH ratio, which could ultimately be responsible for the stress observed during CO2 limitation. Clear overexpression of an alcohol dehydrogenase (adhE) was observed, which may explain the increased ethanol production, while no changes were seen for PFL and PFOR expression that could explain the anticipated switch based on the fermentation results.

中文翻译：

---

CO 2限制对热琥珀酸假单胞菌代谢的影响。

生物基琥珀酸有望成为一种可持续的平台化学品。其通过微生物发酵的生产与通过烯醇式丙酮酸磷酸酯的羧化作用的CO 2固定同时存在。在这里，我们研究了可利用的二氧化碳对假单胞菌热琥珀酸假单胞菌（已知的唯一产生琥珀酸酯的嗜热菌）代谢的影响。在充满1％和20％CO2的生物反应器中进行了分批培养，这使我们能够仔细研究CO2限制的影响。在低CO2浓度下，甲酸酯产量会大大降低，这表明从丙酮酸甲酸酯裂解酶（PFL）转换为丙酮酸：铁氧还蛋白氧化还原酶（PFOR）可形成乙酰辅酶A。内源性CO2产生的相应增加（通过PFOR）使得琥珀酸的产生得以很大程度上维持，因为其产率仅降低了26％，因此，还可以维持伴随的NADH重氧化，这对于糖酵解再生NAD +必不可少。乙酸盐的产率也略有降低，而乳​​酸盐的产率略有​​提高。CO 2限制还促使形成大量乙醇，乙醇仅在过量CO 2期间少量产生。总之，发酵产物产量的变化会导致铁氧还蛋白的增加和NAD +的减少，以及CO2限制期间NADPH氧化的增加，这必须与这些辅因子的改组（反式）氢化机制联系起来，以使其保持平衡。RNA测序，以调查CO2限制的转录效应，仅产生关于已知的（反式）氢化机制的模棱两可的结果。结果提示NAD + / NADH比例降低，这可能最终导致二氧化碳限制期间观察到的压力。观察到明显的乙醇脱氢酶（adhE）过表达，这可以解释乙醇产量的增加，而PFL和PFOR表达没有变化，这可以解释基于发酵结果的预期转换。