Kobayashi Shunsuke, Kato Junya, Wada Keisuke, Takemura Kaisei, Kato Setsu, Fujii Tatsuya, Iwasaki Yuki, Aoi Yoshiteru, Morita Tomotake, Matsushika Akinori, Murakami Katsuji, Nakashimada Yutaka
Graduate School of Integrated Sciences for Life, Hiroshima University, Higashihiroshima, Japan.
National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Front Microbiol. 2022 May 12;13:897066. doi: 10.3389/fmicb.2022.897066. eCollection 2022.
Hydrogen (H) converted to reducing equivalents is used by acetogens to fix and metabolize carbon dioxide (CO) to acetate. The utilization of H enables not only autotrophic growth, but also mixotrophic metabolism in acetogens, enhancing carbon utilization. This feature seems useful, especially when the carbon utilization efficiency of organic carbon sources is lowered by metabolic engineering to produce reduced chemicals, such as ethanol. The potential advantage was tested using engineered strains of that produce ethanol. By adding H to the fructose-supplied culture, the engineered strains produced increased levels of acetate, and a slight increase in ethanol was observed. The utilization of a knockout strain of the major acetate production pathway, aimed at increasing the carbon flux to ethanol, was unexpectedly hindered by H-mediated growth inhibition in a dose-dependent manner. Metabolomic analysis showed a significant increase in intracellular NADH levels due to H in the ethanol-producing strain. Higher NADH level was shown to be the cause of growth inhibition because the decrease in NADH level by dimethyl sulfoxide (DMSO) reduction recovered the growth. When H was not supplemented, the intracellular NADH level was balanced by the reversible electron transfer from NADH oxidation to H production in the ethanol-producing strain. Therefore, reversible hydrogenase activity confers the ability and flexibility to balance the intracellular redox state of . Tuning of the redox balance is required in order to benefit from H-supplemented mixotrophy, which was confirmed by engineering to produce acetone.
氢气（H）转化为还原当量后被产乙酸菌用于固定和代谢二氧化碳（CO）以生成乙酸盐。氢气的利用不仅能实现自养生长，还能使产乙酸菌进行混合营养代谢，提高碳的利用率。这一特性似乎很有用，特别是当通过代谢工程降低有机碳源的碳利用效率以生产还原型化学品（如乙醇）时。利用产乙醇的工程菌株对这一潜在优势进行了测试。通过向供应果糖的培养物中添加氢气，工程菌株产生的乙酸盐水平增加，并且观察到乙醇略有增加。旨在增加碳流向乙醇的主要乙酸盐产生途径的基因敲除菌株的利用，却意外地受到氢气介导的剂量依赖性生长抑制的阻碍。代谢组学分析表明，产乙醇菌株中由于氢气导致细胞内NADH水平显著增加。较高的NADH水平被证明是生长抑制的原因，因为通过二甲基亚砜（DMSO）还原降低NADH水平可恢复生长。当不添加氢气时，产乙醇菌株中细胞内NADH水平通过从NADH氧化到氢气产生的可逆电子传递得以平衡。因此，可逆氢化酶活性赋予了平衡细胞内氧化还原状态的能力和灵活性。为了从添加氢气的混合营养中获益，需要调节氧化还原平衡，这一点在生产丙酮的工程中得到了证实。
