Graduate School of Science, Technology, and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
Metab Eng. 2020 Sep;61:225-237. doi: 10.1016/j.ymben.2020.06.010. Epub 2020 Jul 2.
Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and lactate and in anabolic metabolism in the cytosol, or is transported into the mitochondria for use as a substrate in the tricarboxylic acid (TCA) cycle. In this study, we focused on controlling pyruvate metabolism in aerobic microorganisms for the biological production of various chemicals. We successfully improved productivity by redirecting pyruvate metabolism in the aerobic filamentous fungus Aspergillus oryzae via the deletion of two genes that encode pyruvate decarboxylase and mitochondrial pyruvate carriers. Production of ethanol as a major byproduct was completely inhibited, and the limited translocation of pyruvate into the mitochondria shifted the metabolism from respiration for energy conversion to the effective production of lactate or 2,3-butandiole, even under aerobic conditions. Metabolomic and transcriptomic analyses showed an emphasis on glycolysis and a repressed TCA cycle. Although the dry mycelial weights of the deletion mutants were reduced compared with those of wild type, the titer and yields of the target products were drastically increased. In particular, the redirection of pyruvate metabolism shifted from anabolism for biomass production to catabolism for the production of target chemicals. Conclusively, our results indicate that the redirection of pyruvate metabolism is a useful strategy in the metabolic engineering of aerobic microorganisms.
丙酮酸是生物合成各种化学品的重要代谢中间产物。在真核生物中，糖酵解产生的丙酮酸用于转化为乙醇和乳酸以及细胞质中的合成代谢，或者被转运到线粒体中作为三羧酸 (TCA) 循环的底物使用。在这项研究中，我们专注于控制好需氧微生物中的丙酮酸代谢，以用于各种化学品的生物合成。我们通过删除编码丙酮酸脱羧酶和线粒体丙酮酸载体的两个基因，成功地改善了好氧丝状真菌米曲霉中的丙酮酸代谢，从而提高了生产力。乙醇作为主要副产物的生产被完全抑制，并且由于丙酮酸有限地转运到线粒体中，代谢从呼吸作用（用于能量转换）转变为有效生产乳酸或 2,3-丁二醇，即使在需氧条件下也是如此。代谢组学和转录组学分析表明，糖酵解受到重视，而 TCA 循环受到抑制。尽管与野生型相比，缺失突变体的干燥菌丝体重量减少，但目标产物的浓度和产率却大大提高。特别是，丙酮酸代谢的重定向从生物合成（用于生物量生产）转变为分解代谢（用于生产目标化学品）。总之，我们的结果表明，丙酮酸代谢的重定向是好氧微生物代谢工程的一种有用策略。
