Soma Yuki, Yamaji Taiki, Matsuda Fumio, Hanai Taizo
Laboratory for Bioinformatics, Graduate School of Systems Lifesciences, Kyushu University, 804 Westwing, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; Research Center for Transomics Medicine, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Laboratory for Bioinformatics, Graduate School of Systems Lifesciences, Kyushu University, 804 Westwing, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
J Biosci Bioeng. 2017 May;123(5):625-633. doi: 10.1016/j.jbiosc.2016.12.009. Epub 2017 Feb 14.
Almost all synthetic pathways for biofuel production are designed to require endogenous metabolites in glycolysis, such as phosphoenolpyruvate, pyruvate, and acetyl-CoA. However, such metabolites are also required for bacterial cell growth. To reduce the metabolic imbalance between cell growth and target chemical production, we previously constructed a metabolic toggle switch (MTS) as a conditional flux redirection tool controlling metabolic flux of TCA cycle toward isopropanol production. This approach succeeded to improve the isopropanol production titer and yield while ensuring sufficient cell growth. However, excess accumulation of pyruvate, the precursor for acetyl-CoA synthesis, was also observed. In this study, for efficient conversation of pyruvate to acetyl-CoA (pyruvate oxidation), we designed a synthetic metabolic bypass composed of poxB and acs with the MTS for acetyl-CoA supply from the excess pyruvate. When this designed bypass was expressed at the appropriate expression level associated with the conditional metabolic flux redirection, pyruvate accumulation was prevented, and the isopropanol production titer and yield were improved. Final isopropanol production titer of strain harboring MTS with the synthetic metabolic bypass improved 4.4-fold compared with strain without metabolic flux regulation, and it was 1.3-fold higher than that of strain harboring the conventional MTS alone. Additionally, glucose consumption was also improved 1.7-fold compared with strain without metabolic flux regulation. On the other hand, introduction of the synthetic metabolic bypass alone showed no improvement in isopropanol production and glucose consumption. These results showed that the improvement in bio-production process caused by synergy between the MTS and the synthetic metabolic bypass.
几乎所有生物燃料生产的合成途径都设计为需要糖酵解中的内源性代谢物，如磷酸烯醇丙酮酸、丙酮酸和乙酰辅酶A。然而，细菌细胞生长也需要这些代谢物。为了减少细胞生长与目标化学品生产之间的代谢失衡，我们之前构建了一种代谢切换开关（MTS）作为一种条件性通量重定向工具，控制三羧酸循环的代谢通量以用于异丙醇生产。这种方法成功提高了异丙醇的生产滴度和产量，同时确保了足够的细胞生长。然而，也观察到了乙酰辅酶A合成前体丙酮酸的过量积累。在本研究中，为了将丙酮酸高效转化为乙酰辅酶A（丙酮酸氧化），我们设计了一种由poxB和acs组成的合成代谢旁路，并结合MTS从过量的丙酮酸中供应乙酰辅酶A。当这种设计的旁路以与条件性代谢通量重定向相关的适当表达水平表达时，丙酮酸积累得到了防止，异丙醇的生产滴度和产量也得到了提高。与没有代谢通量调控的菌株相比，带有合成代谢旁路的MTS菌株的最终异丙醇生产滴度提高了4.4倍，比仅带有传统MTS的菌株高1.3倍。此外，与没有代谢通量调控的菌株相比，葡萄糖消耗也提高了1.7倍。另一方面，单独引入合成代谢旁路在异丙醇生产和葡萄糖消耗方面没有显示出改善。这些结果表明，MTS与合成代谢旁路之间的协同作用导致了生物生产过程的改善。
