Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.
Biotechnol Bioeng. 2019 Dec;116(12):3292-3300. doi: 10.1002/bit.27148. Epub 2019 Sep 2.
An engineering tool for controlling flux distribution on metabolic pathways to an appropriate state is highly desirable in bioproduction. An optogenetic switch, which regulates gene expression by light illumination is an attractive on/off switchable system, and is a promising way for flux control with an external stimulus. We demonstrated a light-inducible flux control between glycolysis and the methylglyoxal (MGO) pathway in Escherichia coli using a CcaS/CcaR system. CcaR is phosphorylated by green light and is dephosphorylated by red light. Phosphorylated CcaR induces gene expression under the cpcG2 promoter. The tpiA gene was expressed under the cpcG2 promoter in a genomic tpiA deletion strain. The strain was then cultured with glucose minimum medium under green or red light. We found that tpiA messenger RNA level under green light was four times higher than that under red light. The repression of tpiA expression led to a decrease in glycolytic flux, resulting in slower growth under red light (0.25 hr ) when compared to green light (0.37 hr ). The maximum extracellular MGO concentration under red light (0.2 mM) was higher than that under green light (0.05 mM). These phenotypes confirm that the MGO pathway flux was enhanced under red light.
在生物生产中，非常需要一种能够控制代谢途径通量分布到适当状态的工程工具。光遗传学开关是一种通过光照调节基因表达的吸引人的开/关可切换系统，是一种利用外部刺激进行通量控制的有前途的方法。我们使用 CcaS/CcaR 系统在大肠杆菌中演示了糖酵解和甲基乙二醛 (MGO) 途径之间的光诱导通量控制。CcaR 被绿光磷酸化，被红光去磷酸化。磷酸化的 CcaR 在 cpcG2 启动子下诱导基因表达。在基因组 tpiA 缺失菌株中，tpiA 基因在 cpcG2 启动子下表达。然后，将该菌株在含有葡萄糖的最小培养基中在绿光或红光下培养。我们发现，绿光下的 tpiA 信使 RNA 水平是红光下的四倍。tpiA 表达的抑制导致糖酵解通量减少，导致在红光下（0.25 小时）比绿光下（0.37 小时）生长更慢。红光下（0.2 mM）的最大细胞外 MGO 浓度高于绿光下（0.05 mM）。这些表型证实了 MGO 途径通量在红光下增强。
