Singapore Institute of Food and Biotechnology Innovation (SIFBI), Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos, Singapore, 138669, Singapore.
Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), 30 Biopolis St, Matrix, Singapore, 138671, Singapore.
NPJ Syst Biol Appl. 2024 Oct 1;10(1):109. doi: 10.1038/s41540-024-00440-7.
Engineered microorganisms have emerged as viable alternatives for limonene production. However, issues such as low enzyme abundance or activities, and regulatory feedback/forward inhibition may reduce yields. To understand the underlying metabolism, we adopted a systems biology approach for an engineered limonene-producing Escherichia coli strain K-12 MG1655. Firstly, we generated time-series metabolomics data and, secondly, developed a dynamic model based on enzyme dynamics to track the native metabolic networks and the engineered mevalonate pathway. After several iterations of model fitting with experimental profiles, which also included C-tracer studies, we performed in silico knockouts (KOs) of all enzymes to identify bottleneck(s) for optimal limonene yields. The simulations indicated that ALDH/ADH (aldehyde dehydrogenase/alcohol dehydrogenase) and LDH (lactate dehydrogenase) suppression, and HK (hexokinase) enhancement would increase limonene yields. Experimental confirmation was achieved, where ALDH-ADH and LDH KOs, and HK overexpression improved limonene yield by 8- to 11-fold. Our systems biology approach can guide microbial strain re-engineering for optimal target production.
工程微生物已成为生产柠檬烯的可行替代品。然而，酶丰度或活性低、调控反馈/前馈抑制等问题可能会降低产量。为了了解潜在的代谢情况，我们采用系统生物学方法对工程化的柠檬烯生产大肠杆菌菌株 K-12 MG1655 进行了研究。首先，我们生成了时间序列代谢组学数据，其次，基于酶动力学开发了一个动态模型来跟踪天然代谢网络和工程甲羟戊酸途径。在对实验谱进行了几次模型拟合迭代，包括 C 示踪研究之后，我们对所有酶进行了计算机模拟敲除（KO），以确定最佳柠檬烯产量的瓶颈。模拟表明，抑制 ALDH/ADH（醛脱氢酶/醇脱氢酶）和 LDH（乳酸脱氢酶），增强 HK（己糖激酶）可以提高柠檬烯的产量。实验证实，ALDH-ADH 和 LDH 的 KO 和 HK 的过表达可使柠檬烯的产量提高 8 到 11 倍。我们的系统生物学方法可以指导微生物菌株的重新设计，以实现最佳目标产量。
