Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH, acpS, acpT, acs, and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2S)-naringenin production (523.7 ± 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production.

代谢异质性和代谢通量的动态变化是微生物发酵的两个固有特征，它们限制了对代谢的精确控制，通常会导致细胞生长受损和生产力低下。动态代谢工程通过设计多层和多基因动态调节网络 (DRN) 来解决这些挑战，该网络允许单个细胞根据其生长和代谢物积累条件自主调整代谢通量。在这里，我们开发了一种增长耦合 NCOMB（柚皮素-香豆酸-丙二酰辅酶 A-平衡）DRN，系统优化了（2S）-柚皮素和p用于实时控制细胞内丙二酰辅酶A供应的香豆酸反应调节途径。在这种情况下，酰基载体蛋白被用作微调丙二酰辅酶A消耗的新型关键节点，而不是以前研究中常用的脂肪酸合酶的直接抑制。为此，我们首先设计了一个多层 DRN，使单个细胞能够同时调节acpH、acpS、acpT、acs和ACC在丙二酰辅酶A分解代谢和合成代谢途径中。接下来，NCOMB DRN 被优化以通过基于生物传感器的定向进化策略增强不同动态调节层之间的协同作用。最后，与使用静态应变工程的基础菌株相比，从 NCOMB DRN 方法获得的高生产者使 (2 S )-柚皮素产量提高了 8.7 倍（523.7 ± 51.8 mg/L），同时细胞生长增加了 20%方法，从而证明了该系统在改善通路生产方面的高效率。