School of Environmental Science and Engineering, Tianjin University, Tianjin, China.
Department of Microbiology, Miami University, Oxford, Ohio, USA.
mBio. 2021 Jun 29;12(3):e0088121. doi: 10.1128/mBio.00881-21.
Understanding carbon flux controlling mechanisms in a tangled metabolic network is an essential question of cell metabolism. Secondary metabolism, such as terpene biosynthesis, has evolved with low carbon flux due to inherent pathway constraints. Thraustochytrids are a group of heterotrophic marine unicellular protists and can accumulate terpenoids under the high-salt conditions in their natural environment. However, the mechanism behind terpene accumulation is not well understood. Here, we show that terpene biosynthesis in Thraustochytrium sp. ATCC 26185 is constrained by local thermodynamics in the mevalonate pathway. Thermodynamic analysis reveals metabolite limitation in the nondecarboxylative Claisen condensation of acetyl-coenzyme A (CoA) to the acetoacetyl-CoA step, catalyzed by the acetyl-CoA acetyltransferase (ACAT). Through a sodium-elicited mechanism, higher respiration leads to increased ATP investment into the mevalonate pathway, providing a strong thermodynamic driving force for enhanced terpene biosynthesis. Proteomic and metabolomic analyses further show that the increased ATP demands are fulfilled by shifting energy generation from carbohydrate to lipid oxidation. This study demonstrates a unique strategy in nature that uses ATP to drive a low-flux metabolic pathway, providing an alternative solution for efficient terpene metabolic engineering. Terpenoids are a large class of lipid molecules with important biological functions and diverse industrial and medicinal applications. Metabolic engineering for terpene production has been hindered by the low-flux distribution to its biosynthesis pathway. In practice, a high substrate load is generally required to reach high product titers. Here, we show that mevalonate-derived terpene biosynthesis is constrained by local pathway thermodynamics, which can only be partially relieved by increasing substrate levels. Through comparative omics and biochemical analyses, we discovered a unique mechanism for high terpene accumulation in marine protist thraustochytrids. Through a sodium-induced mechanism, thraustochytrids shift their energy metabolism from carbohydrate to lipid oxidation for enhanced ATP production, providing a strong thermodynamic driving force for efficient terpene biosynthesis. This study reveals an important mechanism in eukaryotes to overcome the thermodynamic constraint in low-flux pathways by increased ATP consumption. Engineering energy metabolism thus provides an important alternative to relieve flux constraints in low-flux and energy-consuming pathways.
理解代谢网络中碳通量控制机制是细胞代谢的一个基本问题。次生代谢物，如萜类生物合成，由于固有途径的限制，其碳通量较低。硫球藻是一组异养海洋单细胞原生生物，在其天然高盐环境中可以积累萜类化合物。然而，萜类化合物积累的机制尚不清楚。在这里，我们表明，硫球藻属 sp.ATCC 26185 的萜类生物合成受到甲羟戊酸途径中局部热力学的限制。热力学分析表明，在非脱羧克莱森缩合反应中，乙酰辅酶 A（CoA）与乙酰乙酰 CoA 的步骤中存在代谢物限制，该反应由乙酰辅酶 A 乙酰转移酶（ACAT）催化。通过钠诱导的机制，较高的呼吸作用导致更多的 ATP 投入到甲羟戊酸途径中，为增强的萜类生物合成提供了强大的热力学驱动力。蛋白质组学和代谢组学分析进一步表明，增加的 ATP 需求通过将能量生成从碳水化合物转移到脂质氧化来满足。这项研究展示了自然界中一种独特的策略，即利用 ATP 驱动低通量代谢途径，为高效萜类代谢工程提供了一种替代解决方案。萜类化合物是一类具有重要生物学功能的大型脂类分子，具有广泛的工业和药用应用。萜类化合物生产的代谢工程受到其生物合成途径通量低的限制。在实践中，通常需要高底物负荷才能达到高产物滴度。在这里，我们表明，甲羟戊酸衍生的萜类生物合成受到局部途径热力学的限制，只能通过增加底物水平来部分缓解。通过比较组学和生化分析，我们发现了海洋原生质体硫球藻中高萜类化合物积累的一种独特机制。通过钠诱导的机制，硫球藻将其能量代谢从碳水化合物转移到脂质氧化，以增强 ATP 的产生，为高效萜类生物合成提供了强大的热力学驱动力。这项研究揭示了真核生物中一种重要的机制，通过增加 ATP 消耗来克服低通量途径中的热力学限制。因此，工程化的能量代谢为缓解低通量和能量消耗途径中的通量限制提供了一个重要的替代方案。
