Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
ACS Synth Biol. 2022 Jul 15;11(7):2348-2360. doi: 10.1021/acssynbio.2c00075. Epub 2022 Apr 4.
The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biology. Synthetic cellular systems are envisioned as out-of-equilibrium enzymatic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metabolism. Importantly, gaining tight control over the external medium is essential to avoid thermodynamic equilibrium due to nutrient depletion or waste buildup in a closed compartment (, a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, , of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymatic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable l-arginine breakdown. In addition, we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium composition and to achieve sustainable glycerol 3-phosphate synthesis.
自下而上构建自主生长、自我复制的细胞对合成生物学来说是一个巨大的挑战。合成细胞系统被设想为一种非平衡的酶网络，被选择性开放的磷脂双层所包围，允许蛋白质介导的通讯；内部代谢物的再循环是可持续代谢的另一个关键方面。重要的是，对外部介质的严格控制对于避免由于在封闭隔室（如试管）中营养物质耗尽或废物积累而导致的热力学平衡是至关重要的。实施可持续的磷脂生物合成策略是扩大细胞边界的关键。然而，磷脂生物合成目前受到甘油 3-磷酸底物可用性的限制，而甘油 3-磷酸是磷脂头部基团的必需核心。在这里，我们在大单层囊泡中重新构建了可持续的甘油 3-磷酸合成酶网络。我们利用甘油激酶 GlpK 从外部供应的甘油合成甘油 3-磷酸。我们通过可持续的 l-精氨酸分解来为磷脂头部基团的形成提供燃料。此外，我们设计并表征了一种用于合成细胞的优化动态透析装置，用于控制外部介质组成并实现可持续的甘油 3-磷酸合成。
