Recent advances in synthetic biology have enabled the in vitro operation of the central dogma in the reconstituted cell-free protein synthesis system (i.e., the PURE system), which represents a convenient platform to address molecular-level biochemical questions and a robust workhorse for biomanufacturing of noncanonical peptides, polyketides, and enzymes that are difficult to express in vivo. However, unlike living cells regenerating their building blocks from substrates, PURE systems require an extra supply of 20 amino acids (AAs) for protein synthesis. Cell-free protein synthesis would be more cost-effective and environmentally friendly if the PURE systems could self-regenerate the protein building blocks (i.e., AAs) from a renewable feedstock, such as plastic waste. Here, we developed a renovated PURE system capable of self-regenerating aspartate, asparagine, glutamate, and glutamine using polylactate (PLA) plastics and α-ketoglutarate, CO₂, and NH₄⁺ as the AAs precursors. We first established a one-pot, cofactor self-sufficient multienzyme cascade to oxidize dl-PLA to (i) produce pyruvate as the precursor of aspartate and asparagine and (ii) regenerate NADH (reducing equivalents) for the reductive amination of α-ketoglutarate to yield glutamate and subsequent glutamine, the shared amine group donors for most AAs. Subsequently, the PLA-metabolic multienzyme cascade was introduced into the PURE system devoid of the four PLA-derived AAs. The PLA hydrolase-coding mRNA was translated in the modified PURE system, producing PLA hydrolase incorporating PLA-derived AAs. This enzyme further metabolizes PLA into more AAs for mRNA translation, forming a closed-loop circuit that seamlessly couples mRNA translation to AA metabolism. This process resembles a simplified heterotrophic life form, utilizing PLA both as building blocks and as reducing equivalents. Therefore, the “PLA-eating” PURE system established here offers a bioeconomy platform for valorizing PLA plastic for the future production of peptidyl biochemicals.

中文翻译：

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以 PLA 塑料、CO2、铵和 α-酮戊二酸为原料的氨基酸自我再生无细胞蛋白质合成系统


合成生物学的最新进展使得重建的无细胞蛋白质合成系统（即 PURE 系统）中的中心法则能够在体外运行，这代表了解决分子水平生化问题的便捷平台和生物制造的强大主力难以在体内表达的非经典肽、聚酮化合物和酶。然而，与活细胞从底物再生其构建模块不同，PURE 系统需要额外供应 20 种氨基酸 (AA) 来进行蛋白质合成。如果 PURE 系统能够从塑料废物等可再生原料中自我再生蛋白质构建块（即 AA），那么无细胞蛋白质合成将更具成本效益且更加环保。在这里，我们开发了一种翻新的 PURE 系统，能够使用聚乳酸 (PLA) 塑料和 α-酮戊二酸、CO ₂ 和 NH ₄ 自我再生天冬氨酸、天冬酰胺、谷氨酸和谷氨酰胺 ⁺ 作为 AA 前体。我们首先建立了一个一锅、辅因子自给自足的多酶级联，以氧化 dl-PLA，(i) 产生丙酮酸作为天冬氨酸和天冬酰胺的前体，以及 (ii) 再生 NADH（还原当量）用于 α-酮戊二酸的还原胺化产生谷氨酸和随后的谷氨酰胺，这是大多数氨基酸的共享胺基供体。随后，PLA 代谢多酶级联被引入不含四种 PLA 衍生 AA 的 PURE 系统中。 PLA 水解酶编码 mRNA 在改良的 PURE 系统中翻译，产生掺有 PLA 衍生 AA 的 PLA 水解酶。这种酶进一步将 PLA 代谢成更多的 AA，用于 mRNA 翻译，形成闭环回路，将 mRNA 翻译与 AA 代谢无缝耦合。 这个过程类似于简化的异养生命形式，利用 PLA 既作为构建块又作为还原当量。因此，这里建立的“吃PLA”PURE系统为未来生产肽基生物化学品提供了一个生物经济平台，可以使PLA塑料增值。