Amino Acid Self-Regenerating Cell-Free Protein Synthesis System that Feeds on PLA Plastics, CO2, Ammonium, and α-Ketoglutarate

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.