Work

Amino Acid Self-Regenerating Cell-Free Protein Synthesis System that Feeds on PLA Plastics, CO 2 , Ammonium, and α-Ketoglutarate

Year: 2024

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 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, CO 2, and NH 4 + as the AAs precursors. We first established a one-pot, cofactor self-sufficient multienzyme cascade to oxidize d l -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.

Source: ACS Catalysis

DOI: 10.1021/acscatal.4c00992