Microalgal Polyphosphate Drives One-Pot Complete Enzymatic Generation of Flavin Adenine Dinucleotide from Adenosine and Riboflavin

Flavin adenine dinucleotide (FAD) is a universal cellular cofactor involved in biological redox and radical metabolism reactions. FAD biosynthesis from riboflavin typically proceeds through two ATP-dependent enzymatic reactions, with flavin mononucleotide (FMN) as the intermediate. Traditional in vivo methods employ microorganisms for FAD synthesis at an industrial scale; however, these approaches often suffer from complex purification processes. Considering the atomic economy and percentage yield, in vitro enzymatic FAD synthesis using enzymes could be a more efficient and sustainable alternative. While catalytically efficient, the requirements of expensive ATP (substrate) limit the industrialization of enzymatic FAD synthesis. To overcome the ATP requirements, here we develop a two-enzyme cascade for ATP regeneration from adenosine using wastewater microalgal polyphosphate as the P-donor. With the ATP regeneration system, the bifunctional riboflavin kinase/FAD synthetase and pyrophosphatase completely convert saturated riboflavin into FAD within 2 h with a titer of ∼1.2 g/L (1.5 mmol/L). Notably, orthophosphate, the only byproduct of this enzymatic process, can be recycled to synthesize polyphosphate by wastewater microalgae, which can then be fed back into the system as the P-donor in the ATP regeneration step, resulting in a FAD synthesis process with almost net-zero waste generation

<i>N</i>‑Carboxyanhydride-Mediated Fatty Acylation of Amino Acids and Peptides for Functionalization of Protocell Membranes

Early protocells are likely to have arisen from the self-assembly of RNA, peptide, and lipid molecules that were generated and concentrated within geologically favorable environments on the early Earth. The reactivity of these components in a prebiotic environment that supplied sources of chemical energy could have produced additional species with properties favorable to the emergence of protocells. The geochemically plausible activation of amino acids by carbonyl sulfide has been shown to generate short peptides via the formation of cyclic amino acid <i>N</i>-carboxyanhydrides (NCAs). Here, we show that the polymerization of valine-NCA in the presence of fatty acids yields acylated amino acids and peptides via a mixed anhydride intermediate. Notably, <i>N</i>^α-oleoylarginine, a product of the reaction between arginine and oleic acid in the presence of valine-NCA, partitions spontaneously into vesicle membranes and mediates the association of RNA with the vesicles. Our results suggest a potential mechanism by which activated amino acids could diversify the chemical functionality of fatty acid membranes and colocalize RNA with vesicles during the formation of early protocells

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