LAUSR

Crude extract-based cell-free expression systems have been used to produce natural products by reconstitution of their biosynthetic pathways in vitro. However, the chemical scope of cell-free synthesized natural compounds is still limited, which is partially due to the length of biosynthetic gene clusters. To expand the product scope, here, we report cell-free biosynthesis of several lysine-derived unnatural amino acids with functional moieties such as chloro, alkene, and alkyne groups. Specifically, five related enzymes (i.e., halogenase, oxidase, lyase, ligase, and hydroxylase) involved in β-ethynylserine biosynthesis are selected for cell-free expression. These enzymes can be expressed in single, in pairs, or in trios to synthesize different compounds, including, for instance, 4-Cl-l-lysine, 4-Cl-allyl-l-glycine, and l-propargylglycine. The final product of γ-l-glutamyl-l-β-ethynylserine (a dipeptide with an alkyne group) can also be synthesized by cell-free expression of the full biosynthetic pathway (i.e., five enzymes). Our results demonstrate the flexibility of cell-free systems, enabling easy regulation and rational optimization for target compound formation. Overall, this work expands not only the type of enzymes (e.g., halogenase) but also the scope of natural products (e.g., terminal-alkyne amino acid) that can be rapidly produced in cell-free systems. With the development of cell-free biotechnology, we envision that cell-free strategies will create a new frontier for natural product biosynthesis.