Automated Flow Synthesis of Nature-Derived, Engineered, and De Novo-Designed Functional Luciferases.

Luciferases are widely used bioluminescent reporters, yet access to these enzymes has historically relied on recombinant expression. Using automated fast-flow peptide synthesis (AFPS), we synthesized Gaussia luciferase (GLuc, 18.1 kDa, 168 amino acids), picALuc (13 kDa, 120 amino acids), and LuxSit-i (13.8 kDa, 118 amino acids), representing a nature-derived disulfide-rich luciferase, an engineered luciferase, and a de novo-designed luciferase, respectively. Synthetic GLuc was purified, oxidatively folded, and recovered as a monomeric, enzymatically active enzyme with biophysical properties comparable to its recombinant counterpart. picALuc likewise required oxidative folding, whereas cysteine-free LuxSit-i was folded by denaturant removal using size-exclusion chromatography. Biochemical and biophysical analyses confirmed the structural integrity and activity of the three synthetic enzymes. Notably, LuxSit-i represents, to our knowledge, the original total chemical synthesis of a fully de novo-designed enzyme. Together, these proteins expand the known repertoire of fully synthetic enzymes produced by solid-phase peptide synthesis by approximately 14% and establish the initial examples of fully synthetic luciferases produced by solid-phase peptide synthesis that demonstrate enzymatic activity. These findings position AFPS as a platform for accessing functional enzymes across evolutionary and design spaces and may enable the development of luciferases incorporating noncanonical residues, site-specific modifications, or mirror-image sequences.