Ancestral Sequence\nReconstruction to Enable Biocatalytic\nSynthesis of Azaphilones

Biocatalysis can be powerful in organic\nsynthesis but\nis often\nlimited by enzymes’ substrate scope and selectivity. Developing\na biocatalytic step involves identifying an initial enzyme for the\ntarget reaction followed by optimization through rational design,\ndirected evolution, or both. These steps are time consuming, resource-intensive,\nand require expertise beyond typical organic chemistry. Thus, an effective\nstrategy for streamlining the process from enzyme identification to\nimplementation is essential to expanding biocatalysis. Here, we present\na strategy combining bioinformatics-guided enzyme mining and ancestral\nsequence reconstruction (ASR) to resurrect enzymes for biocatalytic\nsynthesis. Specifically, we achieve an enantioselective synthesis\nof azaphilone natural products using two ancestral enzymes: a flavin-dependent\nmonooxygenase (FDMO) for stereodivergent oxidative dearomatization\nand a substrate-selective acyltransferase (AT) for the acylation of\nthe enzymatically installed hydroxyl group. This cascade, stereocomplementary\nto established chemoenzymatic routes, expands access to enantiomeric\nlinear tricyclic azaphilones. By leveraging the co-occurrence and\ncoevolution of FDMO and AT in azaphilone biosynthetic pathways, we\nidentified an AT candidate, CazE, and addressed its low solubility\nand stability through ASR, obtaining a more soluble, stable, promiscuous,\nand reactive ancestral AT (AncAT). Sequence analysis revealed AncAT\nas a chimeric composition of its descendants with enhanced reactivity\nlikely due to ancestral promiscuity. Flexible receptor docking and\nmolecular dynamics simulations showed that the most reactive AncAT\npromotes a reactive geometry between substrates. We anticipate that\nour bioinformatics-guided, ASR-based approach can be broadly applied\nin target-oriented synthesis, reducing the time required to develop\nbiocatalytic steps and efficiently access superior biocatalysts.