From known knowns to unknown unknowns: synthetic biology paths to antimicrobial discovery.

'Structurally known' and 'Structurally unknown' have long served as a binary classification of natural products. However, with the emergence of synthetic biology (SynBio), the boundaries of this simple classification are being redrawn, because SynBio expands both what we can see and what we can build. To reflect this change, we propose a SynBio-centered framework that maps antimicrobial natural products along two axes, chemical novelty and engineering accessibility, yielding four quadrants: (i) known knowns, well-characterized natural products accessed mainly through pathway refactoring and producer strain optimization; (ii) unknown knowns, engineerable scaffolds with established diversification routes but uncertain chemical and functional outcomes; (iii) known unknowns, architectures with precedent but limited realization due to missing transferable engineering routes, often because the relevant coupling logic has not yet been converted into a practical engineering handle; (iv) unknown unknowns, unanticipated scaffolds that need to be recognized through minimal chemical, genetic, or mechanistic anchors before engineering. Since these quadrants are dynamic rather than fixed, compounds may shift across the framework as discovery, route-building, and outcome predictability improve. This two-axis map highlights complementary SynBio strategies, from optimizing existing antibiotics to accessing first-in-class entities, which may help guide efforts to combat current resistance challenges.