<jats:title>Abstract</jats:title><jats:p>Metabolic profiling of <jats:italic>Streptomyces sp</jats:italic>. IB2014/016‐6 led to the identification of three new tetrahydroisoquinoline natural products, perquinolines A–C (<jats:bold>1</jats:bold>–<jats:bold>3</jats:bold>). Labelled precursor feeding studies and the cloning of the <jats:italic>pqr</jats:italic> biosynthetic gene cluster revealed that <jats:bold>1</jats:bold>–<jats:bold>3</jats:bold> are assembled by the action of several unusual enzymes. The biosynthesis starts with the condensation of succinyl‐CoA and <jats:sc>l</jats:sc>‐phenylalanine catalyzed by the amino‐7‐oxononanoate synthase‐like enzyme PqrA, representing rare chemistry in natural product assembly. The second condensation and cyclization events are conducted by PqrG, an enzyme resembling an acyl‐CoA ligase. Last, ATP‐grasp RimK‐type ligase PqrI completes the biosynthesis by transferring a γ‐aminobutyric acid or β‐alanine moiety. The discovered pathway represents a new route for assembling the tetrahydroisoquinoline cores of natural products.