Transcriptome‐Guided Identification of N ‐Acetyltransferases Involved in Melatonin Biosynthesis in a Streptomyces ‐Based Cell Factory

Melatonin is a widely used neurohormone with growing demand in pharmaceuticals and supplements. Microbial production via engineered cell factories offers a sustainable alternative to conventional chemical synthesis. In this study, we employed transcriptome-guided mining to identify endogenous N-acetyltransferases involved in melatonin biosynthesis in an engineered Streptomyces albus strain harboring a de novo synthetic pathway. Three key N-acetyltransferases with distinct substrate specificities toward tryptophan-derived intermediates were identified. By enhancing decarboxylase expression and deleting an N-acetyltransferase that preferentially acts on non-decarboxylated substrates, we developed an improved strain capable of producing approximately 380 mg/L melatonin, representing a ~65% increase over the parental strain. This work advances microbial melatonin biosynthesis and expands the functional repertoire of bacterial N-acyltransferases for synthetic biology applications in diverse microbial hosts.