Khushi, K.; Ganesh, A.; Sharma, A.; Ravindran, F.; Srinivasan, S.; Choudhary, B.

Phyllanthus niruri (Phyllanthaceae) is a medicinally important herb known for producing phyllanthin, a bioactive dibenzylbutane lignan with reported hepatoprotective and antioxidant properties. However, the biosynthetic basis of phyllanthin production remains unresolved, largely due to the absence of a reference genome for the species. We report a chromosome-scale assembly of P. niruri generated by integrating PacBio HiFi long reads and Illumina short reads, followed by reference-guided scaffolding against Phyllanthus cochinchinensis. The assembly has an L50 of 7 and 97.6% BUSCO completeness. Annotation predicted 19,254 protein-coding genes, of which 91.1% were functionally annotated, with phenylpropanoid biosynthesis emerging as the most enriched specialized-metabolism pathway in the genome. Using pathway-guided genome mining, structural similarity analysis, and comparative metabolic reconstruction, we propose a putative biosynthetic pathway for phyllanthin originating from the phenylpropanoid-lignan branch through secoisolariciresinol-like intermediates followed by terminal O-methylation reactions. A total of 305 unique candidate genes associated with the proposed pathway were identified, including expanded families of dirigent proteins, peroxidases, secoisolariciresinol dehydrogenases, and O-methyltransferases. Comparative transcriptomic analyses across related Phyllanthus species further supported the proposed pathway through coordinated expression of lignan-associated genes and tissue-specific enrichment of O-methyltransferases. This work provides the first reference genome for P. niruri and a prioritized candidate gene set for functional characterization of phyllanthin biosynthesis.