BACKGROUND: Colchicine is a natural medicinal alkaloid from Gloriosa superba L. The biosynthetic pathway of colchicine has been nearly completely revealed, but the Pictet-Spenglerase that catalyses the formation of the crucial intermediate 1-phenethylisoquinoline scaffold has not been identified. Identification and characterization of the missing Pictet-Spenglerase will provide the last piece of the puzzle of colchicine biosynthesis in G. superba. RESULTS: Based on the G. superba transcriptome database, orthologues of 1-phenethylisoquinoline scaffold synthase (PSS) were identified and screened, and one candidate gene sequence, GsPSS, was identified. GsPSS has a total length of 480 bp, and multisequence alignment revealed that GsPSS had two common conserved catalytic residues. The subcellular results indicated that GsPSS was localized in the cytoplasm. After heterologous expression in E. coli and purification, in vitro enzyme assays indicated that the recombinant GsPSS protein could catalyse the conversion of 3-(4-Hydroxyphenyl) propanal (4-HDCA) and dopamine to generate a 1-phenethylisoquinoline scaffold. CONCLUSION: In this study, we identified the last previously unknown enzyme involved in the biosynthesis of colchicine. This enzyme belongs to the Pr10/Bet v1 family and catalyses the committed step in colchicine biosynthesis. The knowledge gained here will help to complement and improve the colchicine biosynthetic pathway and facilitate the biosynthesis of colchicine via metabolic engineering.