Epidemiological data have demonstrated a reduced risk of developing cancer with regular consumption of diets rich in cruciferous vegetables.This chemoprevention has been largely attributed to the presence of natural glucosinolate products,particularly methionine-derived glucoraphanin from broccoli.Here we report the first successful engineering of the complete biosynthetic pathway from methionine to glucoraphanin through 14 intermediates catalyzed by 10 enzymes in Escherichia coli via gene mining,pathway design,and protein modification.We usedbranched-chain amino transferase 3 to catalyze two transamination steps to ensure the purity of precursor molecules and used cysteine as a sulfur donor to simplify the synthesis pathway.Two chimeric cytochrome P450 enzymes were constructed and functionally expressed in E.coli.The Neurosporacrassahtrcynylcysttint sulfoxide lyase was used to replace the original plant C-S lyase.Other pathway enzymes were successfully mined from Arabidopsis thaliana ecotype Col-0,Brassica rapa,and Brassica oleracea.Biosynthesis of glucoraphanin after combined expression of the optimized enzymes in vivo was confirmed by liquid chromatography-tandem mass spectrometry analysis.No other glucosinolate analogs(except for glucoiberin) were identified that could facilitate the downstream purification processes.Production of glucoraphanin in this study serves as a proof-of-concept for the large-scale microbial production of specific glucosinolates that can benefit human health.