QS-21 is a potent vaccine adjuvant derived from the Chilean soapbark tree, *Quillaja saponaria*. Its complex structure, consisting of a glycosylated triterpene scaffold and a 18-carbon acyl chain, is crucial for its immunostimulant activity. The acyl chain's biosynthetic route was previously unknown. This study identifies the enzymes required for the synthesis of the acyl chain and reconstitutes the entire 20-step pathway in tobacco plants. The research reveals that the acyl chain is derived from iterative decarboxylative condensations of malonyl-CoA with 2-MB-CoA, a catabolite of l-isoleucine, catalyzed by plant PKSII family members. The study also identifies five additional enzymes needed for the addition of the acyl chain to the triterpene scaffold, including ketoreductases, acyl transferases, and a sugar transferase. The successful reconstitution of QS-21 biosynthesis in a heterologous host opens up opportunities for bioengineering designer saponins with optimal immunostimulatory activity and low toxicity, potentially reducing reliance on natural extraction from the soapbark tree.QS-21 is a potent vaccine adjuvant derived from the Chilean soapbark tree, *Quillaja saponaria*. Its complex structure, consisting of a glycosylated triterpene scaffold and a 18-carbon acyl chain, is crucial for its immunostimulant activity. The acyl chain's biosynthetic route was previously unknown. This study identifies the enzymes required for the synthesis of the acyl chain and reconstitutes the entire 20-step pathway in tobacco plants. The research reveals that the acyl chain is derived from iterative decarboxylative condensations of malonyl-CoA with 2-MB-CoA, a catabolite of l-isoleucine, catalyzed by plant PKSII family members. The study also identifies five additional enzymes needed for the addition of the acyl chain to the triterpene scaffold, including ketoreductases, acyl transferases, and a sugar transferase. The successful reconstitution of QS-21 biosynthesis in a heterologous host opens up opportunities for bioengineering designer saponins with optimal immunostimulatory activity and low toxicity, potentially reducing reliance on natural extraction from the soapbark tree.