This article describes the complete biosynthesis of QS-21, a potent vaccine adjuvant derived from the Chilean soapbark tree. QS-21 is a key component of several human vaccines, including Shingrix, Mosquirix, and NVX-CoV2373. It consists of a glycosylated triterpene scaffold linked to a complex glycosylated 18-carbon acyl chain, which is critical for its immunostimulant activity. While the triterpene scaffold's biosynthesis was previously identified, the acyl chain's origin remained unknown. This study elucidates the biosynthetic pathway for the acyl chain, characterizes the enzymes involved, and reconstitutes the entire 20-step pathway in tobacco, demonstrating QS-21 production in a heterologous system. This breakthrough enables the bioengineering of vaccine adjuvants, investigation of structure-activity relationships, and understanding of immune response mechanisms. QS-21 is produced by the Chilean soapbark tree, Quillaja saponaria, and has been used in veterinary medicine for decades. In 2017, the first saponin adjuvant, AS01, containing QS-21, was approved for use in the Shingrix vaccine. QS-21 is now a key component of several human vaccines. However, its supply is limited, and increased demand is expected with the approval of these vaccines. The QS-21 molecule consists of a triterpene core with branched and linear sugar chains, and a glycosylated 18-carbon acyl chain. The acyl chain is unique to Quillaja saponins and is essential for immunostimulant activity. The study identifies the biosynthetic origin of the acyl chain, showing it is derived from L-isoleucine through a series of enzymatic reactions involving a polyketide synthase (PKSIII). The acyl chain is then transferred to the triterpene scaffold through a series of enzymes, including ketoreductases, acyl transferases, and glycosyl transferases. The study also identifies the remaining genes required for QS-21 biosynthesis, including five additional enzymes necessary for the acyl chain addition. These enzymes, along with previously identified genes, were used to reconstitute the entire QS-21 pathway in tobacco. The study also demonstrates that increasing the availability of L-isoleucine can enhance QS-21 production. Finally, the study confirms the complete biosynthesis of QS-21 in a heterologous host, showing that the produced molecule matches the structure of the naturally occurring QS-21. This research provides a foundation for the future engineering of designer saponins with optimal immunostimulatory activity and low toxicity.This article describes the complete biosynthesis of QS-21, a potent vaccine adjuvant derived from the Chilean soapbark tree. QS-21 is a key component of several human vaccines, including Shingrix, Mosquirix, and NVX-CoV2373. It consists of a glycosylated triterpene scaffold linked to a complex glycosylated 18-carbon acyl chain, which is critical for its immunostimulant activity. While the triterpene scaffold's biosynthesis was previously identified, the acyl chain's origin remained unknown. This study elucidates the biosynthetic pathway for the acyl chain, characterizes the enzymes involved, and reconstitutes the entire 20-step pathway in tobacco, demonstrating QS-21 production in a heterologous system. This breakthrough enables the bioengineering of vaccine adjuvants, investigation of structure-activity relationships, and understanding of immune response mechanisms. QS-21 is produced by the Chilean soapbark tree, Quillaja saponaria, and has been used in veterinary medicine for decades. In 2017, the first saponin adjuvant, AS01, containing QS-21, was approved for use in the Shingrix vaccine. QS-21 is now a key component of several human vaccines. However, its supply is limited, and increased demand is expected with the approval of these vaccines. The QS-21 molecule consists of a triterpene core with branched and linear sugar chains, and a glycosylated 18-carbon acyl chain. The acyl chain is unique to Quillaja saponins and is essential for immunostimulant activity. The study identifies the biosynthetic origin of the acyl chain, showing it is derived from L-isoleucine through a series of enzymatic reactions involving a polyketide synthase (PKSIII). The acyl chain is then transferred to the triterpene scaffold through a series of enzymes, including ketoreductases, acyl transferases, and glycosyl transferases. The study also identifies the remaining genes required for QS-21 biosynthesis, including five additional enzymes necessary for the acyl chain addition. These enzymes, along with previously identified genes, were used to reconstitute the entire QS-21 pathway in tobacco. The study also demonstrates that increasing the availability of L-isoleucine can enhance QS-21 production. Finally, the study confirms the complete biosynthesis of QS-21 in a heterologous host, showing that the produced molecule matches the structure of the naturally occurring QS-21. This research provides a foundation for the future engineering of designer saponins with optimal immunostimulatory activity and low toxicity.