Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species including food crops, such as tomato, potato and eggplant. Unlike true alkaloids, nitrogen is introduced at a late stage of SGA biosynthesis through an unknown transamination reaction. Here, we reveal the mechanism by which GLYCOALKALOID METABOLISM12 (GAME12) directs the biosynthesis of nitrogen-containing steroidal alkaloid aglycone in Solanum. We report that GAME12, a neofunctionalized γ-aminobutyric acid (GABA) transaminase, undergoes changes in both active site specificity and subcellular localization to switch from its renown and generic activity in core metabolism to function in a specialized metabolic pathway. Moreover, overexpression of GAME12 alone in engineered S. nigrum leaves is sufficient for de novo production of nitrogen-containing SGAs. Our results highlight how hijacking a core metabolism GABA shunt enzyme is crucial in numerous Solanum species for incorporating a nitrogen to a steroidal-specialized metabolite backbone and form defensive alkaloids.

类固醇配糖生物碱 (SGA) 是由数百种茄属植物产生的特殊代谢物，包括番茄、马铃薯和茄子等粮食作物。与真正的生物碱不同，氮是在 SGA 生物合成的后期通过未知的氨基转移反应引入的。在这里，我们揭示了糖基生物碱代谢12（GAME12）指导茄属中含氮甾体生物碱苷元生物合成的机制。我们报告说，GAME12是一种新功能化的γ-氨基丁酸（GABA）转氨酶，其活性位点特异性和亚细胞定位都发生了变化，从其在核心代谢中众所周知的通用活性转变为在专门的代谢途径中发挥作用。此外，在工程化的黑龙江叶片中单独过度表达GAME12就足以从头生产含氮 SGA。我们的结果强调了劫持核心代谢 GABA 分流酶在许多茄属物种中对于将氮结合到类固醇专门代谢物主链并形成防御性生物碱至关重要。