Discovery and Engineering of the Cocaine Biosynthetic Pathway.

Cocaine, the archetypal tropane alkaloid from the plant genus , has recently been used clinically as a topical anesthesia of the mucous membranes. Despite this, the key biosynthetic step of the requisite tropane skeleton (methylecgonone) from the identified intermediate 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid (MPOA) has remained, until this point, unknown. Herein, we identify two missing enzymes (CYP81AN15 and MT4) necessary for the biosynthesis of the tropane skeleton in cocaine by transient expression of the candidate genes in . Cytochrome P450 CYP81AN15 was observed to selectively mediate the oxidative cyclization of -MPOA to yield the unstable intermediate ecgonone, which was then methylated to form optically active methylecgonone by methyltransferase MT4 in . The establishment of this pathway corrects the long-standing (but incorrect) biosynthetic hypothesis of MPOA methylation first and oxidative cyclization second. Notably, the de novo reconstruction of cocaine was realized in with the two newly identified genes, as well as four already known ones. This study not only reports a near-complete biosynthetic pathway of cocaine and provides new insights into the metabolic networks of tropane alkaloids (cocaine and hyoscyamine) in plants but also enables the heterologous synthesis of tropane alkaloids in other (micro)organisms, entailing significant implications for pharmaceutical production.