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Cocaine, the archetypal tropane alkaloid from the plant genus Erythroxylum, 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 (EnCYP81AN15 and EnMT4) necessary for the biosynthesis of the tropane skeleton in cocaine by transient expression of the candidate genes in Nicotiana benthamiana. Cytochrome P450 EnCYP81AN15 was observed to selectively mediate the oxidative cyclization of S-MPOA to yield the unstable intermediate ecgonone, which was then methylated to form optically active methylecgonone by methyltransferase EnMT4 in Erythroxylum novogranatense. The establishment of this pathway corrects the longstanding (but incorrect) biosynthetic hypothesis of MPOA methylation first and oxidative cyclization second. Notably, the de novo reconstruction of cocaine was realized in N. benthamiana 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.
Cocaine, the archetypal tropane alkaloid from the plant genus Erythroxylum, 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 (EnCYP81AN15 and EnMT4) necessary for the biosynthesis of the tropane skeleton in cocaine by transient expression of the candidate genes in Nicotiana benthamiana. Cytochrome P450 EnCYP81AN15 was observed to selectively mediate the oxidative cyclization of S-MPOA to yield the unstable intermediate ecgonone, which was then methylated to form optically active methylecgonone by methyltransferase EnMT4 in Erythroxylum novogranatense. The establishment of this pathway corrects the longstanding (but incorrect) biosynthetic hypothesis of MPOA methylation first and oxidative cyclization second. Notably, the de novo reconstruction of cocaine was realized in N. benthamiana 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.
Tropane alkaloids such as scopolamine (C17H21NO4), atropine (C17H23NO3) and hyoscyamine (C17H23NO3) are the most important plant secondary metabolites in the pharmaceutical industry due to anticholinergic activity, competition with muscarinic receptors and also treating different human diseases. Scopolamine, hyoscyamine and atropine are the most important tropane alkaloids used as anticoagulants and spasmolytic drugs in the digestive system and urinary excretion. Tropane alkaloids are natural phytochemical compounds, which are present in different plant families. These compounds are the main secondary metabolites in Solanaceae family plants such as Hyoscyamus niger and Atropa belladonna. The main source of raw material for the production of tropane alkaloids in the world is Duboisia spp. leaves which contain 2-4% alkaloids (more than 60% scopolamine, and 30% hyoscyamine). Common methods of analysis of tropane alkaloids include gas chromatography (GC), high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), in which liquid chromatography is mostly adopted. Various enzymes involved in the biosynthesis of tropane alkaloids, in which N-Putrescine methyltransferase (PMT), tropinone reductase I and II, and hyoscyamine 6-beta-hydroxylase (H6H) have a key role. To increase the biosynthesis of these important alkaloids, many studies were focused on the manipulation of key genes expressing enzymes in the biosynthesis pathway such as pmt and h6h genes. Although many biotechnological and agronomic studies have been done to increase the biosynthesis efficiency of these metabolites, further investigations are necessary. This paper is intended to provide a comprehensive overview of these tropane alkaloids.
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