Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform

被引:16
作者
Chavez, Benjamin G. [1 ]
Srinivasan, Prashanth [2 ]
Glockzin, Kayla [3 ]
Kim, Neill [3 ]
Estrada, Olga Montero [3 ]
Jirschitzka, Jan [4 ]
Rowden, Gage [3 ]
Shao, Jonathan [5 ]
Meinhardt, Lyndel [5 ]
Smolke, Christina D. [2 ,6 ]
D'Auria, John C. [1 ]
机构
[1] Leibniz Inst Plant Genet & Crop Plant Res IPK Ort, Dept Mol Genet, D-06466 Seeland, Germany
[2] Stanford Univ, Dept Bioengn, Stanford, CA 94305 USA
[3] Texas Tech Univ, Dept Chem & Biochem, Lubbock, TX 79409 USA
[4] Max Planck Inst Chem Ecol, Dept Biochem, D-07745 Jena, Germany
[5] USDA ARS, Sustainable Perennial Crops Lab, Beltsville, MD 20705 USA
[6] Chan Zuckerberg Biohub, San Francisco, CA 94158 USA
基金
加拿大自然科学与工程研究理事会;
关键词
convergent evolution; N-methylspermidine; pathway discovery; tropane alkaloid; Erythroxylaceae; CONVERGENT EVOLUTION; PYRROLIDINE RINGS; ORNITHINE; BINDING; RECEPTOR; CYCLASE; PLANTS; STEP;
D O I
10.1073/pnas.2215372119
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.
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页数:12
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