Biosynthesis of the allelopathic alkaloid gramine in barley by a cryptic oxidative rearrangement.

The defensive alkaloid gramine not only protects barley and other grasses from insects but also negatively affects their palatability to ruminants. The key gene for gramine formation has remained elusive, hampering breeding initiatives. In this work, we report that a gene encoding cytochrome P450 monooxygenase CYP76M57, which we name AMI synthase (AMIS), enables the production of gramine in Nicotiana benthamiana, Arabidopsis thaliana, and Saccharomyces cerevisiae. We reconstituted gramine production in the gramine-free barley (Hordeum vulgare) variety Golden Promise and eliminated it from cultivar Tafeno by Cas-mediated gene editing. In vitro experiments unraveled that an unexpected cryptic oxidative rearrangement underlies this noncanonical conversion of an amino acid to a chain-shortened biogenic amine. The discovery of the genetic basis of gramine formation now permits tailor-made optimization of gramine-linked traits in barley by plant breeding.

Plant synthetic biology: from inspiration to augmentation.

Although it is still in its infancy, synthetic biology has the capacity to face scientific and societal problems related to modern agriculture. Innovations in cloning toolkits and genetic parts allow increased precision over gene expression in planta. We review the vast spectrum of available technologies providing a practical list of toolkits that take advantage of combinatorial power to introduce/alter metabolic pathways. We highlight that rational design is inspired by deep knowledge of natural and biochemical mechanisms. Finally, we provide several examples in which modern technologies have been applied to address these critical topics. Future applications in plants include not only pathway modifications but also prospects of augmenting plant anatomical features and developmental processes.

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

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.

Erythroxylum in Focus: An Interdisciplinary Review of an Overlooked Genus.

The genus Erythroxylum contains species used by indigenous people of South America long before the domestication of plants. Two species, E. coca and E. novogranatense, have been utilized for thousands of years specifically for their tropane alkaloid content. While abuse of the narcotic cocaine has impacted society on many levels, these species and their wild relatives contain untapped resources for the benefit of mankind in the form of foods, pharmaceuticals, phytotherapeutic products, and other high-value plant-derived metabolites. In this review, we describe the current state of knowledge of members within the genus and the recent advances in the realm of molecular biology and biochemistry.