Project description:Advanced clinical trials investigate the Psilocybe magic mushroom natural product psilocybin as a treatment against major depressive disorder. Currently, synthetic material is used to meet the demand for legitimate pharmaceutical purposes. Here, we report an in vitro approach to biocatalytically produce psilocybin on a solid-phase matrix charged with five covalently bound biosynthetic enzymes. These enzymes include three Psilocybe enzymes: IasA, an engineered L-tryptophan decarboxylase/aromatic aldehyde synthase, the 4-hydroxytryptamine kinase PsiK and the norbaeocystin methyltransferase PsiM, along with Escherichia coli nucleosidase MtnN and adenine deaminase Ade. In a proof-of-principle experiment, this enzyme-charged resin allowed for quantitative turnover of 4-hydroxy-L-tryptophan into psilocybin. This facile process i) represents a sustainable approach with reusable enzymes, ii) circumvents the drawbacks of in vivo processes while harnessing the selectivity of enzymatic catalysis and iii) helps access an urgently needed drug candidate.
Project description:The human fungal pathogen Candida albicans can switch stochastically and heritably between a “white” phase and an “opaque” phase. Opaque cells are the mating-competent form of the species whereas white cells are essentially “sterile”. Here, we report that glucose depletion, a common nutrient stress, enables C. albicans white cells to undergo efficient sexual mating. The relative expression levels of pheromone-sensing and mating-associated genes (including STE2/3, MFA1, MFalpha1, FIG1, FUS1, and CEK1/2) were increased under glucose depletion conditions, while expression of mating repressors TEC1 and DIG1 was decreased. We show that Cph1 and Tec1, factors that act downstream of the pheromone MAPK pathway, play opposite roles in regulating white cell mating as TEC1 deletion or CPH1 overexpression promoted white cell mating. Moreover, inactivation of the Cph1 repressor Dig1 increased white cell mating ~4,000 fold in glucose-depleted medium relative to that in the presence of glucose. These findings reveal that the white-to-opaque epigenetic switch may not be a prerequisite for sexual mating in C. albicans in nature. Given parallels between C. albicans white cell mating to that of other yeast species, this mechanism of mating could represent a more ancient strategy of sexual reproduction in C. albicans.
Project description:Haploid budding yeast has two mating types, defined by the alleles of the MAT locus, MATa and MATα. Mating occurs when two haploid cells of opposite mating types signal to each other using reciprocal pheromones and receptors, polarize and grow towards each other, and eventually fuse to form a single diploid cell. The pheromones and receptors are necessary and sufficient to define a mating type, but other mating type-specific proteins make mating more efficient. We examined the role of these proteins by genetically engineering “transvestite” cells that swap the pheromone, pheromone receptor, and pheromone processing factors of one mating type for another. These cells can mate with each other, but their mating is inefficient. By characterizing their mating defects and examining their transcriptomes, we found Afb1 (a-factor barrier), a novel MATα-specific protein that interferes with a-factor, the pheromone secreted by MATa cells. We show that strong pheromone secretion is essential for efficient mating and that the weak mating of transvestites can be improved by boosting their pheromone production. Using synthetic biology, it is possible to characterize the factors that control efficiency in biological processes. In the case of budding yeast mating, selection for increased mating efficiency is likely to have continually boosted pheromone levels and the ability to discriminate between partners who make more (potentially fitter) and less (potentially less fit) pheromones. This sensitivity to which partner makes more pheromone comes at a cost: it means mating is not robust in situations where all potential partners make less pheromone.