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Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

BackgroundThe electronic Dietary Intake Assessment (e-DIA), a digital entry food record mobile phone app, was developed to measure energy and nutrient intake prospectively. This can be used in monitoring population intakes or intervention studies in young adults. ObjectiveThe objective was to assess the relative validity of e-DIA as a dietary assessment tool for energy and nutrient intakes using the 24-hour dietary recall as a reference method. MethodsUniversity students aged 19 to 24 years recorded their food and drink intake on the e-DIA for five days consecutively and completed 24-hour dietary recalls on three random days during this 5-day study period. Mean differences in energy, macro-, and micronutrient intakes were evaluated between the methods using paired t tests or Wilcoxon signed-rank tests, and correlation coefficients were calculated on unadjusted, energy-adjusted, and deattenuated values. Bland-Altman plots and cross-classification into quartiles were used to assess agreement between the two methods. ResultsEighty participants completed the study (38% male). No significant differences were found between the two methods for mean intakes of energy or nutrients. Deattenuated correlation coefficients ranged from 0.55 to 0.79 (mean 0.68). Bland-Altman plots showed wide limits of agreement between the methods but without obvious bias. Cross-classification into same or adjacent quartiles ranged from 75% to 93% (mean 85%). ConclusionsThe e-DIA shows potential as a dietary intake assessment tool at a group level with good ranking agreement for energy and all nutrients.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.