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.

Recently, bioactive benzoxazinoids were discovered in cereal grains and bakery products. In this study, we studied the uptake, distribution, and metabolism of these secondary metabolites using a pig model. Twelve benzoxazinoid compounds and their 4 transformation products were quantified in the pigs' diets and biofluids using high-performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry. The 2-β-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) was the most dominant benzoxazinoid (232 nmol/g DM) seconded by the double-hexose derivative of DIBOA (provisionally characterized here as DIBOA-glc-hex) in the rye-based diet. DIBOA-glc (derived from the diet and intestinal deglycosylation of DIBOA-glc-hex) was apparently reduced to 2-β-D-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-glc), the most dominant benzoxazinoid in the blood (829 nmol/L). The benzoxazinoid compounds were excreted in the urine, with HBOA-glc (18 μmol/L) as a major metabolite. In this study, we determined for the first time the bioavailability of dietary benzoxazinoids that have high digestibility, distribution, and metabolism in mammals. These findings could be a milestone for the exploitation of healthful and pharmacological properties of benzoxazinoids.

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Benzoxazinoids (BXs) are a group of natural chemical compounds with putative pharmacological and health-protecting properties. BXs were formerly identified in and isolated from selected dicot medicinal plants and young cereal plants. Recently, BXs were found to be present in mature cereal grains and bakery products, such that knowledge about the pharmacological properties of BXs, which until now have unknowingly been consumed through the daily bread and breakfast cereals, has come into new focus. This review discusses published results from in vitro studies and a few human and animal model studies on the health effects and pharmacological responses of various BX compounds. Many of these studies have reported antimicrobial, anticancer, reproductive system stimulatory, central nervous system stimulatory, immunoregulatory, and appetite- and weight-reducing effects of BXs and/or BX derivatives. The health benefits of wholegrain intake may be associated with the solitary and/or overlapping biological effects of fibers, lignans, phenolic acids, alkylresorcinols, BXs, and other bioactive compounds. In the context of BXs as dietary ingredients, further comprehensive investigations are required to understand their biological functions, to elucidate the underlying mechanisms, to explore their potential contribution on the health effects associated with wholegrain consumption, and to examine their potential as functional food ingredients.

These results indicate that following ingestion in healthy humans, a range of unmetabolized bioactive dietary benzoxazinoids and their sulfate and glucuronide conjugates appear in circulation and urine.

Thorough knowledge of the absorption and metabolism of dietary benzoxazinoids is needed to understand their health-promoting effects. In this study, the fates of these bioactive compounds were examined by LC-MS/MS in plasma, urine, and feces after ingesting a daily dose of 4780 ± 68 nmol benzoxazinoids from rye bread using Wistar rats as a model. HBOA-glc (2-β-D-glucopyranosyloxy-1,4-benzoxazin-3-one) was the predominant benzoxazinoid in the plasma (74 ± 27 nmol/L), followed by DIBOA-glc (2-β-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one) and HBOA. The total level of benzoxazinoids in the urine was 1176 ± 66 nmol/d, which corresponds to approximately 25% of the total dietary intake. The urinary benzoxazinoid profile differed from that of plasma with HBOA-glc and DIBOA-glc (647 ± 31 and 466 ± 33 nmol/d, respectively) as the major urinary components. The glucuronide conjugates of HBOA and DIBOA were detected in both the plasma and urine. N-dehydroxylation was found to be a critical step in the absorption of hydroxamic acids. This unprecedented study will trigger future interest in the biological effects of benzoxazinoids in whole grain rye and wheat diets in humans and other animals.

The dietary BX treatment correlated well with the plasma and urine levels, illustrating strong dose-dependent BX absorption, which also had a rapid washout, especially from the plasma compartment.

Rye bread contains high amounts of benzoxazinoids, and in vitro studies have shown suppressive effects of selected benzoxazinoids on prostate cancer cells. Thus, research into benzoxazinoids as possible suppressors of prostate cancer is demanded. A pilot study was performed in which ten prostate cancer patients received a rye-enriched diet 1 week prior to prostatectomy. Plasma and urine samples were collected pre- and postintervention. Ten prostate biopsies were obtained from each patient and histologically evaluated. The biopsies exhibited concentrations above the detection limit of seven benzoxazinoids ranging from 0.15 to 10.59 ng/g tissue. An OPLS-DA analysis on histological and plasma concentrations of benzoxazinoids classified the subjects into two clusters. A tendency of higher benzoxazinoid concentrations toward the benign group encourages further investigations. Benzoxazinoids were quantified by an optimized LC-MS/MS method, and matrix effects were evaluated. At low concentrations in biopsy and plasma matrices the matrix effect was concentration-dependent and nonlinear. For the urine samples the general matrix effects were small but patient-dependent.