Anthocyanins (ACNs) are plant secondary metabolites responsible for most of the red, purple and blue colors of flowers, fruits and vegetables. They are increasingly used in the food and beverage industry as natural alternative to artificial colorants. Production of these compounds by fermentation of microorganisms would provide an attractive alternative. In this study, Saccharomyces cerevisiae was engineered for de novo production of the three basic anthocyanins, as well as the three main trans-flavan-3-ols. Enzymes from different plant sources were screened and efficient variants found for most steps of the biosynthetic pathway. However, the anthocyanidin synthase was identified as a major obstacle to efficient production. In yeast, this enzyme converts the majority of its natural substrates leucoanthocyanidins into the off-pathway flavonols. Nonetheless, de novo biosynthesis of ACNs was shown for the first time in yeast and for the first time in a single microorganism. It provides a framework for optimizing the activity of anthocyanidin synthase and represents an important step towards sustainable industrial production of these highly relevant molecules in yeast.

Highlights d Acinetobacter baylyi cells elongate while killing competing bacteria d Uptake of DNA from lysed cells triggers SOS response and division arrest d Unregulated DNA uptake can be costly during bacterial competition

The incidence of melanoma, the most fatal dermatological cancer, has dramatically increased over the last few decades. Modern targeted therapy with kinase inhibitors induces potent clinical responses, but drug resistance quickly develops. Combination therapy improves treatment outcomes. Therefore, novel inhibitors targeting aberrant proliferative signaling in melanoma via the MAPK/ ERK and PI3K/AKT pathways are urgently needed. Biosensors were combined that report on ERK/AKT activity with image-based highcontent screening and HPLC-based activity profiling. An in-house library of 2576 plant extracts was screened on two melanoma cell lines with different oncogenic mutations leading to pathological ERK/AKT activity. Out of 140 plant extract hits, 44 were selected for HPLC activity profiling. Active thymol derivatives and piperamides from Arnica montana and Piper nigrum were identified that inhibited pathological ERK and/or AKT activity. The pipeline used enabled an efficient identification of natural products targeting oncogenic signaling in melanoma.

The discovery of bioactive natural products remains a time-consuming and challenging task. The ability to link high-confidence metabolite annotations in crude extracts with activity would be highly beneficial to the drug discovery process. To address this challenge, HPLC-based activity profiling and advanced UHPLC-HRMS/MS metabolite profiling for annotation were combined to leverage the information obtained from both approaches on a crude extract scaled down to the submilligram level. This strategy was applied to a subset of an extract library screening aiming to identify natural products inhibiting oncogenic signaling in melanoma. Advanced annotation and data organization enabled the identification of compounds that were likely responsible for the activity in the extracts. These compounds belonged to two different natural product scaffolds, namely, brevipolides from a Hyptis brevipes extract and methoxylated flavonoids identified in three different extracts of Hyptis and Artemisia spp. Targeted isolation of these prioritized compounds led to five brevipolides and seven methoxylated flavonoids. Brevipolide A (1) and 6-methoxytricin (9) were the most potent compounds from each chemical class and displayed AKT activity inhibition with an IC50 of 17.6 ± 1.6 and 4.9 ± 0.2 μM, respectively.

A library of more than 2500 plant extracts was screened for activity on oncogenic signaling in melanoma cells. The ethyl acetate extract from the aerial parts of Artemisia argyi displayed pronounced inhibition of the PI3K/AKT pathway. Active compounds were tracked with the aid of HPLC-based activity profiling, and altogether 21 active compounds were isolated, including one novel dimerosequiterpenoid (1), one new disesquiterpenoid (2), three new guaianolides (3–5), 12 known sesquiterpenoids (6–17), and four known flavonoids (19–22). A new eudesmanolide derivative (13b) was isolated as an artifact formed by methanolysis. Compound 1 is the first adduct comprising a sesquiterpene lactone and a methyl jasmonate moiety. The absolute configurations of compounds 1 and 3–18 were established by comparison of their experimental and calculated ECD spectra. The absolute configuration for 2 was determined by X-ray diffraction analysis. Guaianolide 8 was the most potent sesquiterpene lactone, inhibiting the PI3K/AKT pathway with an IC50 value of 8.9 ± 0.9 μM.