De novo biosynthesis of the hops bioactive flavonoid xanthohumol in yeast

The flavonoid xanthohumol is an important flavor substance in the brewing industry that has a wide variety of bioactivities. However, its unstable structure is easily oxidized during the brewing process, resulting in its low content in beer. Moreover, its extraction and purification from plants requires laborious and expensive procedures. Microbial biosynthesis is considered a sustainable and economically viable alternative to supply natural products. However, the complex structures of natural products and regulation of the biosynthetic pathways make it challenging to construct and optimize the xanthohumol pathway in microbes. Here, we harnessed the brewing yeast Saccharomyces cerevisiae for the de novo biosynthesis of xanthohumol from glucose by modular fine-tuning of the competitive metabolic pathways, prenyltransferase engineering, enhancing precursor supply, substrate channeling, and peroxisomal engineering. These strategies significantly improved the production of the key xanthohumol precursor demethylxanthohumol (DMX) by 83-fold and achieved the de novo biosynthesis of xanthohumol for the first time in a microbial cell factory. We also revealed that prenylation is the key limiting step in DMX biosynthesis and that enhancing the supply of dimethylallyl pyrophosphate (DMAPP) and substrate channeling helps to drive the metabolic flux toward DMX biosynthesis, which should be helpful for improving the production of prenylated natural products. Our work provides feasible approaches for systematically engineering yeast cell factories for the de novo biosynthesis of complex natural products.