Heterologous naringenin production in the filamentous fungus Penicillium rubens

Naringenin is a natural product with several reported bioactivities and is the key intermediate for the entire class of plant flavonoids. The translation of flavonoids into modern medicine as pure compounds is often hampered by their low abundance in nature and difficult chemical synthesis. Here, we investigated new avenues toward producing high levels of naringenin in microbial hosts. Penicillium rubens is a well characterized and highly engineered traditional "workhorse" for the production of {beta}-lactam antibiotics and cholesterol-lowering statins. We explored a secondary metabolite deficient P. rubens strain, P. rubens 4xKO, that was derived from an earlier industrial production strain as a promising microbial host for a recombinant flavonoid pathway. By integrating two plant genes encoding for enzymes in the naringenin biosynthesis pathway into the genome of this strain, we achieved a high naringenin titer in flask fermentations 36 h after feeding the precursor p-coumaric acid. Along with the rapid product accumulation of up to an 88% molar yield, we also observed rapid degradation of naringenin. Based on high-resolution mass spectrometric analysis, we identified the degradation products and proposed a naringenin degradation pathway in P. rubens 4xKO, which is distinct from other flavonoid-converting pathways reported in fungi. Our approach combines fundamental research with application-oriented microbial engineering, and our findings will pave the way to the more sustainable and economically feasible production of flavonoids for pharmaceutical and nutraceutical applications.