Rawoof, A.; Lin, Y. T.; Rajendran, S.; Antoine, G.; Jayasundara, S.; Cai, Y.; Singh, D.; Whitehead, P.; Dornberger, H.; Mall, S.; Alonso, A. P.; Carroll, M. C.; Skellam, E.; Chapman, K. D.

Commercial penicillin production has relied on microbial fermentation for more than 80 years. Here, we engineered the plant, Nicotiana benthamiana, to produce penicillin G in its leaves by transient expression of up to seven fungal biosynthetic genes. Remarkably, all recombinant proteins localize to the analogous subcellular compartments without engineering signal peptide sequences or post-translational modification sites. Although non-ribosomal peptide synthetases occur widely in fungi and bacteria to produce a plethora of specialized metabolites, their evolutionary distribution does not extend to plants. Our results now open a new metabolic frontier for natural product synthesis, and offer possibilities to address global health concerns through an alternative biotechnology platform for fungal-derived pharmaceutical production.