Due to the worsening antimicrobial resistance crisis, there is a clear urgent need to identify new antimicrobials, revisit underexplored antimicrobials and explore new platforms to effectively produce antimicrobials. Pleuromutilin is an antibiotic, predominantly active against Gram-positive bacteria, produced by the fungus Clitopilus passeckerianus. The pleuromutilin antibiotics have previously shown low rates of resistance development, and minimal cross-resistance with other antibiotics, however, the pleuromutilin compound is expensive to produce in high titres due to the slow-growing nature of its native host. Previous studies have elucidated the biosynthetic pathway and achieved pleuromutilin production in the heterologous host Aspergillus oryzae. This project attempts to further exploit this heterologous host for production of novel pleuromutilin derivatives by heterologous expression of combinations of biosynthetic enzymes that have not previously been explored. These compounds can then be assessed for antibacterial activity. This project also investigates the feasibility of using plants as heterologous hosts to improve pleuromutilin titres, by heterologous expression of biosynthetic enzymes in the plant host Nicotiana benthamiana. Since N. benthamiana has been used as a heterologous host for various plant diterpenes, we are looking to identify whether it could be engineered to produce this fungal diterpene. If successful, this opens up an avenue for production of other useful antibacterials using plant hosts. Overall, this project aims to identify novel pleuromutilin antibacterials that could be explored as treatment options for bacterial pathogens, and to assess the feasibility of using plants as non-native hosts for efficient production of useful antibacterial compounds.