Development of a platform yeast strain for cytochrome P450-containing pathway

Research Project, 2022 – 2025

Natural products are an immense source of bioactive compounds for the pharmaceutical, cosmetic and food industry. As synthesis levels in their native hosts are often low, microorganisms have been established as cell factories for natural products such as terpenoids and flavonoids.
However, the functional expression of these multi-step pathways in hosts like yeast can be intricate. A specific challenge are cytochrome P450s (CYPs), a superfamily of enzymes containing a heme-iron core and anchored in the membrane of the endoplasmic reticulum (ER) that often show low initial activity.In this project, we aim to create a platform yeast strain optimized for expression of heterologous CYPs. Different engineering strategies such as increasing co-factor supply and enhancing the ER surface area will be combined.In addition, we will use a multiplex CRISPRi approach to further modify ER architecture and phospholipid composition. A strain library will be screened with the help of metabolite biosensors indicating CYP activity using a double emulsion FACS approach. Cells with increased CYP activity will be analysed by next-generation sequencing and the underlying mechanisms elucidated.The final strain combining all strategies will be used to improve the production of abscisic acid, a plant hormone conferring drought tolerance and two flavonoid antioxidants in yeast. In addition, this project will also lead to a better fundamental understanding of factors influencing CYP activity in yeast.