Real cells are complex systems where the various constituents interact in different ways such as regulation feedback and cascade reactions. The next step for the bioPISA GUVs towards artificial cells was to co-encapsulate enzymes acting in parallel to Mb but with an orthogonal activity (Figure ). β-Galactosidase (β-gal) is a robust, widely used enzyme for industrial biotransformations and prodrug activation, and was an ideal candidate as it does not interfere with Mb activity. Similarly to Mb, β-gal activity (the hydrolysis of fluorescein di-β-Dgalactopyranoside, FDG) was lower in the vesicles than for free enzyme (36 vs. 1730 RFU/minute), which could be due to a certain level of deactivation during the bioPISA, or more likely, due to diffusion limitation by the membrane (Figure ), the latter hypothesis being supported by the observation that the resulting fluorescein concentrated within the lumen of the GUVs (Figure ). Finally, a glucose oxidase (GOx) -Mb enzyme cascade 65 was established in the GUVs where the formation of the final product requires the synergy of two coencapsulated enzymes (Figure ). Driven by the GOx-catalysed reaction of glucose and oxygen into glucono-1,5lactone and H2O2, followed by a peroxidase reaction catalysed by Mb, the cascade led to the production of resorufin in the bioPISA-derived GUVs (Figure ). Thus, the bioPISA GUVs served as microreactors for enzyme cascades and were able to develop both fluorescence based on the substrates provided.