Fig. 5.

Dynamic inducible bioproduction in S. cerevisiae ELMs. a) Schematic of bioproducing S.cerevisiae ELMs. These ELMs are fabricated by seeding BSA-PEGDA hydrogels with engineered S. cerevisiae. The ELMs are printed using a SLA 3-D printer and continuously cultured with media exchange every 2 days. For scheduled bioproduction, ELMs were dynamically induced to control expression of native and heterologous enzymes. b) Multi-input genetic programs in S. cerevisiae ELMs for multi-product biosynthesis: 1) copper (II)-inducible expression of dioxygenase enzyme (mjDOD gene product) with constitutive expression of P450 enzyme (CYP76AD5 gene product) for betaxanthins production, and 2) galactose-inducible expression of secreted proteinase A enzyme (scPEP4 gene). c) Bioproduction Induction cycle 1 consists of 3 phases (Phase 1 ​= ​7 days, Phase 2 ​= ​8 days, Phase 3 ​= ​12 days). Top: Alternating induction by selective addition of copper (II) inducer (orange) in Phases 1 and 3, and galactose inducer (green) in Phase 2. Bottom: Time-dependent betaxanthins and proteinase A production levels. Values represent the mean ​± ​standard deviation from n ​= ​3 technical replicates. d) Bioproduction Induction cycle 2 with 3 phases (phase lengths are identical to c). Top: Alternating induction by selective addition of galactose inducer (green) in Phases 1 and 3, and copper (II) inducer in Phase 2. Bottom: Time-dependent betaxanthins and proteinase A production levels. Values represent the mean ​± ​standard deviation from n ​= ​3 technical replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)