Fig. 1: A comprehensive experimental setup for systematic characterization of heterologous protein secretion.

a The platform uses 8 bioreactors for continuous culture of yeast cells. An accessory strain (blue cells) is co-cultured with the strain of interest (white cells) in an initial 1:10 ratio. Output flow lines of reactors are connected to a pipetting robot that prepares the samples and loads them into the cytometer every 45 min. b To monitor internal and secreted levels of the heterologous protein under study, the protein is fused to a “bright tag” that comprises of a green fluorescent reporter, mNeonGreen, followed by three copies of the FLAG tag. c All modules are integrated in yeast chromosomes. The optogenetic transcription factor EL222 is constitutively expressed (TDH3 promoter, pConst) and activates the pLight promoter upon blue light stimulation. Then, the POI is expressed and may produce secretory-associated stress, activating the unfolded protein response that regulates the expression of genes under the control of the pStress promoter. We introduced a red fluorescent reporter, mScarlet-I, under the control of pStress to monitor secretory-associated stress levels (pUPR). d To quantify secretion levels using a cytometer, we developed a methodology based on immuno-magnetic beads capturing only the secreted POI. The distinction between beads and remaining cells is made thanks to their different light scatter properties (Supplementary Note 4) e Schematic representation of our strategy to study protein trafficking and secretion capabilities of cells. The production demand is controlled by blue light illumination of the cell culture. The production of the protein is followed by its translocation into the secretory compartments. Internal protein levels in these compartments (iPOI levels) can be followed by measuring the green fluorescence of cells. The protein can be secreted in the media or can accumulate in the cell. Protein accumulation triggers a secretory stress (UPR stress levels) that can be quantified by measuring the red fluorescence of the cells. The cell stress responses have antagonistic effects with respect to bioproduction: increasing trafficking capabilities or targeting the accumulated proteins for degradation. f Example of data obtained by the proposed pipeline. The intensity of the blue color corresponds to the intensity of the light stimulation received by cells in the bioreactor, as reported by the induction level of the co-cultured accessory strain. This corresponds to the protein production demand. RPU: relative promoter units. Source data are provided as a Source Data file.