Regulatory control circuits for stabilizing long-term anabolic product formation in yeast

Abstract

Engineering living cells for production of chemicals, enzymes and therapeutics can burden cells due to use of limited native co-factor availability and/or expression burdens, totalling a fitness deficit compared to parental cells encoded through long evolutionary trajectories to maximise fitness. Ultimately, this discrepancy puts a selective pressure against fitness-burdened engineered cells under prolonged bioprocesses, and potentially leads to complete eradication of high-performing engineered cells at the population level. Here we present the mutation landscapes of fitness-burdened yeast cells engineered for vanillin-{beta}-glucoside production. Next, we design synthetic control circuits based on transcriptome analysis and biosensors responsive to vanillin-{beta}-glucoside pathway intermediates in order to stabilize vanillin-{beta}-glucoside production over [~]55 generations in sequential passage experiments. Furthermore, using biosensors with two different modes of action we identify control circuits linking vanillin-{beta}-glucoside pathway flux to various essential cellular functions, and demonstrate control circuits robustness and 92% higher vanillin-{beta}-glucoside production, including 5-fold increase in total vanillin-{beta}-glucoside pathway metabolite accumulation, in a fed-batch fermentation compared to vanillin-{beta}-glucoside producing cells without control circuits.