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

Vasil D'ambrosio, Eleonora Dore, Roberto Di Blasi, Marcel van den Broek, Suresh Sudarsan, Jolanda ter Horst, Francesca Ambri, Morten Otto Alexander Sommer, Peter Rugbjerg, Jay D. Keasling, Robert Mans, Michael Krogh Jensen

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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-β-glucoside production. Next, we design synthetic control circuits based on transcriptome analysis and biosensors responsive to vanillin-β-glucoside pathway intermediates in order to stabilize vanillin-β-glucoside production over ~55 generations in sequential passage experiments. Furthermore, using biosensors with two different modes of action we identify control circuits linking vanillin-β-glucoside pathway flux to various essential cellular functions, and demonstrate control circuits robustness and almost 2-fold higher vanillin-β-glucoside production, including 5-fold increase in total vanillin-β-glucoside pathway metabolite accumulation, in a fed-batch fermentation compared to vanillin-β-glucoside producing cells without control circuits.
Original languageEnglish
JournalMetabolic Engineering
Pages (from-to)369-380
Publication statusPublished - 2020


  • Stability
  • Yeast
  • Production
  • Biosensor
  • Essential genes
  • Control circuits


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