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

  • D'Ambrosio, Vasil
  • Dore, Eleonora
  • Di Blasi, Roberto
  • van den Broek, Marcel
  • Sudarsan, Suresh
  • Horst, Jolanda Ter
  • Ambri, Francesca
  • Sommer, Morten OA
  • Rugbjerg, Peter
  • Keasling, Jay D
  • Mans, Robert
  • Jensen, Michael K
Publication Date
Sep 01, 2020
eScholarship - University of California
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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.

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