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 92% 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.

中文翻译：

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稳定酵母中长期合成代谢产物形成的调节控制电路

由于使用有限的天然辅因子可利用性和/或表达负担，用于生产化学药品，酶和治疗剂的工程化活细胞会给细胞造成负担，与通过长进化轨迹编码的亲代细胞使适应性最大化相比，适应性不足。最终，这种差异给生物适应性工程细胞在长期的生物过程中施加了选择性压力，并有可能导致在种群水平上彻底根除高性能工程细胞。在这里，我们介绍了为香草醛-β-葡萄糖苷生产而设计的适合健身的酵母细胞的突变情况。下一个，我们基于转录组分析和对香草醛-β-葡萄糖苷途径中间体有反应的生物传感器设计了合成控制电路，以便在连续传代实验中稳定香草醛-β-葡萄糖苷的生产约55代。此外，使用具有两种不同作用方式的生物传感器，我们确定了将香草醛-β-葡萄糖苷途径通量与各种基本细胞功能联系起来的控制电路，并证明了控制电路的鲁棒性和香草醛-β-葡萄糖苷产量提高了92％，其中包括5倍的增加。与没有对照回路的产生香草醛-β-葡萄糖苷的细胞相比，在补料分批发酵中香草醛-β-葡萄糖苷途径的总代谢物积累。