We have developed a novel selection circuit based on carbon source utilization that establishes and sustains growth–production coupling over several generations in a medium with maltose as the sole carbon source. In contrast to traditional antibiotic resistance-based circuits, we first proved that coupling of cell fitness to metabolite production by our circuit was more robust with a much lower escape risk even after many rounds of selection. We then applied the selection circuit to the optimization of L-tryptophan (l-Trp) production. We demonstrated that it enriched for specific mutants with increased l-Trp productivity regardless of whether it was applied to a small and defined mutational library or a relatively large and undefined one. From the latter, we identified four novel mutations with enhanced l-Trp output. Finally, we used it to select for several high l-Trp producers with randomly generated genome-wide mutations and obtained strains with up to 65% increased l-Trp production. This selection circuit provides new perspectives for the optimization of microbial cell factories for diverse metabolite production and the discovery of novel genotype–phenotype associations at the single-gene and whole-genome levels.

中文翻译：

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麦芽糖利用作为一种新的选择策略，可不断提高微生物代谢产物的产量

我们开发了一种基于碳源利用的新型选择电路，该电路在以麦芽糖为唯一碳源的培养基中建立并维持了几代人的生长－生产耦合。与传统的基于抗生素抗药性的回路相反，我们首先证明了即使经过多轮选择，细胞回路适应性与我们回路产生的代谢物的结合也更牢固，逃逸风险也低得多。然后，我们将选择电路应用于L-色氨酸（l -Trp）生产的优化。我们证明了它可以增加l增加的特定突变体-Trp生产率，无论是应用于小型且定义明确的突变库还是相对大型且未定义的突变库。从后者，我们鉴定出具有增强的1- Trp输出的四个新颖的​​突变。最后，我们用它来选择几个具有随机产生的全基因组突变的高I- Trp产生者，并获得了I- Trp产生最多增加65％的菌株。该选择电路为优化微生物细胞工厂以生产各种代谢物提供了新的见解，并为单基因和全基因组水平的新型基因型-表型关联的发现提供了新的思路。