Recombinant microbes have emerged as promising alternatives to natural sources of naringenin—a key molecular scaffold for flavonoids. In recombinant strains, expression levels of the pathway genes should be optimized at both transcription and the translation stages to precisely allocate cellular resources and maximize metabolite production. However, the optimization of the expression levels of naringenin generally relies on evaluating a small number of variants from libraries constructed by varying transcription efficiency only. In this study, we introduce a systematic strategy for the multi-level optimization of biosynthetic pathways. We constructed a multi-level combinatorial library covering both transcription and translation stages using synthetic T7 promoter variants and computationally designed 5′-untranslated regions. Furthermore, we identified improved strains through high-throughput screening based on a synthetic naringenin riboswitch. The most-optimized strain obtained using this approach exhibited a 3-fold increase in naringenin production, compared with the parental strain in which only the transcription efficiency was modulated. Furthermore, in a fed-batch bioreactor, the optimized strain produced 260.2 mg/L naringenin, which is the highest concentration reported to date using glycerol and p-coumaric acid as substrates. Collectively, this work provides an efficient strategy for the expression optimization of the biosynthetic pathways.

重组微生物已成为柚皮素天然来源的有前途的替代品，柚皮素是黄酮类化合物的关键分子支架。在重组菌株中，通路基因的表达水平应在转录和翻译阶段进行优化，以精确分配细胞资源并最大限度地提高代谢产物的产量。然而，柚皮素表达水平的优化通常依赖于评估来自仅通过不同转录效率构建的文库的少量变体。在这项研究中，我们引入了一种用于生物合成途径多级优化的系统策略。我们使用合成的 T7 启动子变体和计算设计的 5' 非翻译区构建了一个涵盖转录和翻译阶段的多级组合文库。此外，我们通过基于合成柚皮素核糖开关的高通量筛选鉴定了改良菌株。与仅调节转录效率的亲本菌株相比，使用这种方法获得的最优化菌株的柚皮素产量增加了 3 倍。此外，在分批补料生物反应器中，优化的菌株产生了 260.2 mg/L 的柚皮素，这是迄今为止使用甘油和对香豆酸作为底物。总的来说，这项工作为生物合成途径的表达优化提供了一种有效的策略。