Fine-tuning the (2S)-naringenin synthetic pathway using an iterative high-throughput balancing strategy
Fine-tuning the (2S)-naringenin synthetic pathway using an iterative high-throughput balancing strategy
复制标题
使用迭代高通量平衡策略微调 (2S)-柚皮素合成途径
DOI：
10.1002/bit.26941
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发表时间：
2019
影响因子：
3.8
通讯作者：
Zhou Jingwen
中科院分区：
文献类型：
--
作者：
Zhou Shenghu;Lyu Yunbin;Li Huazhong;Koffas Mattheos A G;Zhou Jingwen
代谢工程始终要求产生最大的碳和能量通量的目标化学品。为了平衡代谢通量，人工合成途径的基因表达水平通常使用多模块优化策略进行微调。然而，正向构建是一个工程难题，因为需要构建和分析大量可能的途径组合。在这里，建立了迭代高通量平衡（IHTB）策略，以彻底微调（2S）-柚皮素生物合成途径。将一系列大肠杆菌梯度组成型启动子随机克隆到途径基因的上游，利用基于AlCl 3与（2S）-柚皮素相互作用的紫外-荧光分光光度法高通量筛选文库。分析筛选的高滴度菌株的代谢通量，并根据分析结果进行迭代筛选。几轮后，（2S）-柚皮素合成途径的代谢通量平衡，最终滴度达到191.9 mg/L，其中29.2 mg/Lp-香豆酸蓄积。查尔酮合酶被推测为限速酶，因为其表达水平与（2S）-柚皮素和p-香豆酸的产生密切相关。建立的IHTB策略可用于有效平衡多基因途径，这将加速高效重组菌株的开发。
Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine‐tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high‐throughput balancing (IHTB) strategy was established to thoroughly fine‐tune the (2S)‐naringenin biosynthetic pathway. A series of gradient constitutive promoters fromEscherichia coliwere randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry–fluorescence spectrophotometry high‐throughput method, which was established based on the interactions between AlCl3and (2S)‐naringenin. The metabolic flux of the screened high‐titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)‐naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/Lp‐coumaric acid accumulation. Chalcone synthase was speculated to be the rate‐limiting enzyme because its expression level was closely related to the production of both (2S)‐naringenin andp‐coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains.