As a key molecular scaffold for various flavonoids, naringenin is a value-added chemical with broad pharmaceutical applicability. For efficient production of naringenin from acetate, it is crucial to precisely regulate the carbon flux of the OAA-PEP regulatory node through appropriate pckA expression control, as excessive overexpression of pckA can cause extensive loss of OAA and metabolic imbalance. However, considering the critical impact of pckA on naringenin biosynthesis, the conventional strategy of transcriptional regulation of gene expression is limited in its ability to cover the large and balanced solution space, which could result in suboptimal naringenin production. To overcome this hurdle, in this study, pckA expression was fine-tuned at both the transcriptional and translational levels for the precise exploration of optimal naringenin production from acetate. Specifically, a combinatorial expression library was generated using promoters with different strengths and rationally designed 5′-UTR variants with discrete translation efficiency. Additionally, we identified the effects of regulating pckA expression by validating the correlation between PCK activity and naringenin production. As a result, the flux-optimized strain exhibited a significant increase in naringenin production, with a 49.8-fold increase compared to the unoptimized strain, producing 122.12 mg/L of naringenin. Collectively, this study demonstrated the significance of transcriptional and translational flux rebalancing at the key regulatory node, proposing a pivotal metabolic engineering strategy for the biosynthesis of various flavonoids derived from naringenin using acetate.

中文翻译：

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关键调控节点的转录和翻译通量优化，以在工程大肠杆菌中使用乙酸盐提高柚皮素的产量

作为各种黄酮类化合物的关键分子支架，柚皮素是一种具有广泛药用价值的增值化学品。为了从乙酸盐中高效生产柚皮素，通过适当的 pckA 表达控制来精确调节 OAA-PEP 调节节点的碳通量至关重要，因为 pckA 的过度过度表达会导致 OAA 的广泛损失和代谢失衡。然而，考虑到pckA对柚皮素生物合成的关键影响，传统的基因表达转录调控策略在覆盖大且平衡的溶液空间的能力方面受到限制，这可能导致柚皮素生产不理想。为了克服这一障碍，在本研究中，pckA 表达在转录和翻译水平上进行了微调，以精确探索从乙酸盐中产生柚皮素的最佳方法。具体来说，使用不同强度的启动子和合理设计的具有离散翻译效率的5'-UTR变体生成组合表达文库。此外，我们通过验证 PCK 活性和柚皮素生成之间的相关性来确定调节 pckA 表达的效果。结果，通量优化的菌株柚皮素产量显着增加，与未优化的菌株相比增加了49.8倍，产生122.12 mg/L的柚皮素。总的来说，这项研究证明了关键调控节点转录和翻译通量再平衡的重要性，提出了使用乙酸盐生物合成柚皮素衍生的各种黄酮类化合物的关键代谢工程策略。