Shikimate is a key intermediate in high demand for synthesizing valuable antiviral drugs, such as the anti-influenza drug and oseltamivir (Tamiflu®). Microbial-based shikimate production strategies have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. Although shikimate biosynthesis has been reported in several engineered bacterial species, the shikimate production yield is still unsatisfactory. This study designed an Escherichia coli cell factory and optimized the fed-batch culture process to achieve a high titer of shikimate production. Using the previously constructed dehydroshikimate (DHS)-overproducing E. coli strain, two genes (aroK and aroL) responsible for converting shikimate to the next step were disrupted to facilitate shikimate accumulation. The genes with negative effects on shikimate biosynthesis, including tyrR, ptsG, and pykA, were disrupted. In contrast, several shikimate biosynthetic pathway genes, including aroB, aroD, aroF, aroG, and aroE, were overexpressed to maximize the glucose uptake and intermediate flux. The shiA involved in shikimate transport was disrupted, and the tktA involved in the accumulation of both PEP and E4P was overexpressed. The rationally designed shikimate-overproducing E. coli strain grown in an optimized medium produced approximately 101 g/l of shikimate in 7-l fed-batch fermentation, which is the highest level of shikimate production reported thus far. Overall, rational cell factory design and culture process optimization for microbial-based shikimate production will play a key role in complementing traditional plant-derived shikimate production processes.

中文翻译：

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用于大肠埃希菌莽草酸生产的人工细胞工厂设计

Shikimate 是合成有价值的抗病毒药物（如抗流感药物和奥司他韦 (Tamiflu®)）的关键中间体。已经开发了基于微生物的莽草酸生产策略，以克服源自传统植物提取工艺的莽草酸供应不稳定和昂贵的问题。尽管已经在几种工程菌种中报道了莽草酸的生物合成，但莽草酸的产量仍然不能令人满意。本研究设计了一个大肠杆菌细胞工厂，并优化了补料分批培养工艺，以实现莽草酸的高滴度生产。使用先前构建的脱氢莽草酸 (DHS) 过量生产大肠杆菌菌株，负责将莽草酸转化为下一步的两个基因（aroK 和 aroL）被破坏以促进莽草酸的积累。对莽草酸生物合成具有负面影响的基因，包括 tyrR、ptsG 和 pykA，被破坏。相比之下，一些莽草酸生物合成途径基因，包括 aroB、aroD、aroF、aroG 和 aroE，被过表达以最大化葡萄糖摄取和中间通量。参与莽草酸运输的 shiA 被破坏，参与 PEP 和 E4P 积累的 tktA 被过度表达。合理设计的莽草酸过量生产大肠杆菌菌株在优化培养基中生长，在 7 升补料分批发酵中产生大约 101 g/l 莽草酸，这是迄今为止报道的最高莽草酸产量。全面的，