Flux redistribution of central carbon metabolism for efficient production of l‐tryptophan in Escherichia coli,Biotechnology and Bioengineering

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Flux redistribution of central carbon metabolism for efficient production of l‐tryptophan in Escherichia coli
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2021-01-05 , DOI: 10.1002/bit.27665
Bo Xiong _{1,

2} , Yongduo Zhu _{1,

2} , Daoguang Tian _{1,

2} , Shuai Jiang _{1,

2} , Xiaoguang Fan _{1,

2} , Qian Ma _{1,

2} , Heyun Wu ₃ , Xixian Xie _{1,

2}

Affiliation

Microbial production of l‐tryptophan (l‐trp) has received considerable attention because of its diverse applications in food additives and pharmaceuticals. Overexpression of rate‐limiting enzymes and blockage of competing pathways can effectively promote microbial production of l‐trp. However, the biosynthetic process remains suboptimal due to imbalanced flux distribution between central carbon and tryptophan metabolism, presenting a major challenge to further improvement of l‐trp yield. In this study, we redistributed central carbon metabolism to improve phosphoenolpyruvate (PEP) and erythrose‐4‐phosphate (E4P) pools in an l‐trp producing strain of Escherichia coli for efficient l‐trp synthesis. To do this, a phosphoketolase from Bifidobacterium adolescentis was introduced to strengthen E4P formation, and the l‐trp titer and yield increased to 10.8 g/L and 0.148 g/g glucose, respectively. Next, the phosphotransferase system was substituted with PEP‐independent glucose transport, meditated by a glucose facilitator from Zymomonas mobilis and native glucokinase. This modification improved l‐trp yield to 0.164 g/g glucose, concomitant with 58% and 40% decreases of acetate and lactate accumulation, respectively. Then, to channel more central carbon flux to the tryptophan biosynthetic pathway, several metabolic engineering strategies were applied to rewire the PEP‐pyruvate‐oxaloacetate node. Finally, the constructed strain SX11 produced 41.7 g/L l‐trp with an overall yield of 0.227 g/g glucose after 40 h fed‐batch fermentation in 5‐L bioreactor. This is the highest overall yield of l‐trp ever reported from a rationally engineered strain. Our results suggest the flux redistribution of central carbon metabolism to maintain sufficient supply of PEP and E4P is a promising strategy for efficient l‐trp biosynthesis, and this strategy would likely also increase the production of other aromatic amino acids and derivatives.

中文翻译：

中心碳代谢的通量再分配以在大肠杆菌中高效生产 l-色氨酸

l-色氨酸( l- trp)的微生物生产因其在食品添加剂和药品中的广泛应用而受到广泛关注。限速酶的过度表达和竞争途径的阻断可以有效促进微生物产生l- trp。然而，由于中心碳和色氨酸代谢之间的通量分布不平衡，生物合成过程仍然不理想，这对进一步提高l- trp 产量提出了重大挑战。在这项研究中，我们重新分配了中心碳代谢，以改善大肠杆菌产l- trp 菌株中的磷酸烯醇式丙酮酸 (PEP) 和赤藓糖-4-磷酸 (E4P) 池，以提高效率。l- trp合成。为此，引入了青春双歧杆菌的磷酸酮醇酶以增强 E4P 的形成，l- trp 滴度和产量分别增加到 10.8 g/L 和 0.148 g/g 葡萄糖。接下来，磷酸转移酶系统被 PEP 非依赖性葡萄糖转运取代，由来自运动发酵单胞菌的葡萄糖促进剂和天然葡萄糖激酶调节。此修改改进了l-trp 产量为 0.164 g/g 葡萄糖，同时乙酸和乳酸积累分别减少 58% 和 40%。然后，为了将更多的中心碳通量引导到色氨酸生物合成途径，应用了几种代谢工程策略来重新连接 PEP-丙酮酸-草酰乙酸节点。最后，构建的 SX11 菌株在 5-L 生物反应器中分批补料发酵 40 小时后产生 41.7 g/L l- trp，总产量为 0.227 g/g 葡萄糖。这是从合理工程菌株中报道的l- trp 的最高总产量。我们的研究结果表明，中心碳代谢的通量重新分配以维持 PEP 和E4P的充足供应是一种有前途的有效策略。-trp 生物合成，这种策略也可能会增加其他芳香族氨基酸和衍生物的产量。

更新日期：2021-02-18

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