Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of N-acetylglucosamine.
Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of N-acetylglucosamine.
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通过酶挖掘消除主客体不相容性，实现 N-乙酰氨基葡萄糖的高温生产
DOI：
10.1016/j.isci.2022.105774
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发表时间：
2023-01-20
期刊：
影响因子：
5.8
通讯作者：
Tao, Fei
中科院分区：
文献类型：
--
作者：
Wu, Yutong;Liu, Jiongqin;Han, Xiao;Meng, Xuanlin;Li, Mengke;Wang, Jing;Xue, Hongsong;Yang, Yuhan;Xu, Ping;Tao, Fei
生产宿主和非天然酶之间的主客体不相容性对合成生物学提出了艰巨的挑战，特别是嗜温衍生的酶和嗜热底盘之间的挑战。在本研究中，我们开发了一种嗜热酶挖掘策略，包括基于协同进化的自动筛选管道（http：cem.sjtu.edu.cn），基于计算的酶表征和基于基因合成的功能验证。以葡萄糖胺-6-磷酸乙酰转移酶（GNA 1）为例，我们成功挖掘出四种具有优异热稳定性和催化性能的新型GNA 1。还进行了基于AlphaFold 2生成的结构的计算和分析，以揭示其优异性能背后的机制。最后，在系统代谢工程和温度编程的帮助下，我们开采的GNA 1用于实现高温N-乙酰葡萄糖胺（GlcNAc）生产，其滴度高达119.3 g/L。该研究证明了酶挖掘策略的有效性，突出了从海量数据库中挖掘新酶的应用前景，为解决主客体不相容性问题提供了有效的解决方案。酶工程;合成生物学
The host-guest incompatibility between a production host and non-native enzymes has posed an arduous challenge for synthetic biology, particularly between mesophile-derived enzymes and a thermophilic chassis. In the present study, we develop a thermophilic enzyme mining strategy comprising an automated co-evolution-based screening pipeline (http://cem.sjtu.edu.cn), computation-based enzyme characterization, and gene synthesis-based function validation. Using glucosamine-6-phosphate acetyltransferase (GNA1) as an example, we successfully mined four novel GNA1s with excellent thermostabilities and catalytic performances. Calculation and analysis based on AlphaFold2-generated structures were also conducted to uncover the mechanism underlying their excellent properties. Finally, our mined GNA1s were used to enable the high-temperature N-acetylglucosamine (GlcNAc) production with high titers of up to 119.3 g/L, with the aid of systems metabolic engineering and temperature programming. This study demonstrates the effectiveness of the enzyme mining strategy, highlighting the application prospects of mining new enzymes from massive databases and providing an effective solution for tackling host-guest incompatibility. Enzyme engineering; Synthetic biology
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