高通量筛选技术在菌种进化中的研究进展

doi:10.16085/j.issn.1000-6613.2018-1637

化工进展 ›› 2019, Vol. 38 ›› Issue (05): 2402-2412.DOI: 10.16085/j.issn.1000-6613.2018-1637

高通量筛选技术在菌种进化中的研究进展

杨祖明^1,²(),王颖^1,²,姚明东^1,²(),肖文海^1,²

1. 天津大学系统生物工程教育部重点实验室，天津 300072
2. 天津化学化工协同创新中心合成生物学平台，天津 300072

收稿日期:2018-08-09 修回日期:2018-10-16 出版日期:2019-05-05 发布日期:2019-05-05
通讯作者: 姚明东
作者简介:<named-content content-type="corresp-name">杨祖明</named-content>（1993—），男，硕士研究生，研究方向为合成生物学。E-mail：<email>zumingyang@tju.edu.cn</email>。|姚明东，副研究员，研究方向为细胞工程与酶工程。E-mail：<email>mingdong.yao@tju.edu.cn</email>。
基金资助:
国家自然科学基金青年基金项目(31600052);天津市创新平台与人才项目(16PTSYJC00050和16PTGCCX00140)

High-throughput screening technology in strain evolution

Zuming YANG^1,²(),Ying WANG^1,²,Mingdong YAO^1,²(),Wenhai XIAO^1,²

1. Key Laboratory of Systems Bioengineering （Ministry of Education）, Tianjin University, Tianjin 300072, China
2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China

Received:2018-08-09 Revised:2018-10-16 Online:2019-05-05 Published:2019-05-05
Contact: Mingdong YAO

摘要/Abstract

摘要：

菌种进化工程是绿色生物制造的重要策略，利用高效的高通量筛选方法和技术可以快速地获得理想的实用菌株。针对菌种进化工程中的高通量筛选方法，本文重点综述了基于颜色或荧光、基于细胞生长、基于生物传感器以及基于液滴微流体平台等4个方面的高通量筛选技术的重要进展，同时也介绍了各种高通量筛选技术的应用范围和特点，为研究人员从不同进化文库中获得生理特性或者代谢能力显著提高的目标菌株提供了理论指导，极大地提高进化文库的筛选效率，降低了菌株筛选的时间和成本。最后展望了人工智能、合成生物学以及生物信息学的发展对高通量筛选技术的重要影响，以期提高高通量筛选技术的精度、效率和应用范围，进而加速菌种进化过程和工业化进程。

关键词: 菌种进化工程, 绿色生物制造, 高通量筛选, 进化文库

Abstract:

Strain evolution engineering is an important strategy for green bio-manufacturing. High-throughput screening methods and techniques can be used to quickly obtain ideal strains. In view of the high-throughput screening methods in strain evolution engineering, this paper focuses on the important advances in high-throughput screening techniques based on color or fluorescence, cell growth, biosensor, and droplet microfluidic platforms. At the same time, the application range and characteristics of various high-throughput screening technologies were introduced, providing theoretical guidance for researchers to obtain ideal strain having physiological or metabolic capacity significantly improved from different evolutionary libraries, so the screening efficiency can be greatly improved and the time and cost can be reduced. Finally, to the future studies shall focus on the important influence of the development of artificial intelligence, synthetic biology and bioinformatics on high-throughput screening technology, in order to improve the accuracy, efficiency and application range of high-throughput screening technology, and accelerate the evolution process and industrialization process of strains.

Key words: strain evolution engineering, green biomanufacture, high-throughput screening, evolutionary library

中图分类号:

Q819

杨祖明, 王颖, 姚明东, 肖文海. 高通量筛选技术在菌种进化中的研究进展[J]. 化工进展, 2019, 38(05): 2402-2412.

Zuming YANG, Ying WANG, Mingdong YAO, Wenhai XIAO. High-throughput screening technology in strain evolution[J]. Chemical Industry and Engineering Progress, 2019, 38(05): 2402-2412.

图/表 13

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菌株	进化手段	进化目的	突变文库大小	筛选方法	参考文献
莱茵衣藻	基因重组	提高细胞脂质积累	6×10⁴	基于细胞颜色筛选	[8]
酵母	UV诱变	提高菌株α-淀粉酶的分泌能力	10⁵	液滴微流体平台筛选	[9]
谷氨酸棒杆菌	ARTP诱变	提升L-丝氨酸的生产能力	1.2?×?10⁵	生物传感器筛选	[10]
莱茵衣藻	甲磺酸乙酯化学诱变	增加细胞生长和脂质积累	2×10⁵	液滴微流体平台筛选	[11]
大肠杆菌	ARTP诱变	提高菌株L-赖氨酸的生产能力	10⁷	生物传感器筛选	[12]
大肠杆菌	ARTP诱变	提高菌株苏氨酸的生产能力	2 × 10⁷	生物传感器筛选	[13]

菌株	进化手段	进化目的	突变文库大小	筛选方法	参考文献
莱茵衣藻	基因重组	提高细胞脂质积累	6×10⁴	基于细胞颜色筛选	[8]
酵母	UV诱变	提高菌株α-淀粉酶的分泌能力	10⁵	液滴微流体平台筛选	[9]
谷氨酸棒杆菌	ARTP诱变	提升L-丝氨酸的生产能力	1.2?×?10⁵	生物传感器筛选	[10]
莱茵衣藻	甲磺酸乙酯化学诱变	增加细胞生长和脂质积累	2×10⁵	液滴微流体平台筛选	[11]
大肠杆菌	ARTP诱变	提高菌株L-赖氨酸的生产能力	10⁷	生物传感器筛选	[12]
大肠杆菌	ARTP诱变	提高菌株苏氨酸的生产能力	2 × 10⁷	生物传感器筛选	[13]

宿主细胞	响应物质	生物传感器筛选结果或作用	参考文献
谷氨酸棒杆菌	L-缬氨酸	目标突变体L-缬氨酸的产量提升了25％，副产物减少了3～4倍	[30]
酿酒酵母	木糖	开发了一套木糖转运蛋白高通量筛选方法，并获得了一种优良的突变体，其木糖转运能力提高了6.5倍	[31]
大肠杆菌	酪氨酸	目标突变体酪氨酸产量提升5倍	[32]
大肠杆菌	1-丁醇	目标突变体1-丁醇产量提升35%	[33]
大肠杆菌	葡萄糖二酸和柚皮素	目标突变体葡萄糖二酸产量提升22倍，柚皮素产量提升36倍	[34]
大肠杆菌	乙醇脱氢酶	乙醇脱氢酶对底物的特异性地得到提高	[35]
大肠杆菌	三乙酸内酯	成功突变天然转录因子 AraC特异性响应细胞内三乙酸内酯	[41,42]
大肠杆菌	甲羟戊酸	成功突变天然转录因子 AraC特异性响应细胞内甲羟戊酸	[43]
大肠杆菌	D-阿拉伯糖	成功突变天然转录因子 AraC特异性响应细胞内D-阿拉伯糖信号	[44]
大肠杆菌	四氢嘧啶	成功突变天然转录因子 AraC特异性响应细胞内四氢嘧啶，并成功筛选到高产四氢嘧啶的目标突变体	[45]
酿酒酵母	丙二酰辅酶A	目标突变体丙二酰辅酶A表达提高，下游产物3-羟基丙酸的产量提升了120%	[46]

宿主细胞	响应物质	生物传感器筛选结果或作用	参考文献
谷氨酸棒杆菌	L-缬氨酸	目标突变体L-缬氨酸的产量提升了25％，副产物减少了3～4倍	[30]
酿酒酵母	木糖	开发了一套木糖转运蛋白高通量筛选方法，并获得了一种优良的突变体，其木糖转运能力提高了6.5倍	[31]
大肠杆菌	酪氨酸	目标突变体酪氨酸产量提升5倍	[32]
大肠杆菌	1-丁醇	目标突变体1-丁醇产量提升35%	[33]
大肠杆菌	葡萄糖二酸和柚皮素	目标突变体葡萄糖二酸产量提升22倍，柚皮素产量提升36倍	[34]
大肠杆菌	乙醇脱氢酶	乙醇脱氢酶对底物的特异性地得到提高	[35]
大肠杆菌	三乙酸内酯	成功突变天然转录因子 AraC特异性响应细胞内三乙酸内酯	[41,42]
大肠杆菌	甲羟戊酸	成功突变天然转录因子 AraC特异性响应细胞内甲羟戊酸	[43]
大肠杆菌	D-阿拉伯糖	成功突变天然转录因子 AraC特异性响应细胞内D-阿拉伯糖信号	[44]
大肠杆菌	四氢嘧啶	成功突变天然转录因子 AraC特异性响应细胞内四氢嘧啶，并成功筛选到高产四氢嘧啶的目标突变体	[45]
酿酒酵母	丙二酰辅酶A	目标突变体丙二酰辅酶A表达提高，下游产物3-羟基丙酸的产量提升了120%	[46]

宿主细胞	响应物质	生物传感器筛选结果或作用	参考文献
大肠杆菌	茶碱	鉴定出了增加茶碱生产的最佳生物元件组合	[49]
大肠杆菌	氰尿二酰胺	改造设计正交核糖体开关，不再响应细胞内天然配体分子，而特异性响应非天然小分子氰尿二酰胺	[50]
酿酒酵母	6-磷酸葡糖胺	能够有效分离高产n-乙酰氨基葡糖的突变体	[51]
苜蓿根瘤菌	VB12	获得了一株优良的阳性突变体，VB12产量比野生型提高了21.9％	[52]

高通量筛选技术在菌种进化中的研究进展

High-throughput screening technology in strain evolution

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宿主细胞	响应物质	生物传感器筛选结果或作用	参考文献
大肠杆菌	6-磷酸海藻糖	实时监测细胞内的6-磷酸海藻糖浓度	[59]
大肠杆菌和酵母	蛋氨酸	细胞内蛋氨酸监测	[60]
大肠杆菌	亮氨酸	细胞内亮氨酸监测	[61]
大肠杆菌	抗生素	建立了高通量筛选潜在抗生素分子的方法	[62]
大肠杆菌	肌醇1,4,5-三磷酸	建立了高通量筛选小分子肌醇1,4,5-三磷酸的方法	[63]
酿酒酵母	蛋白酶	成功筛选出一种对底物选择性提高30倍的突变体	[64]