细胞编程技术：助力高效细胞工厂的设计

doi:10.13560/j.cnki.biotech.bull.1985.2023-1227

摘要/Abstract

摘要：

合成生物学是近年来蓬勃发展的一门涉及分子生物学、生物工程、微生物学、系统生物学等多个学科新型交叉学科，旨在利用生物学原理和工程方法创造全新的生物学系统和生物产品。合成生物学的发展也受到了高效“细胞工厂”理念的推动，这使得生物工程技术朝着工业化应用的方向迈出了重要一步。然而，受制于生产效率低、遗传不稳定、调控过程难等问题，如何获得转化效率高、鲁棒性强的“细胞工厂”仍然是合成生物学领域面临的重要任务。近年来，细胞工程和基因工程领域发展迅速，新型细胞元件、细胞底盘以及基因回路构造方式等技术逐渐成熟，其通过精确的基因编辑和调控，可实现对细胞的特定功能进行编程，例如增强细胞的代谢能力、改变细胞的分化路径以及设计新的细胞功能模块等，具有广泛的应用前景。本文从新型细胞元件、底盘细胞以及基因回路构造方式等角度，综述了近年来在细胞工程和基因工程领域发展迅速的细胞编程技术，这些技术的进步已经或者将要用于合成生物学的发展中，将会赋予工程菌更加强大的工作能力。

关键词: 合成生物学, 细胞编程, 细胞元件, 底盘细胞

Abstract:

Synthetic biology, an emerging interdisciplinary field, involves molecular biology, bioengineering, microbiology, and system biology, aiming to create entirely new biological systems and products using principles of biology and engineering methods. The conceptualization of the “cell factory” has propelled synthetic biology towards industrial applications, allowing the bioengineering technology having a big step towards industrial application. However, challenges such as low production efficiency, genetic instability, and intricate regulatory processes persist, hindering the creation of highly efficient and robust “cell factories” for transformation. In recent years, the fields of cell engineering and genetic engineering have developed rapidly, with the maturation of technologies such as new cell elements, cell chassis, and gene circuit construction methods. Through precise gene editing and regulation, these technologies can enable the programming of specific functions in cells, such as enhancing cell metabolism, altering cell differentiation pathways, and designing new cell functional modules, with broad application prospects.This review provides a comprehensive overview of rapidly evolving cell programming technologies. These technologies, situated within the realms of cell engineering and genetic engineering, encompass novel cellular components, cellular chassis concepts, and gene circuit construction methods. The strategic integration of these advancements aims to address the existing challenges in synthetic biology. The utilization of these technologies is poised to empower engineered bacteria with enhanced working capacities, thus paving the way for the development of more efficient and resilient “cell factories.”

Key words: synthetic biology, cell programming, cellular components, cell chassis

琚康辉, 田晓雅, 王立, 陈晶瑜. 细胞编程技术：助力高效细胞工厂的设计[J]. 生物技术通报, 2024, 40(10): 160-171.

JU Kang-hui, TIAN Xiao-ya, WANG Li, CHEN Jing-yu. Cell Programming Technology: Paving the Way for Efficient Cell Factories[J]. Biotechnology Bulletin, 2024, 40(10): 160-171.

图/表 5

参考文献 55

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细胞编程 Cell reprogramming		描述 Description	来源 Reference
遗传元件 Genetic elements	振荡器
	抑制振荡器-RFL	初代“repressilator”，3个转录抑制子建立的振荡网络，开发早、研究范围广、独立控制尚未实现	[8]
	双反馈振荡器-DFO	由两个诱导操纵子控制，可以通过控制诱导剂的浓度实现振荡器电路的独立控制，使得振荡器能够用于更复杂的电路设计	[10]
	存储器
	功能稳态型存储器	利用λ噬菌体的Cro蛋白诱导的CI/Cro转录开关作为“记忆元件”使得工程菌能够维持功能稳定至少6个月之久	[11]
	状态机	使用重组酶特有的DNA切除和反转功能在大肠杆菌中构建状态机（State machines），读取基因表达的状态，记录所有输入的时序，并对基因表达进行多输入、多输出的控制	[12]
	生物磁带记录器	使用基于CRISPR的适应系统，构建了一种“生物磁带记录器”，将其用来描述细胞动态和环境变化，从而相对准确地描述时间间隔生物信号和调节程序的能力	[13]
	生物传感器
	产物响应型生物传感器	基于产物响应的生物传感器，产物生成量越高，细胞传感荧光水平越强，以此建立了超高通量的筛选方式	[14]
	温敏型生物传感器	利用温敏型生物传感器控制启动子表达，解偶联细胞生长与合成的关系，极大地提高了类胡萝卜的产生	[15]
细胞底盘 Cell chassis	枯草芽孢杆菌 Bacillus subtilis	模式化的细胞工厂，生产工业用酶的优良底盘	[16]
	酪丁酸梭菌 Clostridium tyrobutyricum	在生产丁酸及丁醇的生产上有极高的生产效率，短链脂肪酸潜在的理性“细胞工厂”	[17]
	米曲霉 Aspergillus oryzae	在天然产物的生物合成领域有重要的研究价值	[18]
	嗜盐碱单胞菌 Natronomonas kamekura	用于高分子材料、化学品和燃料的生物制造	[19]
	解脂耶氏酵母 Yarrowia lipolytica	适合用于油脂及其衍生物的生物合成	[20]
	蓝藻	与上转换纳米粒子一起封装，能够创建工程化的微氧细胞工厂	[21]
	Minicell	无染色体细胞，药物递送的优良载体	[22]
	Simcell	无染色体细胞，安全可编程的通用平台	[23]
复杂基因逻辑回路设计Complex gene logic circuit design	AND、NOT联用型遗传电路设计	利用AND和NOT联用的基因电路，巧妙实现了细胞生长过渡到生物合成时间点的精确把控，减少外源电路引入对宿主菌株的代谢负担	[24]
复杂基因逻辑回路设计Complex gene logic circuit design	CASwitch	将CRISPR-Cas内环核酸酶与Tet-On3G诱导基因系统相结合而设计的高性能基因电路，用于哺乳动物系统中高性能诱导表达	[25]

细胞编程 Cell reprogramming		描述 Description	来源 Reference
遗传元件 Genetic elements	振荡器
	抑制振荡器-RFL	初代“repressilator”，3个转录抑制子建立的振荡网络，开发早、研究范围广、独立控制尚未实现	[8]
	双反馈振荡器-DFO	由两个诱导操纵子控制，可以通过控制诱导剂的浓度实现振荡器电路的独立控制，使得振荡器能够用于更复杂的电路设计	[10]
	存储器
	功能稳态型存储器	利用λ噬菌体的Cro蛋白诱导的CI/Cro转录开关作为“记忆元件”使得工程菌能够维持功能稳定至少6个月之久	[11]
	状态机	使用重组酶特有的DNA切除和反转功能在大肠杆菌中构建状态机（State machines），读取基因表达的状态，记录所有输入的时序，并对基因表达进行多输入、多输出的控制	[12]
	生物磁带记录器	使用基于CRISPR的适应系统，构建了一种“生物磁带记录器”，将其用来描述细胞动态和环境变化，从而相对准确地描述时间间隔生物信号和调节程序的能力	[13]
	生物传感器
	产物响应型生物传感器	基于产物响应的生物传感器，产物生成量越高，细胞传感荧光水平越强，以此建立了超高通量的筛选方式	[14]
	温敏型生物传感器	利用温敏型生物传感器控制启动子表达，解偶联细胞生长与合成的关系，极大地提高了类胡萝卜的产生	[15]
细胞底盘 Cell chassis	枯草芽孢杆菌 Bacillus subtilis	模式化的细胞工厂，生产工业用酶的优良底盘	[16]
	酪丁酸梭菌 Clostridium tyrobutyricum	在生产丁酸及丁醇的生产上有极高的生产效率，短链脂肪酸潜在的理性“细胞工厂”	[17]
	米曲霉 Aspergillus oryzae	在天然产物的生物合成领域有重要的研究价值	[18]
	嗜盐碱单胞菌 Natronomonas kamekura	用于高分子材料、化学品和燃料的生物制造	[19]
	解脂耶氏酵母 Yarrowia lipolytica	适合用于油脂及其衍生物的生物合成	[20]
	蓝藻	与上转换纳米粒子一起封装，能够创建工程化的微氧细胞工厂	[21]
	Minicell	无染色体细胞，药物递送的优良载体	[22]
	Simcell	无染色体细胞，安全可编程的通用平台	[23]
复杂基因逻辑回路设计Complex gene logic circuit design	AND、NOT联用型遗传电路设计	利用AND和NOT联用的基因电路，巧妙实现了细胞生长过渡到生物合成时间点的精确把控，减少外源电路引入对宿主菌株的代谢负担	[24]
复杂基因逻辑回路设计Complex gene logic circuit design	CASwitch	将CRISPR-Cas内环核酸酶与Tet-On3G诱导基因系统相结合而设计的高性能基因电路，用于哺乳动物系统中高性能诱导表达	[25]

回路类型 Loop type	原理 Principle	来源 Reference
OR	Input1和Input2分别控制S273和Q271R的形成，二者均可作为甘氨酸合成的前体	[46]
NOT	Lac I和IPTG共同作用于Ptac启动子，作为OR门电路的两个输入。当有高电平输出信号时，会刺激cI和ECFP的表达，进而通过非门抑制EYFP的表达	[47]
AND	Input1控制T7 RNA聚合酶基因的转录，Input2控制琥珀酸抑制剂t RNA sup D。当两种成分转录后，合成T7 RNA聚合酶，实现后续的基因表达	[48]
NAND	Phlf和Lmr是两个非门，当IPTG和HSL同时诱导时，黄色荧光蛋白的表达会停止，其余情况正常	[49]
NOR	Input1和Input2合成信号分子，翻转两个终止子的状态，当两个输入信号都表达时，关闭输出启动子	[50-51]

回路类型 Loop type	原理 Principle	来源 Reference
OR	Input1和Input2分别控制S273和Q271R的形成，二者均可作为甘氨酸合成的前体	[46]
NOT	Lac I和IPTG共同作用于Ptac启动子，作为OR门电路的两个输入。当有高电平输出信号时，会刺激cI和ECFP的表达，进而通过非门抑制EYFP的表达	[47]
AND	Input1控制T7 RNA聚合酶基因的转录，Input2控制琥珀酸抑制剂t RNA sup D。当两种成分转录后，合成T7 RNA聚合酶，实现后续的基因表达	[48]
NAND	Phlf和Lmr是两个非门，当IPTG和HSL同时诱导时，黄色荧光蛋白的表达会停止，其余情况正常	[49]
NOR	Input1和Input2合成信号分子，翻转两个终止子的状态，当两个输入信号都表达时，关闭输出启动子	[50-51]