功能拓扑的理性设计及其在合成生物学中的应用

doi:10.12211/2096-8280.2023-003

合成生物学 ›› 2023, Vol. 4 ›› Issue (3): 444-463.DOI: 10.12211/2096-8280.2023-003

功能拓扑的理性设计及其在合成生物学中的应用

孙智¹, 杨宁¹, 娄春波², 汤超¹, 杨晓静¹

^1.北京大学定量生物学中心，北京大学-清华大学生命科学联合中心，北京大学前沿学科交叉研究院，北京大学，北京 100871
^2.中国科学院深圳先进技术研究院，细胞与基因线路设计中心，广东深圳 518000

收稿日期:2023-01-02 修回日期:2023-02-09 出版日期:2023-06-30 发布日期:2023-07-05
通讯作者: 汤超,杨晓静
作者简介:孙智（1994—），男，博士后。研究方向为定量合成生物学，人工生命系统的理性设计。 E-mail：sunz@pku.edu.cn
杨宁（1995—），男，博士研究生。研究方向为复杂系统、定量生物学。 E-mail：yn_biophy@pku.edu.cn
汤超（1958—），男，中国科学院院士，讲席教授，博士生导师。研究方向为物理生物学，系统生物学，统计物理和复杂系统。 E-mail：tangc@pku.edu.cn
杨晓静（1978—），女，副研究员。研究方向为系统生物学，定量生物学，合成生物学。 E-mail：xiaojing_yang@pku.edu.cn
基金资助:
国家重点研发计划(2018YFA0900700);北京大学-清华大学生命科学联合中心博士后基金

Rational design for functional topology and its applications in synthetic biology

Zhi SUN¹, Ning YANG¹, Chunbo LOU², Chao TANG¹, Xiaojing YANG¹

^1.Center for Quantitative Biology，Peking-Tsinghua Center for Life Sciences，Academy for Advanced Interdisciplinary Studies，Peking University，Beijing 100871，China
^2.Cell and Gene Circuit Design Center，Shenzhen Institute of Advanced Technology，Chinese Academy of Sciences，Shenzhen 518000，Guangdong，China

Received:2023-01-02 Revised:2023-02-09 Online:2023-06-30 Published:2023-07-05
Contact: Chao TANG, Xiaojing YANG

摘要/Abstract

摘要：

生物网络以超乎寻常的精度、可靠性和鲁棒性执行着各种各样复杂的功能。网络的拓扑结构、动力学性质与功能之间密切相关。如何定量刻画这种关系，找到复杂多样的生物网络的底层设计规律是系统生物学和合成生物学的巨大挑战。本文对功能拓扑的理性设计及其在合成生物学中的应用进行了综述。生物网络在统计性质上不同于随机网络，其结构呈现出模块化的趋势，本文首先总结了自然生物系统中出现的高频模块及其功能，回顾了最近系统生物学对于功能拓扑设计原理的探索，包括目前搜索功能拓扑的两种常用计算方法，同时对理论获得的典型功能拓扑进行了总结。继而总结了近年来实际合成生物学系统中功能拓扑的设计和构建，以基于转录调控的基因回路为主，按照其内部调控节点的数目，系统地介绍了不同拓扑结构被用来实现的具体功能及其典型实例。最后，介绍了近期自动化设计集成基因线路的发展、非转录多层次调控机制以及网络鲁棒性的设计原理，并简单探讨了对于复杂功能拓扑设计的机遇和挑战。

关键词: 合成生物学, 系统生物学, 功能拓扑, 基因回路

Abstract:

Biological networks are capable of performing complex functions with accuracy, reliability, and robustness. In topology, the dynamic property and function of a network are closely related. How to depict this relationship quantitatively and discover design principles for complex and diverse biological networks are great challenges. In this review, we comment progress in this regard and its applications in synthetic biology. Biological networks are different statistically from random ones, which show a characteristic of modularization. There are recurrent network motifs linked to particular functions, such as temporally programed expression, reliable cell decisions, and robust biological oscillations, suggesting that despite the apparent complexity of cellular networks, there may only be a limited number of topological networks for executing particular biological functions robustly. Indeed, by enumerating all possible topological networks with two or three nodes, systems biology studies have shown that only a handful of topological networks can perform a given function. Here, we first summarize the high-frequency modules and their functions in natural biological systems, review progress in systems biology to find design principles for functional topology, including two current methods (i.e., enumeration and optimization) to search functional topology computationally, and highlight the typical functional topological networks that have been developed theoretically so far. Then, we focus on the functional topology that has been constructed and used in synthetic biology, such as genetic circuits developed based on transcriptional regulation. Organized by the number of nodes, we show typical examples and applications of different functional topological networks, including single-node positive/negative feedback loops, two-node positive/negative feedback loops, multi-node negative feedback/feedforward loops, and the combinational logic gates etc. Finally, we address frontiers in functional topology, including automated design of integrated gene circuits, other regulatory mechanisms beyond transcription, design of network robustness. We end by briefly discussing opportunities and challenges for designing complex functional topological networks.

Key words: synthetic biology, systems biology, functional topology, genetic circuit

中图分类号:

孙智, 杨宁, 娄春波, 汤超, 杨晓静. 功能拓扑的理性设计及其在合成生物学中的应用[J]. 合成生物学, 2023, 4(3): 444-463.

Zhi SUN, Ning YANG, Chunbo LOU, Chao TANG, Xiaojing YANG. Rational design for functional topology and its applications in synthetic biology[J]. Synthetic Biology Journal, 2023, 4(3): 444-463.

图/表 3

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功能	表型	拓扑	备注
适应性			酶促反应网络 Me, et al., 2009^[18]
振荡			Wagner, 2005^[22] Elowitz and Leibler, 2000^[28]
双稳态			Shah and Sarkar, 2011^[21]
持续性检测			Alon, 2007^[8]
抗噪			Nie, et al., 2020^[13]
体节发育			Ma, et al., 2006^[17]
细胞极化			Chau,et al., 2012^[23]
适应性/振荡双功能			Zhang, et al., 2019^[24]
调频/调幅响应			转录调控网络 Gao, et al., 2018^[25]

功能	表型	拓扑	备注
适应性			酶促反应网络 Me, et al., 2009^[18]
振荡			Wagner, 2005^[22] Elowitz and Leibler, 2000^[28]
双稳态			Shah and Sarkar, 2011^[21]
持续性检测			Alon, 2007^[8]
抗噪			Nie, et al., 2020^[13]
体节发育			Ma, et al., 2006^[17]
细胞极化			Chau,et al., 2012^[23]
适应性/振荡双功能			Zhang, et al., 2019^[24]
调频/调幅响应			转录调控网络 Gao, et al., 2018^[25]

线路类型	节点数/设计层次	拓扑结构/线路特点	可执行功能	主要基因元件或模块
基本转录调控线路	单节点	自抑制	降低表达噪声；加速响应完成时间；剂量响应线性化	TetR自抑制；aTc剂量效应^[5,11,35-36] TetR自抑制结合光感元件LOV2响应光强度剂量^[37]
	单节点	自激活	双稳态自维持； “双模态”切换动力学；参数分岔的“磁滞现象”	DBD-VP64自激活，pGal-DBD-VP64记忆瞬时乳糖浓度^[38] ZF-VP64自激活，响应多种瞬时刺激^[39] rtTA自激活线路，测试切换动力学^[40] tTA-VP16自激活，红霉素响应元件E-KRAB调控^[41] σ因子SigW自激活，利用RsiW调谐“磁滞区间”^[42]
	双节点	“互抑制”型正反馈	双稳态自维持； “双模态”切换动力学；参数分岔的“磁滞现象”	LacⅠ与CI构建toggle switch拨动开关^[43] RecA裂解CI控制双稳态切换，控制TraA表达诱发生物膜^[44] 利用mfLon蛋白酶控制拨动开关^[45] 利用UAS与pGal设计主开关，控制toggle switch开启^[46] PIP/E-KRAB构建toggle switch^[47] TALE元件设计toggle switch，耦联TALE自激活线路^[48] 正交TALE元件库，shRNA控制拨动开关，细胞分类器^[49] 定量RBS折叠自由能可预测设计toggle switch二态比例^[50]
	双节点	“激活-抑制”型负反馈	内源性自主振荡；受迫振荡； “锁相”行为；适应性响应	LacⅠ-AraC，节点间负反馈结合自调控，设计自主振荡^[51] 温敏LacⅠ，补偿LacⅠ-AraC振荡线路的温度变化影响^[52] LacⅠ-AraC，周期性外源Arabinose驱动下的“锁相”振荡^[53] sense/anti-sense mRNA或siRNA设计负反馈振荡^[54-55] T7 RNAP与CRISPRi/dCpf1设计正负反馈耦合，实现RPA^[56]
	多节点	负反馈环路	三节点互抑制振荡	TetR-LacⅠ-λCI的三节点互抑制负反馈环^[28] TetR-LacⅠ-λCI的三节点负反馈环简化，实现超稳定振荡^[57] 微流与光阱捕获技术，线路建库，筛选稳定振荡菌株^[58]
		前馈线路	脉冲发生器（iFFL）；带通滤波器（iFFL）；信号加速器（cFFL）	天然系统中的前馈实现信号加速与延迟的理论分析^[59-64] AHL扩散，LuxR-CI-GFP构建iFFL实现pulse generator^[65] LuxR-CI-GFP iFFL的空间动力学行为^[66] 光感pompC-LuxI-CI控制下游pLux-λ构建iFFL，边界探测^[67] tTA-Pip-E-KRAB构建iFFL，响应aTc剂量的band-pass filter^[68] 转录元件精确定量表征，构建iFFL的定量可预测设计^[69]
		组合逻辑线路	各类逻辑门线路	利用TetR-LacⅠ-λCI元件设计一系列逻辑门线路^[70] 双启动子控制阻遏蛋白的NOR Gate基本框架^[71] TetR family的正交NOT Gate阻遏蛋白挖掘^[72]
		时序逻辑线路	触点开关；条件记忆学习线路；锁存器线路	LexA-LacⅠ NOR Gate，CI-CI434双稳态，组合构建触点开关^[73] tag-supD AND Gate、CI-CI434双稳态，巴甫洛夫样学习线路^[74] 双稳态与NOR Gate组合锁存结构，时序切换控制^[75]
集成基因线路	多节点	组合前馈逻辑线路	逻辑门线路自动化设计；逻辑切换动力学预测；个性化线路设计	Cello实现基因线路三输入逻辑真值表自动化CAD设计^[76] 四输入自动化设计，动力学过程表征与预测^[77] 基因组上Landing Pad超稳定低负载基因线路CAD设计^[78] 非模式菌的基础元件表征与线路CAD设计^[79] S. cerevisiae的基础元件设计表征与线路CAD设计^[80]
多层次调控线路	DNA层次	重组酶线路	带通滤波器；表达顺序控制线路	Bxb1、φC31、TP901重组酶受H₂O₂浓度调控，构建iFFL^[81] Cre、Flp、φC31受GIB、ABA诱导二聚化的自移除设计^[82]
	RNA层次	RNA元件	RNA结合蛋白； RNA Replicon系统	L7Ae、MS2结合蛋白的互抑制设计，RNA Replicon双稳态^[83] 使用TMP-DHFR可诱导降解标签调控L7Ae结合蛋白浓度^[84]
	RNA层次	CRISPR Circuit	CRISPR/Cas元件设计； Cas主蛋白中枢型线路	TetR-CI-CRISPR/dCas9构建repressilator^[85] CRISPR/dCas9的转录抑制调控元件设计与表征^[86-87] dCas9*-PhlF融合构建减毒CRISPR调控系统^[88] 以dCas9、dCas12a为中枢蛋白设计的sgRNA调控线路等^[89-90]
	蛋白质层次	蛋白酶线路	降解肽切割调控；分裂蛋白多聚化调控	TEV/TVMV/SuMMV Protease移除/暴露降解肽标签调控设计^[91] Split TEV/TVMV/HCV Protease调控结合leucine zipper二聚化调控，设计logic gate、iFFL、protease toggle等功能^[92-93]
		转录因子互作	多稳态基因调控线路	GCN4、FKBP等结构域调控ZF结合蛋白二聚化多稳态线路^[94]
		翻译后修饰	超快速响应切换线路	CRE1/STAT5设计磷酸化OR Gate，PTP-pHOG1对HOT1-pJH1去磷酸化NOT Gate，组合设计信号转导toggle switch^[95]
鲁棒性控制线路	模块间“追溯活性”	线路问题检查	负载表达动力学干扰；元件模块化组装干扰	pLac与lacO负载位点对LacⅠ的竞争，干扰表达动力学^[96] LuxR-NahR-GFP顺序组装后系统行为偏离预期^[97]
	模块间“追溯活性”	功能修复线路	转录因子磷酸化线路；激酶/磷酸酶元件开发；减弱资源竞争模块设计；公共资源自调节线路	NRII(L16R)-NRII(H139N)，STAT5-HKRR/YPD1/SKN7的磷酸化级联，屏蔽负载位点竞争干扰^[98-99] EnvZ激酶/磷酸酶活性改造，对OmpR-VP64调控活性控制，磷酸化水平负反馈线路控制表达^[100] ECF32 σ因子表达sRNA-mRNA负反馈；CasE切割mRNA 5′-UTR、miR-FF4设计iFFL线路等，减弱资源竞争效应^[101-103] 设计sgRNA靶向dCas9的自负反馈，公共中枢蛋白资源表达自调节，减弱下游模块间竞争^[104]
	“线路-底盘”互作	线路问题检查	拓扑对生长的敏感性；资源占用导致生长停滞	AraC自激活与TetR-LacI互抑制对生长速率的不同敏感性^[105] pT7-T7 RNAP的自激活线路产生显著的表达量-生长异质性^[106]
	“线路-底盘”互作	功能修复线路	低拷贝基因线路设计；表达负担负反馈线路；毒性转录因子自负反馈；生长速率补偿调控	基因组单拷贝整合的TetR-LacⅠ toggle switch^[107] 负担敏感phtpG1控制CRISPR/dCas9抑制的负反馈控制^[108] 转录因子CymR的自抑制诱导系统设计，减弱生长抑制毒性^[7] SpoTH元件降低ppGpp含量，维持生长速率恒定^[109]
	扰动噪声屏蔽	目的基因表达控制	稳定基因拷贝数变异；组合iFFL/负反馈线路	TALE设计线性抑制功能，稳定启动子表达受拷贝数影响^[110] rtTA-LacI/miR-FF3设计iFFL，排除转染量影响^[111] tTA-miR-FF4的iFFL与负反馈，组合控制^[112]
		积分反馈控制线路	σ/anti-σ； sense/anti-sense mRNA	SigW-RsiW的σ/anti-σ互作设计积分反馈控制^[113] tTA sense/anti-sense mRNA互作设计反馈控制^[114]
		鲁棒性功能拓扑发掘	基于拓扑约束的线路构建与扰动测试	基于拓扑穷举理论计算结果，T7 RNAP与CRISPR/dCpf1设计负反馈耦合线性弱自激活的基因线路，在输入信号改变、拓扑参数变异及底盘生理条件等进行系统扰动下均可实现RPA功能^[56]

线路类型	节点数/设计层次	拓扑结构/线路特点	可执行功能	主要基因元件或模块
基本转录调控线路	单节点	自抑制	降低表达噪声；加速响应完成时间；剂量响应线性化	TetR自抑制；aTc剂量效应^[5,11,35-36] TetR自抑制结合光感元件LOV2响应光强度剂量^[37]
	单节点	自激活	双稳态自维持； “双模态”切换动力学；参数分岔的“磁滞现象”	DBD-VP64自激活，pGal-DBD-VP64记忆瞬时乳糖浓度^[38] ZF-VP64自激活，响应多种瞬时刺激^[39] rtTA自激活线路，测试切换动力学^[40] tTA-VP16自激活，红霉素响应元件E-KRAB调控^[41] σ因子SigW自激活，利用RsiW调谐“磁滞区间”^[42]
	双节点	“互抑制”型正反馈	双稳态自维持； “双模态”切换动力学；参数分岔的“磁滞现象”	LacⅠ与CI构建toggle switch拨动开关^[43] RecA裂解CI控制双稳态切换，控制TraA表达诱发生物膜^[44] 利用mfLon蛋白酶控制拨动开关^[45] 利用UAS与pGal设计主开关，控制toggle switch开启^[46] PIP/E-KRAB构建toggle switch^[47] TALE元件设计toggle switch，耦联TALE自激活线路^[48] 正交TALE元件库，shRNA控制拨动开关，细胞分类器^[49] 定量RBS折叠自由能可预测设计toggle switch二态比例^[50]
	双节点	“激活-抑制”型负反馈	内源性自主振荡；受迫振荡； “锁相”行为；适应性响应	LacⅠ-AraC，节点间负反馈结合自调控，设计自主振荡^[51] 温敏LacⅠ，补偿LacⅠ-AraC振荡线路的温度变化影响^[52] LacⅠ-AraC，周期性外源Arabinose驱动下的“锁相”振荡^[53] sense/anti-sense mRNA或siRNA设计负反馈振荡^[54-55] T7 RNAP与CRISPRi/dCpf1设计正负反馈耦合，实现RPA^[56]
	多节点	负反馈环路	三节点互抑制振荡	TetR-LacⅠ-λCI的三节点互抑制负反馈环^[28] TetR-LacⅠ-λCI的三节点负反馈环简化，实现超稳定振荡^[57] 微流与光阱捕获技术，线路建库，筛选稳定振荡菌株^[58]
		前馈线路	脉冲发生器（iFFL）；带通滤波器（iFFL）；信号加速器（cFFL）	天然系统中的前馈实现信号加速与延迟的理论分析^[59-64] AHL扩散，LuxR-CI-GFP构建iFFL实现pulse generator^[65] LuxR-CI-GFP iFFL的空间动力学行为^[66] 光感pompC-LuxI-CI控制下游pLux-λ构建iFFL，边界探测^[67] tTA-Pip-E-KRAB构建iFFL，响应aTc剂量的band-pass filter^[68] 转录元件精确定量表征，构建iFFL的定量可预测设计^[69]
		组合逻辑线路	各类逻辑门线路	利用TetR-LacⅠ-λCI元件设计一系列逻辑门线路^[70] 双启动子控制阻遏蛋白的NOR Gate基本框架^[71] TetR family的正交NOT Gate阻遏蛋白挖掘^[72]
		时序逻辑线路	触点开关；条件记忆学习线路；锁存器线路	LexA-LacⅠ NOR Gate，CI-CI434双稳态，组合构建触点开关^[73] tag-supD AND Gate、CI-CI434双稳态，巴甫洛夫样学习线路^[74] 双稳态与NOR Gate组合锁存结构，时序切换控制^[75]
集成基因线路	多节点	组合前馈逻辑线路	逻辑门线路自动化设计；逻辑切换动力学预测；个性化线路设计	Cello实现基因线路三输入逻辑真值表自动化CAD设计^[76] 四输入自动化设计，动力学过程表征与预测^[77] 基因组上Landing Pad超稳定低负载基因线路CAD设计^[78] 非模式菌的基础元件表征与线路CAD设计^[79] S. cerevisiae的基础元件设计表征与线路CAD设计^[80]
多层次调控线路	DNA层次	重组酶线路	带通滤波器；表达顺序控制线路	Bxb1、φC31、TP901重组酶受H₂O₂浓度调控，构建iFFL^[81] Cre、Flp、φC31受GIB、ABA诱导二聚化的自移除设计^[82]
	RNA层次	RNA元件	RNA结合蛋白； RNA Replicon系统	L7Ae、MS2结合蛋白的互抑制设计，RNA Replicon双稳态^[83] 使用TMP-DHFR可诱导降解标签调控L7Ae结合蛋白浓度^[84]
	RNA层次	CRISPR Circuit	CRISPR/Cas元件设计； Cas主蛋白中枢型线路	TetR-CI-CRISPR/dCas9构建repressilator^[85] CRISPR/dCas9的转录抑制调控元件设计与表征^[86-87] dCas9*-PhlF融合构建减毒CRISPR调控系统^[88] 以dCas9、dCas12a为中枢蛋白设计的sgRNA调控线路等^[89-90]
	蛋白质层次	蛋白酶线路	降解肽切割调控；分裂蛋白多聚化调控	TEV/TVMV/SuMMV Protease移除/暴露降解肽标签调控设计^[91] Split TEV/TVMV/HCV Protease调控结合leucine zipper二聚化调控，设计logic gate、iFFL、protease toggle等功能^[92-93]
		转录因子互作	多稳态基因调控线路	GCN4、FKBP等结构域调控ZF结合蛋白二聚化多稳态线路^[94]
		翻译后修饰	超快速响应切换线路	CRE1/STAT5设计磷酸化OR Gate，PTP-pHOG1对HOT1-pJH1去磷酸化NOT Gate，组合设计信号转导toggle switch^[95]
鲁棒性控制线路	模块间“追溯活性”	线路问题检查	负载表达动力学干扰；元件模块化组装干扰	pLac与lacO负载位点对LacⅠ的竞争，干扰表达动力学^[96] LuxR-NahR-GFP顺序组装后系统行为偏离预期^[97]
	模块间“追溯活性”	功能修复线路	转录因子磷酸化线路；激酶/磷酸酶元件开发；减弱资源竞争模块设计；公共资源自调节线路	NRII(L16R)-NRII(H139N)，STAT5-HKRR/YPD1/SKN7的磷酸化级联，屏蔽负载位点竞争干扰^[98-99] EnvZ激酶/磷酸酶活性改造，对OmpR-VP64调控活性控制，磷酸化水平负反馈线路控制表达^[100] ECF32 σ因子表达sRNA-mRNA负反馈；CasE切割mRNA 5′-UTR、miR-FF4设计iFFL线路等，减弱资源竞争效应^[101-103] 设计sgRNA靶向dCas9的自负反馈，公共中枢蛋白资源表达自调节，减弱下游模块间竞争^[104]
	“线路-底盘”互作	线路问题检查	拓扑对生长的敏感性；资源占用导致生长停滞	AraC自激活与TetR-LacI互抑制对生长速率的不同敏感性^[105] pT7-T7 RNAP的自激活线路产生显著的表达量-生长异质性^[106]
	“线路-底盘”互作	功能修复线路	低拷贝基因线路设计；表达负担负反馈线路；毒性转录因子自负反馈；生长速率补偿调控	基因组单拷贝整合的TetR-LacⅠ toggle switch^[107] 负担敏感phtpG1控制CRISPR/dCas9抑制的负反馈控制^[108] 转录因子CymR的自抑制诱导系统设计，减弱生长抑制毒性^[7] SpoTH元件降低ppGpp含量，维持生长速率恒定^[109]
	扰动噪声屏蔽	目的基因表达控制	稳定基因拷贝数变异；组合iFFL/负反馈线路	TALE设计线性抑制功能，稳定启动子表达受拷贝数影响^[110] rtTA-LacI/miR-FF3设计iFFL，排除转染量影响^[111] tTA-miR-FF4的iFFL与负反馈，组合控制^[112]
		积分反馈控制线路	σ/anti-σ； sense/anti-sense mRNA	SigW-RsiW的σ/anti-σ互作设计积分反馈控制^[113] tTA sense/anti-sense mRNA互作设计反馈控制^[114]
		鲁棒性功能拓扑发掘	基于拓扑约束的线路构建与扰动测试	基于拓扑穷举理论计算结果，T7 RNAP与CRISPR/dCpf1设计负反馈耦合线性弱自激活的基因线路，在输入信号改变、拓扑参数变异及底盘生理条件等进行系统扰动下均可实现RPA功能^[56]

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功能拓扑的理性设计及其在合成生物学中的应用

Rational design for functional topology and its applications in synthetic biology

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