基于细菌双组分系统的生物传感器的研究进展

doi:10.12211/2096-8280.2023-016

合成生物学 ›› 2024, Vol. 5 ›› Issue (1): 38-52.DOI: 10.12211/2096-8280.2023-016

基于细菌双组分系统的生物传感器的研究进展

赵静宇, 张健, 祁庆生, 王倩

山东大学，国家糖工程技术研究中心，微生物技术国家重点实验室，山东青岛 266237

收稿日期:2023-02-22 修回日期:2023-07-01 出版日期:2024-02-29 发布日期:2024-03-20
通讯作者: 祁庆生,王倩
作者简介:赵静宇（1998—），女，硕士研究生。研究方向为微生物代谢工程与合成生物学。 E-mail：1504362801@qq.com
祁庆生（1966—），男，教授，山东大学微生物技术国家重点实验室副主任。研究方向为代谢工程与合成生物学，废弃塑料降解及生物可降解塑料的合成等。 E-mail：qiqingsheng@sdu.edu.cn
王倩（1983—），女，博士，教授。研究方向为微生物代谢工程与合成生物学。 E-mail：qiqi20011983@gmail.com
基金资助:
国家重点研发计划“合成生物学专项”(2019YFA0904900);国家自然科学基金面上项目(32270089)

Research progress in biosensors based on bacterial two-component systems

Jingyu ZHAO, Jian ZHANG, Qingsheng QI, Qian WANG

National Glycoengineeing Research Center，State Key Laboratory of Microbial Technology，Shandong University，Qingdao 266237，Shandong，China

Received:2023-02-22 Revised:2023-07-01 Online:2024-02-29 Published:2024-03-20
Contact: Qingsheng QI, Qian WANG

摘要/Abstract

摘要：

细菌双组分系统能够感知和响应细胞内外的物理、化学和生物刺激，通过耦合传感和调节机制从而引起一系列的细胞反应，是一个普遍存在的信号转导通路家族。当前越来越多的合成生物学家已开始利用双组分系统的特异属性来工程化设计微生物传感系统，并应用于光遗传学、材料科学、肠道微生物组工程、生物炼制和土壤改良等领域。本综述重点介绍了开发基于双组分系统的生物传感器的最新研究进展以及在各个领域中的潜在应用。同时探讨了如何运用新的工程方法提高双组分系统传感器性能的可靠性，包括遗传重构、DNA结合结构域交换、检测阈值调节和磷酸化串扰隔离，以及如何根据特定应用的要求定制双组分系统信号特性。在未来，研究者可以将这些方法与大规模的基因合成、高通量筛选相结合，以加速和帮助发现更多未确定特征输入的双组分系统，并开发新的对广泛的刺激做出反应的基因编码生物传感器，拓展双组分生物传感器在不同领域的应用。

关键词: 合成生物学, 双组分系统, 细胞生物传感器, 代谢工程, 光遗传学, 细菌诊断

Abstract:

Two-component systems (TCSs) in bacteria, are capable of sensing and making responses to physical, chemical, and biological stimuli within and outside the cells, and subsequently induce a wide range of cellular processes through the role played by the regulatory component and the response component in combination, which is a ubiquitous signal transduction pathway. At present, an growing number of synthetic biologists have devoted their effort to using the specific and irreplaceable properties of TCSs to design biosensors with the aim of applying in optogenetics, materials science, engineering of gut microbiome, biorefining and soil improvement, and the like. The purpose of this review is to focus on the most recent research advances in the development of biosensors based on TCSs and their potential applications. At the same time, topics of great importance are discussed on how to use novel engineering methods with synthetic biology to improve the reliability and robustness of the performance of the biosensors, such as genetic remodeling, DNA-binding domain swapping, tuning of the detection threshold and isolation of phosphorylation crosstalk as well as on how to customize the signal characteristics of TCSs to meet particular needs according to the requirements of specific applications. It would be possible in the future for scientists to combine these methods with gene synthesis on a large scale and high-throughput screening in order to speed up and give synthetic biologists a hand in the discovery of TCSs with numerous uncharacterized signal inputs and the development of genetically encoded novel biosensors that may be capable of responding to a broad range of stimuli. This allows for extending the applications of the biosensors in different fields.

Key words: synthetic biology, two-component systems, whole-cell biosensors, metabolic engineering, optogenetics, bacterial diagnostics

中图分类号:

赵静宇, 张健, 祁庆生, 王倩. 基于细菌双组分系统的生物传感器的研究进展[J]. 合成生物学, 2024, 5(1): 38-52.

Jingyu ZHAO, Jian ZHANG, Qingsheng QI, Qian WANG. Research progress in biosensors based on bacterial two-component systems[J]. Synthetic Biology Journal, 2024, 5(1): 38-52.

图/表 5

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输入	天然宿主	SK	RR	输出启动子	表现特点	参考文献
光
紫外光	聚球藻PCC6803	UirS	UirR	P _csiR1	激活5倍	[12]
蓝光	枯草芽孢杆菌，日本血吸虫	YF1	FixJ	P _fixK2	嵌合体与pDusk系统偶联时激活460倍	[13]
绿光	聚球藻PCC6803	CcaSmini#10	CcaR	P _cpcG2-172	激活600倍	[14]
红光	聚球藻PCC6803 大肠杆菌	Cph8*	OmpR	P _ompF112	抑制80倍	[15]
近红外光	大豆根瘤菌	BphP1	PpsR2	PBr_ _crtE	激活2倍	[16]
pH
酸性pH（<6.2）	奥奈登斯链球菌枯草芽孢杆菌	SO-4387	SO_4388_REC-PsdR_DBD137	P _psdA110	用于检测小鼠的肠道炎症	[17]
金属离子
As³⁺（胞外）	根癌土壤杆菌	AioS	AioR	P _aioB	需要 AioX 内膜辅助蛋白	[18]
Ca²⁺（胞外）	铜绿假单胞菌PAO1	CarS	CarR	P _carO	CARS与PhoQ有关	[19]
Cu⁺（胞外）	大肠杆菌	CusS	CusR	P _cusC	也被Ag⁺激活	[20]
Cu²⁺（胞内和胞外）	聚球藻PCC6803	CopS	CopR	P _couM	CopS位于类囊体膜	[21]
Cu²⁺（胞外）	黄曲霉	CorS	CorR	P _couA	—	[22]
Fe²⁺，Fe³⁺（胞外）	黏质沙雷氏菌	RssA	RssB	P _pvcA	活性受天然产物2-异氰基-6,7-二羟基香豆素调节	[23]
K⁺（胞内及胞外）	大肠杆菌	KdpD	KdpE	P _kdpF	K⁺抑制	[24]
U	月柄杆菌	UzcS	UzcR	P _urcA	通过与门耦合到UrpRS提高了灵敏度和特异性	[25]
Zn²⁺	大肠杆菌	ZraS	ZraR	P _zraP	P _zraP 依赖σ⁵⁴	[26]
养分可利用性
硫代硫酸盐	S. halifaxensis	ThsS	ThsR	P _phsA342	成比例地被与DSS诱导的小鼠结肠炎症所激活	[27]
连四硫酸盐	S. baltica	TtrS	TtrR	P _ttrB185-269	动态范围为100倍	[27]
硝酸盐	大肠杆菌	NarX	NarL_REC-Ydfl_DBD¹³¹	P _ydfJ115	在枯草芽孢杆菌中被激活1300倍	[28]
三甲胺氮氧化物（TMAO）	大肠杆菌	TorS	TorR_REC-PsdR_DBD137	P _psdA110	DBD交换消除了O₂对天然输出启动子的交叉抑制。需要周质TorT辅助蛋白	[28]
氧化剂
O₂，H₂O₂，NO	金黄色葡萄球菌	AirS	AirR	P _crtO	AirS需要一个[2Fe-2S]²⁺簇	[29]
小分子代谢物
α-酮戊二酸（细胞外）	铜绿假单胞菌PAO1	MifS	MitR	P _PA5530	激活10倍。对L-谷氨酸有微弱的响应	[30]
丁醇	乙酰丁酸单胞菌	BtrK	BtrR	P _btrT	参与丁醇的耐受性	[31]
柠檬酸	肺炎克雷伯氏菌	CitA	CitB	P _citC	需厌氧条件	[32]
岩藻糖	大肠杆菌	FusK	FusR	P _z0461	岩藻糖抑制转录输出	[33]
富马酸	大肠杆菌	DcuSZ	OmpR	P _ompC	激活2倍	[34]
葡萄糖-6-磷酸	大肠杆菌	UhpB	UhpA	P _uhpT99	需要UhpC内膜辅助蛋白	[35]
L-谷氨酸	铜绿假单胞菌PAO1	AauS	AauR	P _aatJ	被L-天冬氨酸、谷氨酰胺和天冬酰胺微弱激活	[36]
血红素（细胞外）	金黄色葡萄球菌	HssS	HssR	P _hrtA	激活100倍以上	[34]
吲哚	大肠杆菌	BaeS	BaeR	P _arcD	CpxAR TCS的存在放大了对吲哚的响应	[37]
苹果酸	枯草芽孢杆菌	YufL	YufM	P _maeN381	激活100倍	[38]
甲醇	脱氮假单胞菌大肠杆菌	FlhS-EnvZ	OmpR	P _ompC	激活2倍	[34]
丙酮酸（细胞外）	大肠杆菌	BtsS	BtsR	P _yjiY	泌尿道感染期间在泌尿致病性大肠杆菌中被激活	[39]
D-木糖（细胞外）	拜氏梭菌	LytS	YesN	P _xylF	外膜转运蛋白XylFⅡ识别D-木糖	[40]
苯乙烯	假单胞菌菌株Y2	StyS	StyR	P _styA		[41]
细菌间通信信号
CaI-1[（S）-3羟基十三烷-4-酮]	霍乱弧菌	CqsS	LuxO	P _tpqrr4	需要中间磷酸转移蛋白LuxU	[42]
CSP（能力刺激肽）	格登链球菌	ComD	ComE	P _comC		[43]
ComX（胞外信息素）	枯草芽孢杆菌	ComP	ComA	P _srfA		[44]
抗生素
β-内酰胺	霍乱弧菌	VxrA	VxrB	P _murJ	通常由细胞被膜损伤激活	[45]
线霉菌素（膜内）	枯草芽孢杆菌	LnrJ	LnrK	P _lnrL	还可以检测到抗真菌多烯两性霉素B	[46]
万古霉素（胞外）	腔血链球菌	VanS	VanR	P _vanJ	磷酸化的VanR也激活VanSR操纵子	[47]
抗菌肽
口腔致病菌变形链球菌产生的抗菌肽（胞外）	链球菌A12	PcfK	PcfR	P _pcfF	大约激活100倍	[48]
杆菌肽（胞外）	枯草芽孢杆菌	LiaS	LiaR	P _lial(opt)	激活1000倍。需要辅膜蛋白LiaF	[49]
Nisin（胞外）	乳酸乳杆菌	NisK	NisR	P _nisA	激活1000倍	[50]
枯草蛋白（细胞外）	枯草芽孢杆菌	SpaK	SpaR	P _spaS	激活110倍	[50]
低聚糖
阿拉伯半乳聚糖	双歧杆菌	BT0267	BT0267	P _BT0268	BT0267是一种杂交的TCS，其中SK和RR融合在一起	[50-51]
硫酸软骨素	双歧杆菌	BT3334	BT3334	P _BT3324	BT3334是一种杂交的双组分系统	[51]
黏蛋白多糖	铜绿假单胞菌	GacS	GacA	P _rsmY	黏蛋白多聚糖是通过辅助组氨酸激酶rets感受到的	[52]
	双歧杆菌	BT0366	BT0366	P _BT0365	BT0366是一种杂交的双组分系统	[53]
蛋白质
PilA	铜绿假单胞菌	PilS	PilR	P _pilA	PilA是主要的Ⅳ型菌毛蛋白	[54]
宿主信号
哺乳动物感染过程中产生的抗菌肽、二价阳离子限制和酸性pH	鼠伤寒沙门氏菌	PhoQ	PhoP	P _virK	—	[55]
肾上腺素，去甲肾上腺素	大肠杆菌 O157:H7	QseC	QseB	P _flhD	被肾上腺素激活2倍，去甲肾上腺素抑制1.5倍	[56]
吲哚-3-乙酸（生长素）	P. phytofirmans PsJN	lacS	lacR1	P _iacA	二氧吲哚-3-乙酸放大信号	[57]
2-异戊烯基腺嘌呤（细胞分裂素）	X. campestris	PcrK	PcrR	P _ctrA	激活3倍	[58]
植物伤口中存在的酚类物质、单糖和酸性pH	根癌农杆菌	VirA	VirG	P _vir	对单糖的感应需要周质辅助蛋白ChvE	[59]
反式玉米素（细胞分裂素）	拟南芥，大肠杆菌	AQ₄*	PhoP₄*	P _mgrB	AQ₄*为拟南芥AHK4和大肠杆菌PhoQ的传感域嵌体，经改造与所有大肠杆菌双组分系统绝缘，可防止磷酸化串扰	[60]

输入	天然宿主	SK	RR	输出启动子	表现特点	参考文献
光
紫外光	聚球藻PCC6803	UirS	UirR	P _csiR1	激活5倍	[12]
蓝光	枯草芽孢杆菌，日本血吸虫	YF1	FixJ	P _fixK2	嵌合体与pDusk系统偶联时激活460倍	[13]
绿光	聚球藻PCC6803	CcaSmini#10	CcaR	P _cpcG2-172	激活600倍	[14]
红光	聚球藻PCC6803 大肠杆菌	Cph8*	OmpR	P _ompF112	抑制80倍	[15]
近红外光	大豆根瘤菌	BphP1	PpsR2	PBr_ _crtE	激活2倍	[16]
pH
酸性pH（<6.2）	奥奈登斯链球菌枯草芽孢杆菌	SO-4387	SO_4388_REC-PsdR_DBD137	P _psdA110	用于检测小鼠的肠道炎症	[17]
金属离子
As³⁺（胞外）	根癌土壤杆菌	AioS	AioR	P _aioB	需要 AioX 内膜辅助蛋白	[18]
Ca²⁺（胞外）	铜绿假单胞菌PAO1	CarS	CarR	P _carO	CARS与PhoQ有关	[19]
Cu⁺（胞外）	大肠杆菌	CusS	CusR	P _cusC	也被Ag⁺激活	[20]
Cu²⁺（胞内和胞外）	聚球藻PCC6803	CopS	CopR	P _couM	CopS位于类囊体膜	[21]
Cu²⁺（胞外）	黄曲霉	CorS	CorR	P _couA	—	[22]
Fe²⁺，Fe³⁺（胞外）	黏质沙雷氏菌	RssA	RssB	P _pvcA	活性受天然产物2-异氰基-6,7-二羟基香豆素调节	[23]
K⁺（胞内及胞外）	大肠杆菌	KdpD	KdpE	P _kdpF	K⁺抑制	[24]
U	月柄杆菌	UzcS	UzcR	P _urcA	通过与门耦合到UrpRS提高了灵敏度和特异性	[25]
Zn²⁺	大肠杆菌	ZraS	ZraR	P _zraP	P _zraP 依赖σ⁵⁴	[26]
养分可利用性
硫代硫酸盐	S. halifaxensis	ThsS	ThsR	P _phsA342	成比例地被与DSS诱导的小鼠结肠炎症所激活	[27]
连四硫酸盐	S. baltica	TtrS	TtrR	P _ttrB185-269	动态范围为100倍	[27]
硝酸盐	大肠杆菌	NarX	NarL_REC-Ydfl_DBD¹³¹	P _ydfJ115	在枯草芽孢杆菌中被激活1300倍	[28]
三甲胺氮氧化物（TMAO）	大肠杆菌	TorS	TorR_REC-PsdR_DBD137	P _psdA110	DBD交换消除了O₂对天然输出启动子的交叉抑制。需要周质TorT辅助蛋白	[28]
氧化剂
O₂，H₂O₂，NO	金黄色葡萄球菌	AirS	AirR	P _crtO	AirS需要一个[2Fe-2S]²⁺簇	[29]
小分子代谢物
α-酮戊二酸（细胞外）	铜绿假单胞菌PAO1	MifS	MitR	P _PA5530	激活10倍。对L-谷氨酸有微弱的响应	[30]
丁醇	乙酰丁酸单胞菌	BtrK	BtrR	P _btrT	参与丁醇的耐受性	[31]
柠檬酸	肺炎克雷伯氏菌	CitA	CitB	P _citC	需厌氧条件	[32]
岩藻糖	大肠杆菌	FusK	FusR	P _z0461	岩藻糖抑制转录输出	[33]
富马酸	大肠杆菌	DcuSZ	OmpR	P _ompC	激活2倍	[34]
葡萄糖-6-磷酸	大肠杆菌	UhpB	UhpA	P _uhpT99	需要UhpC内膜辅助蛋白	[35]
L-谷氨酸	铜绿假单胞菌PAO1	AauS	AauR	P _aatJ	被L-天冬氨酸、谷氨酰胺和天冬酰胺微弱激活	[36]
血红素（细胞外）	金黄色葡萄球菌	HssS	HssR	P _hrtA	激活100倍以上	[34]
吲哚	大肠杆菌	BaeS	BaeR	P _arcD	CpxAR TCS的存在放大了对吲哚的响应	[37]
苹果酸	枯草芽孢杆菌	YufL	YufM	P _maeN381	激活100倍	[38]
甲醇	脱氮假单胞菌大肠杆菌	FlhS-EnvZ	OmpR	P _ompC	激活2倍	[34]
丙酮酸（细胞外）	大肠杆菌	BtsS	BtsR	P _yjiY	泌尿道感染期间在泌尿致病性大肠杆菌中被激活	[39]
D-木糖（细胞外）	拜氏梭菌	LytS	YesN	P _xylF	外膜转运蛋白XylFⅡ识别D-木糖	[40]
苯乙烯	假单胞菌菌株Y2	StyS	StyR	P _styA		[41]
细菌间通信信号
CaI-1[（S）-3羟基十三烷-4-酮]	霍乱弧菌	CqsS	LuxO	P _tpqrr4	需要中间磷酸转移蛋白LuxU	[42]
CSP（能力刺激肽）	格登链球菌	ComD	ComE	P _comC		[43]
ComX（胞外信息素）	枯草芽孢杆菌	ComP	ComA	P _srfA		[44]
抗生素
β-内酰胺	霍乱弧菌	VxrA	VxrB	P _murJ	通常由细胞被膜损伤激活	[45]
线霉菌素（膜内）	枯草芽孢杆菌	LnrJ	LnrK	P _lnrL	还可以检测到抗真菌多烯两性霉素B	[46]
万古霉素（胞外）	腔血链球菌	VanS	VanR	P _vanJ	磷酸化的VanR也激活VanSR操纵子	[47]
抗菌肽
口腔致病菌变形链球菌产生的抗菌肽（胞外）	链球菌A12	PcfK	PcfR	P _pcfF	大约激活100倍	[48]
杆菌肽（胞外）	枯草芽孢杆菌	LiaS	LiaR	P _lial(opt)	激活1000倍。需要辅膜蛋白LiaF	[49]
Nisin（胞外）	乳酸乳杆菌	NisK	NisR	P _nisA	激活1000倍	[50]
枯草蛋白（细胞外）	枯草芽孢杆菌	SpaK	SpaR	P _spaS	激活110倍	[50]
低聚糖
阿拉伯半乳聚糖	双歧杆菌	BT0267	BT0267	P _BT0268	BT0267是一种杂交的TCS，其中SK和RR融合在一起	[50-51]
硫酸软骨素	双歧杆菌	BT3334	BT3334	P _BT3324	BT3334是一种杂交的双组分系统	[51]
黏蛋白多糖	铜绿假单胞菌	GacS	GacA	P _rsmY	黏蛋白多聚糖是通过辅助组氨酸激酶rets感受到的	[52]
	双歧杆菌	BT0366	BT0366	P _BT0365	BT0366是一种杂交的双组分系统	[53]
蛋白质
PilA	铜绿假单胞菌	PilS	PilR	P _pilA	PilA是主要的Ⅳ型菌毛蛋白	[54]
宿主信号
哺乳动物感染过程中产生的抗菌肽、二价阳离子限制和酸性pH	鼠伤寒沙门氏菌	PhoQ	PhoP	P _virK	—	[55]
肾上腺素，去甲肾上腺素	大肠杆菌 O157:H7	QseC	QseB	P _flhD	被肾上腺素激活2倍，去甲肾上腺素抑制1.5倍	[56]
吲哚-3-乙酸（生长素）	P. phytofirmans PsJN	lacS	lacR1	P _iacA	二氧吲哚-3-乙酸放大信号	[57]
2-异戊烯基腺嘌呤（细胞分裂素）	X. campestris	PcrK	PcrR	P _ctrA	激活3倍	[58]
植物伤口中存在的酚类物质、单糖和酸性pH	根癌农杆菌	VirA	VirG	P _vir	对单糖的感应需要周质辅助蛋白ChvE	[59]
反式玉米素（细胞分裂素）	拟南芥，大肠杆菌	AQ₄*	PhoP₄*	P _mgrB	AQ₄*为拟南芥AHK4和大肠杆菌PhoQ的传感域嵌体，经改造与所有大肠杆菌双组分系统绝缘，可防止磷酸化串扰	[60]

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基于细菌双组分系统的生物传感器的研究进展

Research progress in biosensors based on bacterial two-component systems

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