摘要
重金属因其毒性、持久性、生物蓄积性以及对抗性基因的潜在贡献等特点,已引起人们越来越多的关注。对环境中重金属进行准确、快速、高效和灵敏的监测,对于保护环境和人体健康具有重要意义。全细胞微生物传感器作为一种集成生物识别模块与传感处理模块的生物监测技术,为环境重金属污染的监测提供了新的解决方案。近年来,基于转录因子的全细胞微生物传感器在重金属监测应用方面取得了显著进展。本文综述了重金属全细胞微生物传感器的基本组成及设计原理,总结了近年来研发的重金属全细胞微生物传感器的构建及其应用情况,分析了通过合成生物学技术优化重金属全细胞微生物传感器检测性能的应用实例,并展望了该领域面临的挑战及未来可能的研究方向。本文有望为环境中重金属污染的高效监测与有效防控提供重要参考。
关键词
重金属一般是指密度大于5 g/c
世界各国和组织均高度重视重金属污染控制问题,针对不同类型的重金属均制定了相应的限制标准和管制措
生物传感器的基本工作原理主要涉及生物识别与信号传感(

图1 生物传感器的工作原理。A:生物传感器的一般组成;B:重金属全细胞微生物传感器的生物识别与信号传输过程。
Figure 1 Working principle of biosensor. A: General composition of biosensors; B: Recognition and sensing of heavy metals by whole-cell microbial biosensors.
全细胞微生物传感器(whole-cell microbial biosensor, WCMB)采用完整的微生物细胞集成生物识别模块与信号传感模块,与酶、核酸生物传感器相比,其制备成本更低、操作性能更稳定,能够在复杂环境中生存和适应,并可重复使用;其中以转录因子作为生物识别模块构建的重金属WCMB数量最多、应用最广,不仅展现出对多种重金属离子的高灵敏性和特异性响应,还具备简单快速、现场检测的能
虽然早在10年前已有研究者对重金属WCMB的信号输出/输入、特异性/耐受性、底盘细胞选择以及环境应用、安全风险及监管等方面进行了详细综
1 基于转录因子的重金属全细胞微生物传感器的构建及应用
1.1 全细胞微生物传感器的基本组成和设计原理
WCMB以完整细胞集成生物识别模块与传感处理模
1.1.1 响应元件的选择
响应元件通常由一组响应重金属的转录因子及其启动子组成;基于转录因子构建的WCMB数量最多、应用最广,其工作原理模仿微生物的重金属抗性系统,依赖重金属与转录因子相互作用,从而驱动报告信号基因表

图2 转录因子对重金属的响应机制。A:转录激活;B:转录抑制。
Figure 2 Responsive mechanisms of transcription factors to heavy metals. A: Transcription activation; B: Transcription inhibition.
Metal-responsive protein family | Family members and responders | References |
---|---|---|
MerR family | MerR (Hg), ZntR (Cd, Pb, Zn), ZccR (Zn, PbrR (Pb), CueR (Cu, Au), GolS (Au), HmrR (Ag), CoaR (Co), CadR (Cd), NimR (Ni) |
[ |
Fur family | Fur (Fe), Zur (Zn), Mur (Mn), Nur (Ni), PerR (Mn/Fe) |
[ |
DtxR family | DtxR, IdeR, SirR (Fe), MntR (Mn, ScaR (Cd), TroR (Mn, Zn) |
[ |
NikR family | NikR (Ni) |
[ |
ArsR/SmtB family | CmtR, ZiaR, AztR (Zn), CzrA (Zn, Co), ArsR (As, Sb, Bi), CadC (Cd, Pb, Zn), SmtB (As, Cd), NmtR (Ni, Co), KmtR (Ni), BxmR (Cu, Ag, Zn, Cd) |
[ |
CsoR-RcnR family | CsoR (Cu), RcnR (Ni、Co) |
[ |
CopY family | CopY (Cu) |
[ |
1.1.2 报告元件的选择
生物发光或荧光等光信号传导方式,因其敏感性及报告基因的多样性而备受青睐。这些报告基因可以是荧光蛋白或荧光素酶;常用的荧光蛋白包括绿色荧光蛋白(green fluorescent protein, GFP)、黄色荧光蛋白(yellow fluorescent protein, YFP)、红色荧光蛋白(red fluorescent protein, RFP)等;这些蛋白在特定波长的光激发下能够发出稳定且明亮的荧光,对细胞无毒害且易于检
1.1.3 基因回路的设计
基因回路主要由调节元件和被调节元件组成;调节元件通常包括启动子和转录因子等,它们负责重金属的识别和转录调控;被调节元件则多为编码信号蛋白的基因,负责产生输出信号;金属WCMB基因回路的设计可以有多种方案,如

图3 重金属全细胞微生物生物传感器基因回路的设计方案。A:单输入信号通路;B:双输入单诱导信号通路;C:双输入双诱导信号通路;D:多输入信号通路。
Figure 3 Design of gene circuits for heavy metal whole-cell microbial biosensor. A: Single-input signal pathway; B: Double-input single-induction signal pathway; C: Double-input double-induction signal pathway; D: Multiple-input signal pathway.
1.1.4 底盘细胞的选择
底盘细胞作为监测重金属浓度及毒性的受体宿主,应具备良好的环境适应能力和遗传稳定性,以提升传感器的可靠性、重复性和稳定性;此外,底盘细胞对目标检测物应具有较高的敏感性,能在低目标物浓度下迅速响应,从而拓宽检测范围并提高精度;底盘细胞还应能够特异性地识别目标检测物,避免交叉反应,减少干扰和误差,进而提高传感器的准确
1.2 基于转录因子的重金属全细胞微生物传感器的构建及应用
转录因子是生物传感器中最常见的响应元件,能够识别并结合特定的DNA序列,从而调控相关基因的表达。由于转录因子具有结合特异性和信号转导能力,因此转录因子的选择与设计决定了WCMB的检测性能。转录因子通常由DNA结合域和配体结合域组成;根据其作用机制可分为转录激活因子和转录抑制因子;根据变构方式又可分为单组分转录因子和双组分转录因
Sensing element | Detection object | Reporter gene | Host cell | Application | Detection range (μmol/L) | References |
---|---|---|---|---|---|---|
Transcription activator | ||||||
MerR |
H | rfp | Escherichia coli DH5α | Rapid and convenient screening of total inorganic mercury in cosmetics | 0.050 0-10 |
[ |
H | rfp, amilcpblue | Escherichia coli DH5α | The extremely wide linear range meets the different monitoring requirements | 0.001 0-1 (Mer-RFP), 0.002 0-0.125 0 (Mer-Blue) |
[ | |
ZntR |
Z | zntR, ribB, oprF | Escherichia coli BL21 | Microbial fuel cell electrobiosensor | 0-400 |
[ |
PbrR |
P | vio ABCDE | Escherichia coli TOP10 |
Detection of biologically available P | 0.187 5-1.500 0 |
[ |
P | Luc | Escherichia coli DH5α |
Detection of P | 1-100 |
[ | |
CadR |
C | vio ABCDE | Escherichia coli TOP10 |
Detection of soluble C | 0.049 0-25 |
[ |
C | rfp | Multiple Gram-negative bacteria | The importance of WCMB testing in different genera of bacteria was emphasized |
0.500 0–2.000 0 μg/mL (E. coli DH5α) 0.100 0 μg/mL (Pseudomonas aeruginosa PAO1) 10 μg/mL (Shewanella oneidensis MR-1) 0.250 0 μg/mL (Enterobacter sp. NCR3) 1 μg/mL (Enterobacter sp. LCR17) |
[ | |
P | Luc | Escherichia coli DH5α |
For the detection of P | 0.010 0-10 |
[ | |
CueR |
A | rfp | Cupriavidus etallidurans |
Monitoring the concentration of A | ≥0.110 0 |
[ |
Transcription inhibitors | ||||||
EcArsR |
AsO | luc | Escherichia coli DH5α | A WCMB that is highly sensitive to arsenite has been developed | 0.100 0-1≥0.040 0 |
[ |
ArsR1 |
AsO | gfp | Escherichia coli TOP10 | A WCMB that is highly sensitive to arsenite has been developed |
0.030 0-0.100 0 (2.250 0-7.500 0 μg/L) ≥0.010 0 |
[ |
SxArsR |
S | luc | Sphingobium xenophagum C1 | A novel subtype of the ArsR family transcription factor, designated as SxArsR, has been identified, which exhibits specific responsiveness to Sb |
0.010 0-6 ≥0.010 |
[ |
Combination of transcription factors | ||||||
MerR, CadR |
H | egfp, mCherry | Escherichia coli TOP10 | Detection of concurrent heavy metal contaminants in the environment |
0-40 (H |
[ |
CadC, CadR |
C | egfp and mCherry | Escherichia coli TOP10 | Detection of high concentrations of biologically available Cd in water | ≥0.050 0 (CadC-eGFP), ≥0.100 0 (CadR-mCherry) |
[ |
MerR, PbrR |
H | Indigoidine biosynthetic module | Escherichia coli TOP10 |
Detection of H | ≥0.008 0 |
[ |
CadR, MerR |
C | vioABE, vioC | Escherichia coli TOP10 | Designed for detecting heavy metal contaminants in seawater |
0.004 9-40, ≥0.004 9 (C |
[ |
UzcRS, UrpRS |
UO | gfp | Caulobacter vibrioides | Detection of uranium (U) in groundwater samples | ≥1 |
[ |
(待续)
≥ indicates the minimum detection limit;
1.2.1 基于转录激活因子的重金属全细胞微生物传感器的构建及应用
MerR、ZntR、PbrR、CadR、CueR、CupR以及GolS是细菌中用于重金属解毒的转录激活因
MerR家族的金属调控蛋白是一种被广泛用于构建WCMB的关键元件,通常具有保守的N端DNA结合域和结构存在差异的C端金属结合域,能够特异性地与金属离子结合,导致蛋白构象扭曲,从而容易被RNA聚合酶识别并转录激活下游报告基因的表达,产生可被检测的信号输
对H
铅响应转录调控因子PbrR的金属结合域能够特异性地结合P
CadR金属调控蛋白具有Cys77、Cys112、Cys119等3个保守的金属结合位点,对C
当以PbrR和CadR作为识别元件,荧光素酶蛋白Luc作为报告元件时构建了2种WCMB,其中pGL3-luc/cad可检测0.010 0-10 μmol/L的P
CueR蛋白作为转录激活因子,能够结合在启动子DNA的-35--10区之间,通过改变DNA构象来激活下游基因的转录;以CueR作为识别元件及红色荧光蛋白(RFP)作为报告元件,在耐重金属贪铜菌(Cupriavidus metallidurans)中构建的受启动子PcopZ驱动的金响应WCMB具有高度的A
尽管这些转录因子作为识别元件对重金属展现出高特异性,但在实际应用中,仍可能受到其他金属离子的影响,进而削弱WCMB的准确性和灵敏度。因此,持续的改进和优化工作仍然尤为重要。
1.2.2 基于转录抑制因子的重金属全细胞微生物传感器的构建及应用
目前,已有多种转录抑制因子被用于构建重金属监测WCMB,其中最典型的是来自细菌砷解毒系统中的砷结合蛋白ArsR;ArsR能够与ars操纵子的启动子序列结合,是ars基因簇的负调控因子;当ArsR与砷结合后从启动子区域释放,从而促使编码相关解毒蛋白的ars操纵子中的基因转
目前ArsR家族分为5种亚型,分别具有独特的As/Sb结合位
基于I型ArsR构建的砷响应WCMB是研究最多的一种,其中Fang
NikR是另一种被广泛用于构建重金属监测生物传感器的转录抑制因子。NikR是Ni依赖的转录调节蛋白,通过负调控Ni ABC型转运体(Nik ABCDE)的表达来应对过量的Ni摄入,在维持细菌细胞中的Ni稳态发挥关键作
1.2.3 基于组合转录因子的重金属全细胞微生物传感器的构建及应用
利用不同转录因子组合可以在单一传感细胞内构建多种传感模块,实现多种重金属的同时检测;目前已有多种基于转录因子组合的重金属WCMBs成功应用于实际环境中的重金属检测(
双组分系统是细菌体内的另一种信号转导系统,由组氨酸激酶与应答受体/转录因子组成,分别负责信号识别和细胞行为调控;基于双组分转录因子构建的WCMB具有复杂的遗传调控网络,其在实际环境监测应用中的普及程度尚不及上述基于单组分转录因子系统构建的WCM
2 基于合成生物学策略的全细胞微生物传感器性能改造与优化
尽管现有的WCMB能够检测多种重金属,但仍面临着部分重金属因毒性机制复杂或环境浓度低而导致检测难度增大的挑战;此外,WCMB在灵敏度、特异性和稳定性方面仍有待提高,以满足更为复杂、精细的环境监测需
Sensing element | Detection object | Optimization strategy | Application | Performance enhancement (μmol/L) | References |
---|---|---|---|---|---|
ZntR |
C | Protein engineering |
Cr and Pb-responsive WCMB were developed from the znt-manipulation subsystem for the determination of P |
New selectivity for C |
[ |
P | Protein engineering |
The content of P |
0-0.010 0, <0.005 |
[ | |
ArsR |
P | Protein engineering |
The content of P | Detection accuracy>95% |
[ |
MerR |
H | Protein engineering |
The content of H | ≥0.020 4 |
[ |
H Z C | Protein engineering | A new chimeric regulator WCMB was developed by replacing the metal-binding domain of MerR with ZntR or CueR |
≥0.001 0 H ≥30 Z ≥10 C |
[ | |
CadR |
C | Protein engineering |
The content of C | ≥0.450 0 μg/L |
[ |
C | Protein engineering |
The interference of H |
0.500 0-100, ≥0.079 |
[ | |
MerR |
H | Protein engineering |
The “Parabola principle” is proposed and a visualized WCMB for H | 0.200 0-0.250 0 |
[ |
H |
Protein engineering Promoter engineering |
A super-sensitive visualized WCMB for the detection of ultra-trace H |
≥0.313 0ng/L. When the fluorescence signal is more than 2.500 0 ng/l, it can be observed directl |
[ | |
ArsR |
AsO | Promoter engineering |
A high sensitivity AsO | 0.100 0-4, ≥0.010 0 |
[ |
AsO | Promoter engineering |
A highly sensitive and specific WCMB for AsO |
≥0.010 |
[ | |
AsO | Promoter engineering |
A functional promoter library screening method was developed, and different AsO | ≥0.040 0 |
[ | |
AsO | Promoter engineering |
A highly sensitive and specific WCMB for AsO | ≥0.100 0 |
[ | |
ArsR MerR |
AsO H | Promoter engineering |
A modular cascaded signal amplification method was developed, and a low-cost, portable and accurate AsO |
≥0.100 0 ppb AsO ≥0.010 0 ppb H |
[ |
CadR |
C | Promoter engineering |
A highly sensitive and specific C | ≥0.010 0 |
[ |
C | Promoter engineering |
A C |
≥0.000 |
[ |
≥ indicates a minimum detection limit; < indicates a lower detection limit;
2.1 蛋白质工程——改变转录因子的蛋白结构
除了对特定重金属的专一性响应外,多数天然转录因子还对其他重金属具有非特异性响应,这在复杂环境应用中常导致多金属干扰问题;转录因子的特异性改造,对于解决实际应用过程中的响应干扰问题具有重要的意
基于已知转录因子的结构,利用人工智能模拟与预测蛋白质结构,通过精确改造关键氨基酸残基的位置、电荷及亲疏水性等,优化转录因子与目标分子的相互作用,从而设计出新型结构的转录因
(待续)
除了定点突变以外,研究者们还通过DNA大片段的替换,改造出性能更为优越的WCMB。Mendoza
此外,通过基因随机诱变与定向进化,可以筛选出与目标分子结合能力显著增强的突变体,从而提升转录因子的结合与调控性
2.2 启动子工程——优化结合位点序列、增强报告信号
2.2.1 优化转录因子结合位点及RNA聚合酶结合位点序列
当了解目标基因的启动子序列及转录因子结合位点信息时,可通过优化启动子与操纵子的调控序列以增强报告元件的表达强度,这对提升WCMB的检测灵敏性具有重要作用。传统重金属WCMB的设计主要依赖于模拟细菌的重金属抗性系统,而Guo
Chen
2.2.2 设计增强报告信号的基因回路
在复杂环境中,目标信号往往微弱且易受干扰;对此,可以通过对基因回路的巧妙设计,包括正反馈和负反馈机制等,来放大报告基因的响应强度,以显著提高检测的灵敏度和准确性,对增加生物传感器的检测范围具有重要的作
Jia
在需要快速、大量检测样本的情况下,通过调整识别元件和报告元件的数量以及增加信号放大模块是一种更为有效地提高WCMB灵敏性的方法;通过简单的组件调整和数量增加,即可实现对信号的放大和检测灵敏度的提升,相较于复杂的基因回路设计,该策略在成本控制方面可能更具优
3 总结与展望
基于不同类型转录因子的WCMB在环境重金属污染检测方面展现出极为广阔的应用潜力。由于重金属如Pb、Hg、Cd、As等对人体健康和生态环境系统构成严重威胁,因此对环境中重金属进行准确、快速、高效且灵敏的检测对于保护环境和人体健康具有重要的意义。这种检测技术不仅能确定重金属污染的种类与浓度,还为重金属污染的防控提供有力支持。此外,借助合成生物学的策略,可以对WCMB的结构和功能进行改造和优化,从而实现对低浓度重金属污染物的精准检测,对于及时发现和解决潜在的重金属环境污染问题具有重要意义。此外,WCMB还具有实时在线监测的潜力,通过将WCMB集成到常规环境监测系统中,可以实现对重金属污染物的实时、连续监测,为环境保护部门提供及时、准确的数据支持,有助于制定科学的污染防控策略。
然而,与一些化学检测仪器(如ICP-MS等)相比,尽管化学仪器成本较高、操作相对复杂,但其准确性高;相比之下,WCMB在成本上更具优势,但在准确性方面仍有待提升。其次,由于现有合成生物学技术的局限性和转基因生物可能带来的安全威胁等问题,目前WCMB的设计过程和实际应用仍存在困难。不同类型转录因子的选择和应用仍需更多实验验证和优化。此外,WCMB的稳定性和可重复性问题也亟待解决,将其从实验室规模转移到商业规模同样面临重大挑战。未来可以继续提高基于转录因子的WCMB的灵敏度和特异性,降低WCMB的制造成本和操作复杂性,并优化其稳定性和再生性,以实现实时连续监测。此外,WCMB的设计可以更加注重创新和智能化。一方面,通过开发新的识别元件,有望为重金属污染的有效防控提供更有力的技术支持;另一方面,随着互联网、人工智能等技术的不断发展,重金属WCMB可以向小巧、便携、灵敏、可重复使用等方向进一步发展。随着相关技术和法规的不断进步与完善,相信这一领域将会取得更加丰硕的成果,为保护环境和人体健康提供更加有力的保障。
作者贡献声明
王明月:综述撰写、表格制作和图形绘制;许玫英:主题选择,提供了该领域内的专业见解和建议;纠敏:文献查阅整理与格式校对;陈杏娟:文献的深入分析和讨论,对综述进行修改和补充。
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