合成生物学研究中的微生物启动子工程策略

doi:10.12211/2096-8280.2020-092

摘要/Abstract

摘要：

合成生物学研究对于我国绿色生物制造产业和可持续发展战略至关重要。启动子是合成生物学核心元件，是在转录水平上实现基因高效、精准表达调控的最关键因素之一。本文重点对原核微生物启动子工程研究的基本内容、研究进展及发展趋势进行了综述。首先概述了启动子序列基本特征及其受RNA聚合酶σ因子识别调控的一般规律；并以大肠杆菌乳糖操纵子为例简要介绍了诱导型启动子的负调控与正调控诱导机制。其次，分别从对靶基因自身内源启动子进行突变改造以及采用高效外源启动子进行替换改造这两个方面入手，阐述了启动子改造的常用策略。进一步对近年来公开报道的不同类型诱导型启动子进行了梳理，小结了代表性化学分子诱导剂以及物理信号诱导方式的种类及基本特征。简述了非模式和模式微生物组成型启动子的研究进展及研究侧重点。结合动态代谢调控技术及人工智能工具的突破性发展，提出具有动态调控功能的特殊启动子的发现与改造、全新性能启动子元件的人工智能设计与改造等将成为启动子工程研究的新方向与新前沿。最后分析了启动子工程领域存在的挑战性问题，展望了今后的研究重点，并结合合成生物学的发展，进一步强调了微生物启动子工程的重要作用。

关键词: 合成生物学, 启动子工程, 调控机制, 改造策略, 诱导信号, 动态代谢调控, 人工智能从头设计

Abstract:

Synthetic biology is of vital importance to the green biomanufacturing industry and sustainable development strategies of our country. Promoter is the core-component of synthetic biology, playing a significant role in highly efficient and fine-tuning expression and regulation of target genes at the transcriptional level. Herein we summarized and discussed the key progress and future frontiers of microbial promoter engineering, particularly for prokaryotic microorganisms. Firstly, we introduced the basic DNA sequence characteristics of promoters and the regular mechanism for promoter recognition and transcription-initiation by RNA polymerase sigma factors. Inducible mechanisms for both negative and positive regulation were particularly highlighted with the typical lac operator of Escherichia coli as an example. Then, effective strategies for obtaining improved-promoters were summarized, which were roughly divided into two categories: endogenous promoter mutation and heterologous promoter replacement. For the endogenous promoter mutation, the following strategies, e.g. point mutation toward sigma factor consensus sequence, coupling optimization of -35 and -10 regions with RBS sequence, random mutation or saturation mutagenesis of UP element or spacer sequences accompanying with promoter library construction and high-throughput screening were emphasized. For the heterologous promoter replacement, strategies such as substituting the native promoter into stronger ones from other microorganisms, introducing phage-source chimeric promoters, tuning the constitutive promoter into inducible pattern and integrating positive regulator(s), were mainly discussed. We further sorted out the representative inducers for inducible promoters reported so far, including both chemical molecules and physical signals. Progress in constitutive promoters of non-model and model microbial organisms were simply summarized as well. Next, arising from the breakthrough development of dynamic metabolic regulation and artificial intelligence (AI), we proposed that the innovative research on identification and evolution of new and unique promoters with dynamic-response features and AI de novo design for promoters with novel/superior functions will be the new frontiers of promoter engineering. Finally, we analyzed the challenging scientific issues in the microbial promoter engineering, from the viewpoint of both basic research and large-scale applications; and further discussed the research priority coupling with the vigorous development of synthetic biology.

Key words: synthetic biology, promoter engineering, regulation mechanism, evolution strategies, induction signals, dynamic metabolic regulation, AI de novo design

中图分类号:

Q812

于慧敏, 郑煜堃, 杜岩, 王苗苗, 梁有向. 合成生物学研究中的微生物启动子工程策略[J]. 合成生物学, 2021, 2(4): 598-611.

Huimin YU, Yukun ZHENG, Yan DU, Miaomiao WANG, Youxiang LIANG. Microbial promoter engineering strategies in synthetic biology[J]. Synthetic Biology Journal, 2021, 2(4): 598-611.

图/表 5

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σ 因子	微生物名称	-35区	-10区	参考文献
σ⁷⁰	大肠杆菌	TTGACA	TATAAT	[6-7]
σ⁷⁰	枯草芽孢杆菌	TTGACA	TATAAT	[8-9]
σ^A	谷氨酸棒杆菌	TTG(A/C)CA	TGNTATAATNG	[12-13]
σ^A	红色红球菌	TTGNNN	(T/C)GNNA(A/C)AAT	[14]
σ^A	运动发酵单胞菌	TTGNNN	TATNNN	[15]
σ^hrdB	阿维链霉菌	TTGACA	tAgATT	[16]

σ 因子	微生物名称	-35区	-10区	参考文献
σ⁷⁰	大肠杆菌	TTGACA	TATAAT	[6-7]
σ⁷⁰	枯草芽孢杆菌	TTGACA	TATAAT	[8-9]
σ^A	谷氨酸棒杆菌	TTG(A/C)CA	TGNTATAATNG	[12-13]
σ^A	红色红球菌	TTGNNN	(T/C)GNNA(A/C)AAT	[14]
σ^A	运动发酵单胞菌	TTGNNN	TATNNN	[15]
σ^hrdB	阿维链霉菌	TTGACA	tAgATT	[16]

诱导类型	诱导剂	相关微生物	启动子特征	参考文献
化学信号	IPTG/乳糖	大肠杆菌；谷氨酸棒杆菌；枯草芽孢杆菌等	Plac、Ptac；广谱性好、诱导强度较高；诱导剂价格高；基于LacI阻遏蛋白（负调控蛋白）的负调控机制	[40-42]
	IPTG	大肠杆菌	T7启动子，噬菌体来源；pET表达系统；常与溶源化宿主菌（即T7 RNA聚合酶基因插入在宿主菌基因组中）联用；诱导强度高、广谱性好；诱导剂价格高；基于阻遏蛋白的负调控机制	[39]
	L-鼠李糖/ L-阿拉伯糖	大肠杆菌；红球菌等	rhaP_BAD/araP_BAD启动子；AraC正/负调控蛋白；pBAD表达系统；诱导剂价格较高；诱导强度较高；广谱性较好；其中rhaP_BAD为基于正调控蛋白的正调控机制；araP_BAD为基于阻遏蛋白的负调控机制	[43-44]
	木糖	枯草芽孢杆菌；酵母菌等	Pxyl；诱导剂价格高；基于阻遏蛋白的负调控机制	[45]
	蔗糖	枯草芽孢杆菌等	PsacB/PsacP；强度低，易泄露表达；基于不依赖于ρ因子的RNA抗终止子序列的负调控机制	[46]
	四环素/脱水四环素	浑浊红球菌；丙酮丁醇梭菌等	Ptet；诱导剂用量低；基于TetR阻遏蛋白的负调控机制	[47-49]
	离子液体	木质素分解肠杆菌；大肠杆菌；酿酒酵母等	eilAR表达盒；EilA表达离子液体自诱导内膜转运蛋白；EilR为负调控蛋白；负调控机制	[50]
	尿酸	耐辐射奇球菌；大肠杆菌等	基于HucR阻遏蛋白的负调控机制；强度与pBAD/pET系统相当	[51]
	巴豆酰胺/ 甲基丙烯酰胺	红球菌等	红球菌适用；基于正负调控蛋白协同作用的复合调控机制	[52]
	联苯	红球菌RHA1；红串红球菌	基于BphS-BphT两组分调控元件的正调控机制	[53]
	ε-己内酰胺/十几种腈类	链霉菌；红球菌等	PnitA-NitR杂合元件；强启动高表达；基于NitR正调控蛋白的正调控机制	[28,54]
	尿素	红色红球菌	Pnh，Pami，Pa2；基于正负调控蛋白协同作用的调控机制，具体调控机制尚未解析	[14,55]
	铁离子	多能硫碱弧菌；大肠杆菌等	P3AF启动子，携带19 bp大肠杆菌铁摄取调节子蛋白基因fur的核心区序列，铁离子严谨调控	[56-57]
	碱性染料	大肠杆菌；恶臭假单胞菌；苜蓿中华根瘤菌；新月柄杆菌等	基于EilR阻遏蛋白-操纵序列的负调控机制；结晶紫等廉价、严谨、高效诱导；微生物广谱适用性；低成本	[58]
	丙烯酸盐/葡萄糖酸/红霉素/柚皮素	大肠杆菌；类球红细菌；恶臭假单胞菌	属于TetR家族阻遏蛋白的AcuR/MphR/TtgR等调控蛋白；CdaR，转录激活蛋白；响应4种代谢物的生物传感器性能，分别为负调控和正调控机制；不同诱导信号的正交效果	[59]
	甲醇	红串红球菌；毕赤酵母等	异柠檬酸裂解酶基因（icl_Re）上游序列；基于RamB_Re阻遏蛋白的负调控机制	[60]
	乙醇	运动发酵单胞菌等	P0405、P0435和P0038启动子，调控机制尚未明确	[61]
	对异丙苯甲酸酯/间苯二酚	链霉菌；放线菌	负调控机制；对异丙苯甲酸酯诱导，P21启动子-CymR调控蛋白；间苯二酚，PA3启动子-RolR调控蛋白	[24,62]
	磷酸盐饥饿	大肠杆菌	磷酸盐饥饿诱导；基于PhoB激活的正调控机制	[63]
	氮饥饿	三角褐指藻	铵转运蛋白AMT基因启动子；氮饥饿条件强诱导；机制尚未报道	[64]
	低溶氧	大肠杆菌；谷氨酸棒杆菌；盐单胞菌等	vgb启动子；nar启动子；响应低溶氧条件启动转录	[65-67]
	低pH	黑曲霉	Pgas，低pH诱导（pH2.0/3.0）；受未知功能双调控蛋白调控	[68]
物理信号	高温	大肠杆菌；枯草芽孢杆菌等	pR/pL，受温敏型阻遏蛋白CI857调控；P2和P7等	[69-70]
	低温(-15 ℃)	大肠杆菌	PcspA耦合LacI负调控；杂合启动子，严谨低温调控，pCold载体效率与pET14相当	[71]
	低温(-30 ℃)	大肠杆菌	整合多个温敏转录开关与蛋白降解模块；从37 ℃到30 ℃严谨调控；复合调控机制	[72]
	光	大肠杆菌；酵母菌等	10余种光敏调控蛋白Cph8/OmpR；YF1/FixJ；EL222等；白光诱导、蓝光诱导、黑暗诱导等	[73-75]

诱导类型	诱导剂	相关微生物	启动子特征	参考文献
化学信号	IPTG/乳糖	大肠杆菌；谷氨酸棒杆菌；枯草芽孢杆菌等	Plac、Ptac；广谱性好、诱导强度较高；诱导剂价格高；基于LacI阻遏蛋白（负调控蛋白）的负调控机制	[40-42]
	IPTG	大肠杆菌	T7启动子，噬菌体来源；pET表达系统；常与溶源化宿主菌（即T7 RNA聚合酶基因插入在宿主菌基因组中）联用；诱导强度高、广谱性好；诱导剂价格高；基于阻遏蛋白的负调控机制	[39]
	L-鼠李糖/ L-阿拉伯糖	大肠杆菌；红球菌等	rhaP_BAD/araP_BAD启动子；AraC正/负调控蛋白；pBAD表达系统；诱导剂价格较高；诱导强度较高；广谱性较好；其中rhaP_BAD为基于正调控蛋白的正调控机制；araP_BAD为基于阻遏蛋白的负调控机制	[43-44]
	木糖	枯草芽孢杆菌；酵母菌等	Pxyl；诱导剂价格高；基于阻遏蛋白的负调控机制	[45]
	蔗糖	枯草芽孢杆菌等	PsacB/PsacP；强度低，易泄露表达；基于不依赖于ρ因子的RNA抗终止子序列的负调控机制	[46]
	四环素/脱水四环素	浑浊红球菌；丙酮丁醇梭菌等	Ptet；诱导剂用量低；基于TetR阻遏蛋白的负调控机制	[47-49]
	离子液体	木质素分解肠杆菌；大肠杆菌；酿酒酵母等	eilAR表达盒；EilA表达离子液体自诱导内膜转运蛋白；EilR为负调控蛋白；负调控机制	[50]
	尿酸	耐辐射奇球菌；大肠杆菌等	基于HucR阻遏蛋白的负调控机制；强度与pBAD/pET系统相当	[51]
	巴豆酰胺/ 甲基丙烯酰胺	红球菌等	红球菌适用；基于正负调控蛋白协同作用的复合调控机制	[52]
	联苯	红球菌RHA1；红串红球菌	基于BphS-BphT两组分调控元件的正调控机制	[53]
	ε-己内酰胺/十几种腈类	链霉菌；红球菌等	PnitA-NitR杂合元件；强启动高表达；基于NitR正调控蛋白的正调控机制	[28,54]
	尿素	红色红球菌	Pnh，Pami，Pa2；基于正负调控蛋白协同作用的调控机制，具体调控机制尚未解析	[14,55]
	铁离子	多能硫碱弧菌；大肠杆菌等	P3AF启动子，携带19 bp大肠杆菌铁摄取调节子蛋白基因fur的核心区序列，铁离子严谨调控	[56-57]
	碱性染料	大肠杆菌；恶臭假单胞菌；苜蓿中华根瘤菌；新月柄杆菌等	基于EilR阻遏蛋白-操纵序列的负调控机制；结晶紫等廉价、严谨、高效诱导；微生物广谱适用性；低成本	[58]
	丙烯酸盐/葡萄糖酸/红霉素/柚皮素	大肠杆菌；类球红细菌；恶臭假单胞菌	属于TetR家族阻遏蛋白的AcuR/MphR/TtgR等调控蛋白；CdaR，转录激活蛋白；响应4种代谢物的生物传感器性能，分别为负调控和正调控机制；不同诱导信号的正交效果	[59]
	甲醇	红串红球菌；毕赤酵母等	异柠檬酸裂解酶基因（icl_Re）上游序列；基于RamB_Re阻遏蛋白的负调控机制	[60]
	乙醇	运动发酵单胞菌等	P0405、P0435和P0038启动子，调控机制尚未明确	[61]
	对异丙苯甲酸酯/间苯二酚	链霉菌；放线菌	负调控机制；对异丙苯甲酸酯诱导，P21启动子-CymR调控蛋白；间苯二酚，PA3启动子-RolR调控蛋白	[24,62]
	磷酸盐饥饿	大肠杆菌	磷酸盐饥饿诱导；基于PhoB激活的正调控机制	[63]
	氮饥饿	三角褐指藻	铵转运蛋白AMT基因启动子；氮饥饿条件强诱导；机制尚未报道	[64]
	低溶氧	大肠杆菌；谷氨酸棒杆菌；盐单胞菌等	vgb启动子；nar启动子；响应低溶氧条件启动转录	[65-67]
	低pH	黑曲霉	Pgas，低pH诱导（pH2.0/3.0）；受未知功能双调控蛋白调控	[68]
物理信号	高温	大肠杆菌；枯草芽孢杆菌等	pR/pL，受温敏型阻遏蛋白CI857调控；P2和P7等	[69-70]
	低温(-15 ℃)	大肠杆菌	PcspA耦合LacI负调控；杂合启动子，严谨低温调控，pCold载体效率与pET14相当	[71]
	低温(-30 ℃)	大肠杆菌	整合多个温敏转录开关与蛋白降解模块；从37 ℃到30 ℃严谨调控；复合调控机制	[72]
	光	大肠杆菌；酵母菌等	10余种光敏调控蛋白Cph8/OmpR；YF1/FixJ；EL222等；白光诱导、蓝光诱导、黑暗诱导等	[73-75]

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