工程噬菌体的合成生物学“智造”

doi:10.12211/2096-8280.2022-070

摘要/Abstract

摘要：

噬菌体，以杀菌特性而闻名的天然病毒，是地球上多样性和丰度最高的生物体。在过去100多年中，噬菌体的研究极大地推动了遗传学、分子生物学和合成生物学的发展。随着耐药超级细菌的流行，噬菌体疗法引人入胜的科学历史也被广为传颂。然而，相对于自然环境中丰富的噬菌体数量（总数为10³¹，比所有其他生物的总和还要多），目前只有少数噬菌体成功应用于对抗耐药性细菌感染及其他工程领域。当前，急需践行合成生物学从“造物致知”到“造物致用”理念，采用高通量测序和基因组精准编辑等先进生物技术来创建具有独特属性的增强变体，提高噬菌体治疗的疗效和可编程性。本文综述了近年来噬菌体基因工程改造方法的技术进展以及合成生物学助力噬菌体应用技术发展的研究动态，如按照实际需求改造噬菌体宿主范围、借助宏基因组挖掘自然界中噬菌体的广泛资源、结合多组学技术揭示噬菌体与宿主互作的分子机制、调控肠道噬菌体组维持肠道稳态以促进人体健康及利用大数据和新型人工智能指导噬菌体理性设计。总之，合成生物学正在跨时代驱动传统实验研究范式转变，结合“设计—构建—测试—学习（DBTL）循环”理性设计目标噬菌体，无论是自上而下的体系优化，还是自下而上的生命体重构，工程噬菌体的合成生物学“智造”都大有可为。

关键词: 噬菌体, 合成生物学, 工程噬菌体, 可编程基因组, 理性设计, 噬菌体治疗

Abstract:

Bacteriophages (phages), natural viruses known for infecting and killing bacteria, are the most diverse and abundant organisms on Earth. Within the past 100 years of research on phages, breakthroughs in genetics, molecular biology, and synthetic biology have been successfully achieved. The fascinating scientific history of phage therapy has been repeatedly reported, as drug-resistant bacteria are becoming increasingly prevalent. Although phages outnumber other species combined in nature (10³¹ in total), only a small fraction of them have been successfully exploited for fighting infections caused by drug-resistant bacteria. Therefore, there is an urgent need for implementing the motto of synthetic biology, "build to learn, build to use", and also using methods such as high-throughput sequencing and precise genome editing to create enhanced variants with unique features, improving efficacy and programmability for phage therapy. In the review, we discuss recent technological advances in phage genome engineering approaches and the potential applications of synthetic biology to engineer phages, such as modifying the host range of phages for practical needs, mining the extensive resources of phages in nature with the help of macro genomes, combining multi-omics technologies to reveal molecular mechanisms underlying phage-host interactions, regulating intestinal phages to maintain intestinal homeostasis for human health, and using big data and artificial intelligence to guide rational phage design. Synthetic biology is driving a paradigm shift in traditional experimental research by combining "Design-Build-Test-Learn (DBTL) cycles" to rational design for phages, making synthetically designed phages promising for both top-down system optimization and bottom-up life-form reconstruction.

Key words: phage, synthetic biology, engineered phage, reprogramming genome, rational design, phage therapy

中图分类号:

Q816

陈青黎, 童贻刚. 工程噬菌体的合成生物学“智造”[J]. 合成生物学, 2023, 4(2): 283-300.

Qingli CHEN, Yigang TONG. Merging the frontiers: synthetic biology for advanced bacteriophage design[J]. Synthetic Biology Journal, 2023, 4(2): 283-300.

图/表 2

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策略	菌株	具体方法	年份	出处
随机诱变	T4噬菌体	紫外线诱变获取突变体	1966	[49]
随机诱变	T3、T7、肠道沙门氏菌噬菌体	改善噬菌体的热稳定性	2020	[51]
经典同源重组	T2噬菌体	改造尾丝蛋白结构	2009	[98].
	T4噬菌体	改变或扩大宿主T4样噬菌体宿主范围	2017	[99]
	T7噬菌体	T7噬菌体基因组的单碱基替换和全基因替换	2020	[55]
	T7噬菌体	获得新的RBP结构	2021	[100]
BRED	分歧杆菌噬菌体Giles	非必需基因缺失、读码框内缺失、点突变、无义突变、外源基因精确插入	2008	[58]
	肠杆菌科噬菌体P1	去除IS元件	2012	[60]
	沙门氏菌噬菌体	实现溶原和裂解性质转换	2014	[61]
	克雷伯氏菌噬菌体	建立了克雷伯氏菌噬菌体基因组的重组系统	2017	[62]
	分枝杆菌噬菌体BPs、ZoeJ	将治疗性温和噬菌体转化为烈性噬菌体	2019	[24]
CRISPR-Cas	链球菌噬菌体2972	Ⅱ-A型CRISPR-Cas实现特定点突变和大片段删除	2014	[64]
	T7噬菌体	Ⅰ-E型CRISPR-Cas系统编辑T7基因组	2014	[65]
	乳酸乳球菌烈性噬菌体P2	点突变，基因缺失和替换	2017	[66]
	克雷伯氏菌噬菌体	点突变，基因缺失和替换	2018	[68]
	金黄色葡萄球菌噬菌体	Ⅲ-A型CRISPR-Cas系统CRISPR-Cas10	2019	[69]
	T4噬菌体	Ⅴ型CRISPR-Cas12a系统构建包含缺失和插入的重组T4	2021	[70]
	T5噬菌体	Ⅱ-A型CRISPR-Cas9系统效果不稳定，提出基于Retron的重组方案	2021	[75]
	需钠弧菌噬菌体TT4	使用CRISPR-Cas9基因的缺失和替换	2022	[71]
	大肠杆菌噬菌体	CRISPR-Cas13a	2022	[72]
	ФKZ、OMKO1和PaMx41噬菌体	CRISPR-Cas13a+正向选择基因acrVIA1	2022	[73]
Genome Reboot	大肠杆菌、克雷伯氏菌噬菌体	YAC	2015	[78]
	PICIs	YAC	2020	[80]
	铜绿假单胞菌噬菌体	YAC	2021	[79]
	大肠杆菌噬菌体	电转重启	2019	[84]
	沙门氏菌噬菌体	电转重启	2022	[85]
	MS2噬菌体	无细胞体系	1996	[89]
	T7噬菌体	无细胞体系	2012	[91]
	T4噬菌体	无细胞体系	2018	[86]
	耶尔森氏菌噬菌体	无细胞体系	2022	[88]
	克雷伯氏菌噬菌体	无细胞体系	2022	[88]
	抗酸性分枝杆菌噬菌体	无细胞体系	2022	[92]
	李斯特氏菌、芽孢杆菌、葡萄球菌噬菌体	L-forms	2018	[96]

策略	菌株	具体方法	年份	出处
随机诱变	T4噬菌体	紫外线诱变获取突变体	1966	[49]
随机诱变	T3、T7、肠道沙门氏菌噬菌体	改善噬菌体的热稳定性	2020	[51]
经典同源重组	T2噬菌体	改造尾丝蛋白结构	2009	[98].
	T4噬菌体	改变或扩大宿主T4样噬菌体宿主范围	2017	[99]
	T7噬菌体	T7噬菌体基因组的单碱基替换和全基因替换	2020	[55]
	T7噬菌体	获得新的RBP结构	2021	[100]
BRED	分歧杆菌噬菌体Giles	非必需基因缺失、读码框内缺失、点突变、无义突变、外源基因精确插入	2008	[58]
	肠杆菌科噬菌体P1	去除IS元件	2012	[60]
	沙门氏菌噬菌体	实现溶原和裂解性质转换	2014	[61]
	克雷伯氏菌噬菌体	建立了克雷伯氏菌噬菌体基因组的重组系统	2017	[62]
	分枝杆菌噬菌体BPs、ZoeJ	将治疗性温和噬菌体转化为烈性噬菌体	2019	[24]
CRISPR-Cas	链球菌噬菌体2972	Ⅱ-A型CRISPR-Cas实现特定点突变和大片段删除	2014	[64]
	T7噬菌体	Ⅰ-E型CRISPR-Cas系统编辑T7基因组	2014	[65]
	乳酸乳球菌烈性噬菌体P2	点突变，基因缺失和替换	2017	[66]
	克雷伯氏菌噬菌体	点突变，基因缺失和替换	2018	[68]
	金黄色葡萄球菌噬菌体	Ⅲ-A型CRISPR-Cas系统CRISPR-Cas10	2019	[69]
	T4噬菌体	Ⅴ型CRISPR-Cas12a系统构建包含缺失和插入的重组T4	2021	[70]
	T5噬菌体	Ⅱ-A型CRISPR-Cas9系统效果不稳定，提出基于Retron的重组方案	2021	[75]
	需钠弧菌噬菌体TT4	使用CRISPR-Cas9基因的缺失和替换	2022	[71]
	大肠杆菌噬菌体	CRISPR-Cas13a	2022	[72]
	ФKZ、OMKO1和PaMx41噬菌体	CRISPR-Cas13a+正向选择基因acrVIA1	2022	[73]
Genome Reboot	大肠杆菌、克雷伯氏菌噬菌体	YAC	2015	[78]
	PICIs	YAC	2020	[80]
	铜绿假单胞菌噬菌体	YAC	2021	[79]
	大肠杆菌噬菌体	电转重启	2019	[84]
	沙门氏菌噬菌体	电转重启	2022	[85]
	MS2噬菌体	无细胞体系	1996	[89]
	T7噬菌体	无细胞体系	2012	[91]
	T4噬菌体	无细胞体系	2018	[86]
	耶尔森氏菌噬菌体	无细胞体系	2022	[88]
	克雷伯氏菌噬菌体	无细胞体系	2022	[88]
	抗酸性分枝杆菌噬菌体	无细胞体系	2022	[92]
	李斯特氏菌、芽孢杆菌、葡萄球菌噬菌体	L-forms	2018	[96]

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