文库构建与基因簇靶向筛选驱动的微生物天然产物高效发现

doi:10.12211/2096-8280.2023-083

摘要/Abstract

摘要：

微生物天然产物作为小分子药物的主要来源，已被广泛应用于医药与农业等领域。随着全球抗生素耐药性与其他公共健康问题的加剧，新结构、新靶点微生物天然产物发现迫在眉睫。大规模（宏）基因组测序揭示微生物蕴含了巨大的生物合成潜力，相继催生了多种不同类型的天然产物挖掘策略。然而，目前仍然缺乏将天然产物合成基因簇与编码产物快速关联的高效方案。近年来，（宏）基因组文库构建在获取批量天然产物合成基因簇方面展现出明显优势，结合高效的基因簇靶向筛选方法，显著加速了新结构天然产物系统发现。本文综述了三类基于（宏）基因组文库构建与靶向筛选驱动天然产物创新发现的策略，主要从克隆载体类型、文库构建方式、基因簇靶向筛选方法等角度进行了阐述，并对Cosmid/Fosmid文库、BAC/PAC文库、FAC/YAC文库等不同文库类型的优缺点及应用范围进行了对比，最后对这些策略的发展前景进行了展望。未来，基于文库构建与基因簇靶向筛选策略将极大驱动不同生境微生物来源的活性天然产物挖掘，预期大量新靶点、新结构天然产物将不断涌现。

关键词: 微生物天然产物, （宏）基因组挖掘, 基因簇, 文库构建, 基因簇靶向筛选, 高效发现

Abstract:

Microbial natural products (NPs) are the main sources of small molecule drugs and have been widely used in medicine, agriculture and other fields. The growing global antimicrobial resistance and other public health problems necessitate the rapid discovery of microbial NPs with novel structures or modes of action. With rapid advances in high-throughput and low-cost DNA sequencing technologies, large-scale microbial (meta)genomic sequencing reveals highly diverse biosynthetic gene clusters (BGCs) in bacteria and fungi, in which characterized compounds represent just the tip of the iceberg, with millions of novel small molecules awaiting discovery. Although diverse strategies have been developed for NP discovery, efficiently linking NP biosynthetic pathways to their encoded products remains a challenge. Recently, (meta)genomic library construction strategies have shown advantages to obtain NP biosynthetic pathways at scale and significantly accelerated the discovery of novel NPs by combining with high-efficient targeted BGC screening approaches. In this review, we summarized the three types of strategies for innovative discovery of microbial NPs based on (meta) genomic library construction with targeted screening. We discussed the types of cloning vectors including Cosmid/Fosmid, BAC/PAC and FAC/YAC, and analyzed the strategies of library construction and the approaches of targeted BGC screening, such as LEXAS and CONKAT-Seq. Furthermore, we compared strengths, limitations, and applicability of different library types. Finally, we prospected the future developments of these strategies for the high-throughput discovery of microbial NPs. Now we are witnessing a renaissance in NP discovery for novel drug development, which will continue to make major contributions to crop protection and human health. In the near future, these strategies of library construction combined with targeted BGC screening will significantly facilitate the discovery of bioactive microbial NPs with novel modes of action or chemical structures from different taxa, ecological niches or host microbiomes.

Key words: microbial natural products, (meta)genomic mining, biosynthetic gene cluster, library construction, targeted BGC screening, high-efficient discovery

中图分类号:

Q819

虞旭昶, 吴辉, 李雷. 文库构建与基因簇靶向筛选驱动的微生物天然产物高效发现[J]. 合成生物学, DOI: 10.12211/2096-8280.2023-083.

XuChang YU, Hui WU, Lei LI. Library construction combined with targeted BGC screening-driven high-efficient discovery of microbial natural products[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2023-083.

图/表 4

图1 土壤宏基因组Cosmid文库构建驱动新化合物高效挖掘。（NPSTs—Natural Products Sequence Tag. 采用基于共现网络的CONKAT-Seq策略或基于eSNaPD软件的谱系分析获取感兴趣的基因簇，通过异源表达或化学合成方法获取新化合物。）

Fig. 1 The construction of soil metagenomic cosmid libraries for high-efficiency discovery of novel compounds.(NPSTs— Natural Products Sequence Tags. The CONKAT-Seq strategy based on co-occurrence networks or phylogenetic analysis strategy based on eSNaPD software were used to identify BGCs of interest. Then, novel NPs were obtained by heterologous expression or chemical synthesis.)

图2 细菌基因组BAC/PAC文库构建加速活性天然产物高通量挖掘。（a）构建娄彻氏链霉菌Streptomyces rochei Sal35基因组BAC文库，建立LEXAS筛选系统，高效挖掘新化合物；（b）构建100株链霉菌基因组PAC文库，采用基于共现网络的CONKAT-Seq策略大规模获取新的非核糖体或聚酮类化合物合成基因簇，并在不同宿主中异源表达，高效获取新化合物。NPSTs—Natural Product Sequence Tags

Fig. 2 The construction of bacterial genome BAC/PAC libraries for the high-throughput discovery of bioactive natural products.(a) The BAC library construction of genome of S. rochei Sal35 combined with the LEXAS screening system were used for the high-efficient disocovery of novel compounds; (b) The PAC library construction of genomes of 100 Streptomyces combined with the CONKAT-Seq strategy based on co-occurrence networks were used to identify novel NRP and PK BGCs at scale and efficiently discover novel compounds by heterologous expression in different hosts. NPSTs—Natural Product Sequence Tags.

图3 真菌基因组FAC文库构建驱动真菌天然产物创新发现。（采用非靶向代谢组学打分系统，精准实现新化合物的筛选与分离。）

Fig. 3 The construction of fungal genome FAC libraries for the innovative discovery of fungal natural products.(An untargeted metabolomics scoring system was used to accurately screen and separate novel compounds.)

表1 不同类型文库构建策略用于新化合物高通量发现的比较

Table 1 Comparison of strategies for constructing different library types for high-throughput discovery of novel natural products

文库类型	插入大小	核心元件	方法筛选	文库针对类型	基因簇获取通量	获得化合物	优点	缺点	参考文献
Cosmid	～40 kb	COS位点质粒元件	CONKAT-seq	宏基因组	10⁷个克隆	Omnipeptin（NRP）	适合宏基因组挖掘	克隆片段大小相对较小，在克隆大尺度BGC（>40 kb）方面存在局限	Libis等(2019)^[51]
Cosmid	～40 kb	COS位点质粒元件	eSNaPD	宏基因组	>2 × 10⁷个克隆	MBA3（NRP）			Li等 (2022)^[52]
Fosmid	～40 kb	COS位点 F因子元件	序列引导的PCR 筛选	（宏）基因组	2880个克隆	Actinonin（NRP）			Felix等(2018)^[59]
BAC	～100 kb	loxP F因子元件	LEXAS	基因组	784个克隆	Lexapeptide （羊毛硫肽）等	①转化效率高 ②克隆100-300 kb片段	不适合宏基因组挖掘	Xu等 (2017)^[78]
PAC	～140 kb	F因子元件 P1噬菌体元件	CONKAT-seq	基因组	60 000个克隆	Prolinolexin（NRP） Cinnamexin（NRP）Conkatamycin（PK）	①转化效率高 ②克隆100-300 kb片段	不适合宏基因组挖掘	Libis等(2022)^[85]
FAC	～100 kb	F因子元件真菌ARS	FAC-MS	基因组	156个FACs	Benzomalvin A/D（NRP） Sesterterpenoid（Terpene） Valactamide A（Hybrid NRP-PK）	①克隆100-300 kb片段 ②适合真菌基因组挖掘	克隆稳定性差	Clevenger等(2017)^[96]
YAC	100-2000 kb	着丝粒端粒酵母ARS	NA	基因组	1896个YACs	NA	克隆100-2000 kb片段	①克隆稳定性差 ②转化效率低 ③重组率、嵌合率高	Saji等 (2001)^[101]

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