合成生物学 ›› 2024, Vol. 5 ›› Issue (3): 492-506.DOI: 10.12211/2096-8280.2023-083
虞旭昶1,2, 吴辉2, 李雷1
收稿日期:
2023-11-27
修回日期:
2024-01-26
出版日期:
2024-06-30
发布日期:
2024-07-12
通讯作者:
吴辉,李雷
作者简介:
基金资助:
Xuchang YU1,2, Hui WU2, Lei LI1
Received:
2023-11-27
Revised:
2024-01-26
Online:
2024-06-30
Published:
2024-07-12
Contact:
Hui WU, Lei LI
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。
中图分类号:
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, 2024, 5(3): 492-506.
Xuchang YU, Hui WU, Lei LI. Library construction and targeted BGC screening for more efficient discovery of microbial natural products[J]. Synthetic Biology Journal, 2024, 5(3): 492-506.
图1 土壤宏基因组Cosmid文库构建驱动新化合物高效挖掘(NPST—天然产物序列标签。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 Construction of soil metagenomic cosmid libraries for the discovery of novel compounds(NPST—Natural Products Sequence Tag. The CONKAT-Seq strategy based on co-occurrence network or phylogenetic analysis strategy based on eSNaPD software is used to identify BGCs of interest. Then, novel NPs are 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
Fig. 2 Construction of bacterial genome BAC/PAC libraries for the high-throughput mining of bioactive natural products.(a) BAC library construction for the genome of S. rochei Sal35 combined with the LEXAS screening system is used for the high-efficient mining of novel compounds; (b) PAC library construction for the genomes of 100 Streptomyces combined with the CONKAT-Seq strategy based on co-occurrence network is used to identify novel BGCs encoding NRP and PK, and then heterologous expression can be performed in different hosts to discover novel compounds.
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 Construction of fungal genome FAC libraries for discovering fungal natural products(An untargeted metabolomics scoring system was used to accurately screen and separate novel compounds.)
文库 类型 | 插入 大小 | 核心 元件 | 筛选方法 | 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 | 约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)[ |
约40 kb | eSNaPD | 宏基因组 | >2×107个 克隆 | MBA3(NRP) | Li等 (2022)[ | ||||
Fosmid | 约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) | Wolf等(2018)[ | ||
BAC | 约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 | 徐敏 (2017)[ |
PAC | 约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) | Libis等(2022)[ | ||
FAC | 约100 kb | F因子元件 真菌ARS | FAC-MS | 基因组 | 156个FAC | 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)[ |
YAC | 100~ 2000 kb | 着丝粒 端粒 酵母ARS | NA | 基因组 | 1896个YAC | 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)[ |
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 libraries for high-throughput screening of novel natural products
文库 类型 | 插入 大小 | 核心 元件 | 筛选方法 | 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 | 约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)[ |
约40 kb | eSNaPD | 宏基因组 | >2×107个 克隆 | MBA3(NRP) | Li等 (2022)[ | ||||
Fosmid | 约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) | Wolf等(2018)[ | ||
BAC | 约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 | 徐敏 (2017)[ |
PAC | 约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) | Libis等(2022)[ | ||
FAC | 约100 kb | F因子元件 真菌ARS | FAC-MS | 基因组 | 156个FAC | 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)[ |
YAC | 100~ 2000 kb | 着丝粒 端粒 酵母ARS | NA | 基因组 | 1896个YAC | 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)[ |
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