合成生物学视角下的基因功能探索与酵母工程菌株文库构建

doi:10.12211/2096-8280.2024-079

摘要/Abstract

摘要：

合成生物学作为一门通过设计、构建和改造生物系统来实现其特定功能的学科，被广泛应用于生物制造、环境保护和药物合成等领域。基因功能的系统性探索和工程菌株文库的构建是推动合成生物学发展的重要手段。本研究重点介绍了不同酵母文库在合成生物学中的构建方法及其应用前景。随着基因组测序和高通量技术的快速进展，酿酒酵母和裂殖酵母等微生物文库在系统性研究中发挥了关键作用。基因缺失文库、过表达文库、转座子插入文库等多种类型的酵母文库为基因组合优化和代谢路径设计提供了重要工具，促进了代谢工程和合成生物学的创新应用。这些文库在工业生产中支持高产菌株的构建，如用于生物燃料和化学品的高效生产；在环境领域，通过基因改造筛选，生成具备污染物降解能力的菌株，为生态修复提供解决方案；在药物合成方面，文库帮助构建高效合成药用化合物的菌株，推动生物制药的发展。然而，当前文库构建和应用仍面临诸如构建成本、基因组编辑的精确度及筛选效率等技术瓶颈。未来，自动化、数字化和新型筛选技术的进步有望突破这些瓶颈，推动酵母文库的快速构建和高效筛选，从而加速合成生物学在可持续发展和生态工程中的应用。

关键词: 酵母, 文库, 功能基因组学, 定向进化, 合成生物学

Abstract:

Synthetic biology, as a discipline that designs, constructs, and modifies biological systems to achieve specific functions, is widely applied in fields such as biomanufacturing, environmental protection, and drug synthesis. Systematic exploration of gene functions and the construction of engineered strain libraries are key drivers of the development of synthetic biology. These libraries serve as foundational tools for understanding complex biological processes and engineering microorganisms for novel applications. This review focuses on the construction methods and application prospects of various yeast libraries in synthetic biology. With the rapid advancement of genome sequencing and high-throughput technologies, microbial libraries, such as those of Saccharomyces cerevisiae and Schizosaccharomyces pombe, play a pivotal role in systematic research. Yeast libraries, including gene knockout libraries, overexpression libraries, and transposon insertion libraries, provide valuable tools for optimizing gene combinations and designing metabolic pathways, thus promoting innovative applications in metabolic engineering and synthetic biology. These libraries facilitate the development of high-yield industrial strains, driving improvements in biofuel production, chemical synthesis, and other biotechnological processes. In the environmental field, gene modification screening generates strains with pollutant degradation capabilities, contributing to ecological restoration. In drug synthesis, these libraries aid in constructing strains for the efficient production of pharmaceutical compounds, advancing the development of biopharmaceuticals. Despite these successes, there remain several challenges in library construction and application, such as the high costs of library generation, difficulties in precise genome editing, and limitations in screening efficiency. In the future, advances in automation, digitization, and novel screening technologies are expected to overcome these limitations, facilitating the rapid construction and efficient screening of yeast libraries. Looking ahead, the future of synthetic biology holds immense promise, with improvements in library construction and screening processes expected to accelerate the development of sustainable solutions in industrial production, environmental protection, and healthcare, thereby driving the next wave of innovations in biotechnology.

Key words: yeast, library, functional genomics, directed evolution, synthetic biology

中图分类号:

Q812

章益蜻, 刘高雯. 合成生物学视角下的基因功能探索与酵母工程菌株文库构建[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-079.

Yiqing ZHANG, Gaowen LIU. Gene function exploration and engineering strain library construction from a synthetic biology perspective[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-079.

图/表 3

图1 经典文库构建方法示意图（本图由biorender绘制）（a）基因缺失文库中，目的基因ORF（橘色片段）被替换成卡那霉素抗性筛选标签KanMX（黄色片段），两侧伴有“分子条形码”barcode（蓝色片段）。Chr.DNA：染色体DNA。（b）Bar-seq原理示意图。在特定条件处理下，目标菌株（红色）的生长量相对低（淡粉色），表现为测序时相应barcode的读数量相对低（红色线条比其他颜色少）。（c）基因过表达文库中，目的基因ORF（橘色片段）除了在基因组上正常表达之外，还额外在质粒上由通用型启动子（淡绿色片段）驱动表达。（d）ORF-Tag文库中，在删除终止密码子的目的基因ORF（橘色片段）C端插入荧光蛋白GFP（深绿色片段）和筛选标签KanMX（黄色片段）。Chr.DNA：染色体DNA。

Fig. 1 Schematics of classical library construction method(created by biorender)

图2 SGA与PEM方法原理图（本图由biorender绘制）（a）双突变体筛选策略。两种交配型的细胞分别在各自的突变位点携带不同抗性标签KanMX（绿色空心圆点）和NatMX（粉色空心圆点）用于筛选双突变体子代（黑线框细胞）。YFG （your favourite gene）：目的基因。（b）反二倍体筛选策略。含有野生型CAN1基因的单倍体细胞（蓝色细胞，a型）或二倍体细胞（紫色细胞）会摄入有毒的刀豆氨酸（canavanine），从而被杀死，而can1Δ突变体无法将刀豆氨酸运转入体内，因此能够存活（黑线框细胞）。（c）单倍体筛选策略。在某一细胞型的母本菌株中，构建另一细胞型特异性启动子与营养缺陷筛选标签的表达盒，用于选择任一性别的单倍体子代细胞。例如在a型细胞（蓝色）中，携带只能在α型细胞中表达的STE3pr-LEU2基因线路（红色实心圆点），只有细胞交配使STE3pr-LEU2基因线路存在于α型细胞中时，该细胞存活（含有红色实心圆点的红线框细胞）。（d）反二倍体与单倍体筛选共实现。通过将一个显性致死的抗性基因cyhS（棕色实心方块）“镶嵌”在裂殖酵母交配位点mat1（在蓝色的h-细胞中为蓝色方块表示的matP）附近，使得某一单倍体表型与抗性基因的表达偶联（matP-cyhS ）。

Fig.2 Schematics of SGA and PEM methods(created by biorender)

图3 条件等位基因文库示意图（本图由biorender绘制）（a）温度敏感型（Temperature Sensitive，TS）等位基因中，目的基因ORF（橘色片段）被替换成相应的温敏突变基因TS mutant（深橘色片段）并携带一个抗性筛选标签KanMX（黄色片段）。Chr.DNA：染色体DNA。（b）启动子替换策略中，携带抗性筛选标签KanMX（黄色片段）的诱导性启动子TetO（绿色片段）被插入目的基因ORF（橘色片段）的起始密码子上游。Chr.DNA：染色体DNA。（c）通过mRNA扰动降低蛋白丰度的DAmP策略中，目的基因ORF（橘色片段）的3’UTR区被插入一个抗性标签KanR（黄色片段）。Chr.DNA：染色体DNA。

Fig.3 Schematics of conditional allele libraries(created by biorender)

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