合成生物学 ›› 2025, Vol. 6 ›› Issue (3): 685-700.DOI: 10.12211/2096-8280.2024-079
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
章益蜻1,2, 刘高雯1
- 1.中国科学院深圳先进技术研究院，深圳合成生物学创新研究院，深圳合成基因组学重点实验室，广东省合成基因组学重点实验室，定量合成生物学重点实验室，广东 深圳 518055
2.中国科学院大学，北京 100049
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收稿日期:2024-11-11修回日期:2025-02-20出版日期:2025-06-30发布日期:2025-06-27 -
通讯作者:刘高雯 -
作者简介:章益蜻 （2001—），女，硕士研究生。研究方向为合成生物学与系统基因组学。E-mail：yq.zhang3@siat.ac.cn刘高雯 （1987—），女，副研究员，博士生导师。研究方向为酵母系统基因组学与适应性进化。E-mail：gaowen.liu@siat.ac.cn -
基金资助:广东省合成基因组学重点实验室资助项目(2023B1212060054);深圳合成基因组学重点实验室资助项目(ZDSYS201802061806209)
Exploration of gene functions and library construction for engineering strains from a synthetic biology perspective
ZHANG Yiqing1,2, LIU Gaowen1
- 1.Shenzhen Institute of Synthetic Biology，Shenzhen Key Laboratory of Synthetic Genomics，Guangdong Provincial Key Laboratory of Synthetic Genomics，Key Laboratory of Quantitative Synthetic Biology，Shenzhen Institute of Advanced Technology，Chinese Academy of Sciences，Shenzhen 518055，Guangdong，China
2.University of Chinese Academy of Sciences，Beijing 100049，China
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Received:2024-11-11Revised:2025-02-20Online:2025-06-30Published:2025-06-27 -
Contact:LIU Gaowen
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。本文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。
中图分类号:
引用本文
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, 2025, 6(3): 685-700.
ZHANG Yiqing, LIU Gaowen. Exploration of gene functions and library construction for engineering strains from a synthetic biology perspective[J]. Synthetic Biology Journal, 2025, 6(3): 685-700.
图1 经典文库构建方法（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）］
Fig. 1 Schematics for constructing classical libraries (created by biorender)[(a) In the gene deletion library, the ORF (orange fragment) of target gene is replaced by the KanMX (kanamycin resistance selection marker, yellow fragment), flanked by unique barcodes (blue fragments). Chr.DNA: chromosomal DNA.(b) Schematic diagram of the Bar-seq principle. Under specific treatment conditions, the target strain (red) shows reduced growth (light pink), which is reflected by a lower read count of its corresponding barcode during sequencing (fewer red lines compared to others).(c) In the gene overexpression library, the ORF (orange fragment) of target gene is not only expressed from its endogenous locus but is also additionally expressed from a plasmid, driven by a constitutive promoter (light green fragment).(d) In the ORF-Tag library, the C-terminus of the target gene ORF (orange fragment), with its stop codon removed, is fused to the fluorescent protein GFP (dark green fragment) and the selection marker KanMX (yellow fragment).]
图1 经典文库构建方法（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）］
Fig. 1 Schematics for constructing classical libraries (created by biorender)[(a) In the gene deletion library, the ORF (orange fragment) of target gene is replaced by the KanMX (kanamycin resistance selection marker, yellow fragment), flanked by unique barcodes (blue fragments). Chr.DNA: chromosomal DNA.(b) Schematic diagram of the Bar-seq principle. Under specific treatment conditions, the target strain (red) shows reduced growth (light pink), which is reflected by a lower read count of its corresponding barcode during sequencing (fewer red lines compared to others).(c) In the gene overexpression library, the ORF (orange fragment) of target gene is not only expressed from its endogenous locus but is also additionally expressed from a plasmid, driven by a constitutive promoter (light green fragment).(d) In the ORF-Tag library, the C-terminus of the target gene ORF (orange fragment), with its stop codon removed, is fused to the fluorescent protein GFP (dark green fragment) and the selection marker KanMX (yellow fragment).]
图2 SGA与PEM方法原理（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 for SGA and PEM methods (created by biorender)[(a) Double mutant selection strategy. Two mating-type cells each carry different resistance markers at their respective mutation sites: KanMX (green hollow circle) and NatMX (pink hollow circle). These allow for the selection of double mutant progeny (outlined in black). YFG (your favorite gene): target gene of interest.(b) Counter-diploid selection strategy. Haploid cells carrying a wild-type CAN1 gene (blue, a-type) and diploid cells (purple) can uptake the toxic analog canavanine and are killed, while can1Δ mutants cannot transport canavanine and thus survive (outlined in black).(c) Haploid selection strategy. A mating-type-specific promoter is used to drive the expression of a nutritional selection marker in the opposite mating type, allowing selection of haploid progeny of a specific mating type. For example, when an a-type parent cell (blue), harboring a gene cassette expressing STE3pr-LEU2 (red solid circle) — which is only active in α-type cell — mates with another cell, only the α-type progeny that inherit this construct (outlined in red with a red solid circle) will survive.(d) Combined counter-diploid and haploid selection strategy. A dominant-lethal resistance gene, cyhS (brown solid square), is inserted near the mat1 mating-type locus to link its expression with a specific haploid phenotype. For example, insertion near matP (blue square) in h- cells (blue) leads to the death of cyhS -containing h- haploids and diploids, enabling selection of h+ haploids.]
图2 SGA与PEM方法原理（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 for SGA and PEM methods (created by biorender)[(a) Double mutant selection strategy. Two mating-type cells each carry different resistance markers at their respective mutation sites: KanMX (green hollow circle) and NatMX (pink hollow circle). These allow for the selection of double mutant progeny (outlined in black). YFG (your favorite gene): target gene of interest.(b) Counter-diploid selection strategy. Haploid cells carrying a wild-type CAN1 gene (blue, a-type) and diploid cells (purple) can uptake the toxic analog canavanine and are killed, while can1Δ mutants cannot transport canavanine and thus survive (outlined in black).(c) Haploid selection strategy. A mating-type-specific promoter is used to drive the expression of a nutritional selection marker in the opposite mating type, allowing selection of haploid progeny of a specific mating type. For example, when an a-type parent cell (blue), harboring a gene cassette expressing STE3pr-LEU2 (red solid circle) — which is only active in α-type cell — mates with another cell, only the α-type progeny that inherit this construct (outlined in red with a red solid circle) will survive.(d) Combined counter-diploid and haploid selection strategy. A dominant-lethal resistance gene, cyhS (brown solid square), is inserted near the mat1 mating-type locus to link its expression with a specific haploid phenotype. For example, insertion near matP (blue square) in h- cells (blue) leads to the death of cyhS -containing h- haploids and diploids, enabling selection of h+ haploids.]
图3 条件等位基因文库（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 Construction of conditional allele libraries (created by biorender)[(a) In the temperature-sensitive (TS) allele strategy, the ORF (orange fragment) of target gene is replaced with a corresponding TS mutant (dark orange fragment) and tagged with a KanMX resistance marker (yellow fragment). Chr.DNA: chromosomal DNA.(b) In the promoter replacement strategy, an inducible promoter (green fragment) and a KanMX resistance marker (yellow fragment) is inserted upstream of the start codon of the target gene ORF (orange fragment). Chr.DNA: chromosomal DNA.(c) In the DAmP (Decreased Abundance by mRNA Perturbation) strategy, the 3′UTR of the target gene ORF (orange fragment) is disrupted by insertion of a KanR resistance cassette (yellow fragment) to reduce transcript stability and protein abundance. Chr.DNA: chromosomal DNA.]
图3 条件等位基因文库（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 Construction of conditional allele libraries (created by biorender)[(a) In the temperature-sensitive (TS) allele strategy, the ORF (orange fragment) of target gene is replaced with a corresponding TS mutant (dark orange fragment) and tagged with a KanMX resistance marker (yellow fragment). Chr.DNA: chromosomal DNA.(b) In the promoter replacement strategy, an inducible promoter (green fragment) and a KanMX resistance marker (yellow fragment) is inserted upstream of the start codon of the target gene ORF (orange fragment). Chr.DNA: chromosomal DNA.(c) In the DAmP (Decreased Abundance by mRNA Perturbation) strategy, the 3′UTR of the target gene ORF (orange fragment) is disrupted by insertion of a KanR resistance cassette (yellow fragment) to reduce transcript stability and protein abundance. Chr.DNA: chromosomal DNA.]
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