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, 刘高雯

Exploration of gene functions and library construction for engineering strains from a synthetic biology perspective

ZHANG Yiqing, LIU Gaowen

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cyh^S（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-cyh^S）］

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, cyh^S (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 cyh^S -containing h^-haploids and diploids, enabling selection of h⁺ haploids.]