The challenge of accurately predicting which genetic alternations lead to the desired phenotype necessitates high-throughput metabolic engineering approaches where numerous hypotheses can be tested simultaneously. We describe the CRISPR-Cas9-based method TUNE YALI that enables high-throughput tuning of gene expression in the common industrial yeast Yarrowia lipolytica . The method is based on replacing the promoters of the target genes with native Y. lipolytica promoters of varying strengths or removing the promoters entirely. To demonstrate the method’s capabilities, we created a plasmid library that targets 56 transcription factors (TFs) and changes the expression of each TF to seven different levels. We transformed this library into reference and betanin-producing strains of Y. lipolytica and screened the resulting clones for changes in morphology, thermotolerance, or improved betanin production. The genetic markup of the yeast clones with the desired phenotypic changes was determined by sequencing the inserted plasmids. We identified multiple TFs whose regulatory changes increased thermotolerance, two TFs that eliminated pseudohyphal growth, and several TFs that increased betanin production. Analogous libraries can be designed to target any chosen group of genes and even all the genes. The libraries can be shared and reused, accelerating applied strain development projects and fundamental functional genomics research (TUNE YALI -TF kit and TUNE YALI -TF library are available via AddGene under catalog numbers #1000000255 and #217744).

中文翻译：

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通过基因表达调控对 Yarrowia lipolytica 进行高通量代谢工程

准确预测哪些基因突变导致所需表型的挑战需要高通量代谢工程方法，其中可以同时测试许多假设。我们描述了基于 CRISPR-Cas9 的方法 TUNE YALI，该方法能够高通量调节常见工业酵母 Yarrowia lipolytica 中的基因表达。该方法基于用不同强度的天然 Y. lipolytica 启动子替换靶基因的启动子或完全去除启动子。为了证明该方法的功能，我们创建了一个靶向 56 种转录因子 （TF） 的质粒文库，并将每个 TF 的表达更改为七个不同的水平。我们将该文库转化为 Y. lipolytica 的参考和甜菜碱生产菌株，并筛选所得克隆的形态变化、耐热性或改善的甜菜碱生成。通过对插入的质粒进行测序来确定具有所需表型变化的酵母克隆的遗传标记。我们确定了多个调节变化增加耐热性的 TFs，两个消除假菌丝生长的 TFs，以及几个增加甜菜碱产生的 TFs。类似的文库可以设计为靶向任何选定的基因组，甚至所有基因。这些文库可以共享和重复使用，加速应用菌株开发项目和基础功能基因组学研究（TUNE YALI -TF 试剂盒和 TUNE YALI -TF 文库可通过 AddGene 获得，目录号为 #1000000255 和 #217744）。