Combinatorial library-based metabolic engineering approaches allow lower cost and faster strain development. We developed a genetic toolbox EXPRESSYALI for combinatorial engineering of the oleaginous yeast Yarrowia lipolytica. The toolbox enables consecutive rounds of engineering, where up to three combinatorially assembled gene expression cassettes can be integrated into each yeast clone per round. The cassettes are integrated into distinct intergenic sites or an open reading frame of a target gene if a simultaneous gene knockout is desired. We demonstrate the application of the toolbox by optimizing the Y. lipolytica to produce the red beet color betanin via six consecutive rounds of genome editing and screening. The library size varied between 24 and 360. Library screening was facilitated by automated color-based colony picking. In the first round, betanin pathway genes were integrated, resulting in betanin titer of around 20 mg/L. Through the following five consecutive rounds, additional biosynthetic genes were integrated, and the precursor supply was optimized, resulting in a titer of 70 mg/L. Three beta-glucosidases were deleted to prevent betanin deglycosylation, which led to a betanin titer of 130 mg/L in a small scale and a titer of 1.4 g/L in fed-batch bioreactors. The EXPRESSYALI toolbox can facilitate metabolic engineering efforts in Y. lipolytica (available via AddGene Cat. Nr. 212682–212704, Addgene kit ID # 1000000245).

中文翻译：

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解紫蓍菌代谢工程的组合迭代方法：在甜菜碱生物合成中的应用


基于组合库的代谢工程方法可以降低成本并加快菌株开发速度。我们开发了一个遗传工具箱 EXPRESSYALI，用于油性酵母 Yarrowia lipolytica 的组合工程。该工具箱支持连续轮次工程设计，每轮最多可将三个组合组装的基因表达盒整合到每个酵母克隆中。如果需要同时敲除基因，将盒整合到不同的基因间位点或靶基因的开放阅读框中。我们通过连续六轮基因组编辑和筛选优化 Y. lipolytica 以产生红甜菜色甜菜素来展示该工具箱的应用。文库大小在 24 到 360 之间变化。基于颜色的自动菌落挑选促进了文库筛选。在第一轮中，整合了甜菜苷通路基因，导致甜菜苷滴度约为 20 mg/L。通过接下来的连续 5 轮，整合了额外的生物合成基因，优化了前驱体供应，滴度达到 70 mg/L。删除 3 种 β-葡萄糖苷酶以防止甜菜碱去糖基化，这导致小规模甜菜苷滴度为 130 mg/L，补料分批生物反应器中的滴度为 1.4 g/L。EXPRESSYALI 工具箱可以促进 Y. lipolytica 的代谢工程工作（可通过 AddGene Cat. Nr. 212682–212704，Addgene 试剂盒 ID # 1000000245 获得）。