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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解脂耶氏酵母代谢工程的组合迭代方法：在甜菜苷生物合成中的应用


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。我们开发了一个遗传工具箱 EXPRESSYALI，用于产油酵母解脂耶氏酵母的组合工程。该工具箱可实现连续的工程设计，每轮最多可以将三个组合组装的基因表达盒整合到每个酵母克隆中。如果需要同时基因敲除，则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解脂耶氏酵母以生产红甜菜色甜菜碱，从而展示了该工具箱的应用。文库大小在 24 到 360 之间变化。基于颜色的自动化菌落挑选促进了文库筛选。在第一轮中，甜菜苷途径基因被整合，导致甜菜苷滴度约为20 mg/L。通过接下来的连续五轮，整合了额外的生物合成基因，并优化了前体供应，产生了 70 mg/L 的效价。删除了三种 β-葡萄糖苷酶以防止甜菜苷去糖基化，从而导致小规模甜菜苷滴度为 130 mg/L，在补料分批生物反应器中滴度为 1.4 g/L。 EXPRESSYALI 工具箱可以促进解脂耶氏酵母中的代谢工程工作（可通过 AddGene Cat. Nr. 212682–212704 获得，Addgene 试剂盒 ID # 1000000245）。