2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus. First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 33-factorial library includes all combinations of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4K221L and native KmPHA2, with the medium expression of feedback insensitive KmARO7G141S, results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway.

中文翻译：

---

CRISPR介导的多基因整合使Shikimate途径重构可增强马克斯克鲁维酵母中的2-苯基乙醇生物合成

2-苯基乙醇（2-PE）是一种玫瑰香型的香料和香精化合物，用于食品，饮料和个人护理产品。与汽油的兼容性也使其成为潜在的生物燃料或燃料添加剂。与目前使用化学合成和/或从植物材料中分离的方法相比，将葡萄糖或其他可发酵糖转化为2-PE的生化过程可能会提供更可持续和经济的生产途径。我们通过在耐应力酵母马克斯克鲁维酵母中设计Shikimate和Ehrlich途径来实现这一目标。首先，我们开发了一种多基因整合工具，该工具使用CRISPR-Cas9诱导的基因组断裂作为对编码一个，两个或三个基因表达盒的插入片段的一步整合的选择。包含三个过表达盒的5-kbp插入片段的整合成功发生，在ABZ1位点的效率为51±9％，被用于创建具有重构的Shikimate途径基因的马克斯克鲁维酵母CBS 6556菌株的文库。具有33个因数的文库包含KmARO4，KmARO7和KmPHA2的所有组合，每个组合均由跨越广泛表达范围的三个不同启动子驱动。对重构途径文库的分析表明，酪氨酸来源的KmARO4K221L和天然KmPHA2的高表达，以及对反馈不敏感的KmARO7G141S的中等表达，导致2-PE生物合成的最高增加，产生684±73 mg / L。通过过量表达KmARO10和破坏KmEAT1进行的Ehrlich途径工程进一步将2-PE产量提高到766±6 mg / L。摇瓶培养120 h分批补料操作后，最佳菌株达到1943±63 mg / L 2-PE。CRISPR介导的多基因整合系统扩展了K. marxianus的基因组编辑工具集，K。marxianus是有前途的多应力耐受宿主，可用于生物合成2-PE和其他源自Shikimate途径的芳族化合物。