Kluyveromyces marxianus, a thermotolerant, fast‐growing, Crabtree‐negative yeast, is a promising chassis for the manufacture of various bioproducts. Although several genome editing tools are available for this yeast, these tools still require refinement to enable more convenient and efficient genetic modification. In this study, we engineered the K. marxianus NBRC 104275 strain by impairing the nonhomologous end joining and enhancing the homologous recombination machinery, which resulted in improved homology‐directed repair effective on homology arms of up to 40 bp in length. Additionally, we simplified the CRISPR‐Cas9 editing system by constructing a strain for integrative expression of Cas9 nuclease and plasmids bearing different selection markers for gRNA expression, thereby facilitating iterative genome editing without the need for plasmid curing. We demonstrated that tRNA was more effective than the hammerhead ribozyme for processing gRNA primary transcripts, and readily assembled tRNA‐gRNA arrays were used for multiplexed editing of at least four targets. This editing tool was further employed for simultaneous scarless in vivo assembly of a 12‐kb cassette from three fragments and marker‐free integration for expressing a fusion variant of fatty acid synthase, as well as the integration of genes for starch hydrolysis. Together, the genome editing tool developed in this study makes K. marxianus more amenable to genetic modification and will facilitate more extensive engineering of this nonconventional yeast for chemical production.

中文翻译：

---

CRISPR-Cas9 介导的马克斯克鲁维酵母基因组编辑，用于迭代、多重基因破坏和途径整合


马克斯克鲁维酵母是一种耐热、快速生长、克拉布特里阴性酵母，是生产各种生物产品的有前途的基础。尽管有多种基因组编辑工具可用于这种酵母，但这些工具仍然需要改进，以实现更方便、更有效的基因修饰。在这项研究中，我们通过损害非同源末端连接并增强同源重组机制来改造马克斯克鲁维酵母 NBRC 104275 菌株，从而改善同源定向修复，对长度高达 40 bp 的同源臂有效。此外，我们通过构建整合表达Cas9核酸酶和带有不同gRNA表达选择标记的质粒的菌株来简化CRISPR-Cas9编辑系统，从而促进迭代基因组编辑而无需质粒固化。我们证明，tRNA 在处理 gRNA 初级转录本方面比锤头核酶更有效，并且易于组装的 tRNA-gRNA 阵列可用于至少四个靶标的多重编辑。该编辑工具进一步用于体内同时无痕组装由三个片段组成的 12 kb 盒，以及用于表达脂肪酸合酶融合变体的无标记整合，以及淀粉水解基因的整合。总之，本研究中开发的基因组编辑工具使 K. marxianus 更适合基因改造，并将促进这种非常规酵母更广泛的工程化用于化学生产。