The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

非常规酵母东方侧柏可以在高酸性条件下生长，并且已经被研究用于生产各种有机酸。然而，由于缺乏有效的遗传工具来进行复杂的代谢操作，其广泛的应用受到了阻碍。我们最近基于东方酵母中的酿酒酵母（ScARS）的自主复制序列（ARS）构建了附加型质粒，并开发了用于多重基因缺失的CRISPR / Cas9系统。在这里，我们报告了三种其他的遗传工具，包括：（1）从东方伊蚊中鉴定出一个0.8 kb着丝粒样（CEN-L）序列利用生物信息学和功能筛选进行基因组研究；（2）通过RNA-Seq分析和荧光报告基因在不同培养条件下发现和鉴定一组组成型启动子和终止子；（3）开发了一种快速，有效的体内东方DNA组装方法，当从0.7 kb至1.7 kb的七个DNA片段组装7 kb质粒时，它表现出〜100％的保真度。作为概念的证明，我们使用了这些遗传工具来快速构建东方伊萨酵母中的功能性木糖利用途径。