Department of Chemical and Biomolecular Engineering, U.S. Department of Energy Center for Bioenergy and Bioproducts Innovation (CABBI), Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States.
Department of Chemical and Biomolecular Engineering, U.S. Department of Energy Center for Bioenergy and Bioproducts Innovation (CABBI), Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, United States; Department of Microbiology, Nankai University, Tianjin, China.
Metab Eng. 2020 May;59:87-97. doi: 10.1016/j.ymben.2020.01.005. Epub 2020 Jan 31.
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.
非传统酵母东方伊萨酵母能够在酸性条件下生长，已被探索用于生产各种有机酸。然而，由于缺乏有效的遗传工具来实现复杂的代谢操作，其更广泛的应用受到了阻碍。我们最近在东方伊萨酵母中构建了一种基于酿酒酵母自主复制序列（ARS）的附加体质粒，并开发了用于多重基因缺失的 CRISPR/Cas9 系统。在这里，我们报告了另外三个遗传工具，包括：（1）通过生物信息学和功能筛选，从东方伊萨酵母基因组中鉴定出一个 0.8kb 的着丝粒样（CEN-L）序列；（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表征了一组组成型启动子和终止子；（3）开发了一种在东方伊萨酵母中进行快速高效的体内 DNA 组装方法，当从 0.7kb 到 1.7kb 的七个 DNA 片段组装一个 7kb 的质粒时，其准确率约为 100%。作为概念验证，我们使用这些遗传工具在东方伊萨酵母中快速构建了一个功能性木糖利用途径。
