Cao Mingfeng, Gao Meirong, Lopez-Garcia Carmen Lorena, Wu Yutong, Seetharam Arun Somwarpet, Severin Andrew Josef, Shao Zengyi
Department of Chemical and Biological Engineering, ‡NSF Engineering Research Center for Biorenewable Chemicals (CBiRC), §Genome Informatics Facility, Office of Biotechnology, ∥Interdepartmental Microbiology Program, and ⊥The Ames Laboratory, Iowa State University , 4140 Biorenewables Research Laboratory, Ames, Iowa 50011, United States.
ACS Synth Biol. 2017 Aug 18;6(8):1545-1553. doi: 10.1021/acssynbio.7b00046. Epub 2017 Apr 25.
Many nonconventional yeast species have highly desirable features that are not possessed by model yeasts, despite that significant technology hurdles to effectively manipulate them lay in front. Scheffersomyces stipitis is one of the most important exemplary nonconventional yeasts in biorenewables industry, which has a high native xylose utilization capacity. Recent study suggested its much better potential than Saccharomyces cerevisiae as a well-suited microbial biomanufacturing platform for producing high-value compounds derived from shikimate pathway, many of which are associated with potent nutraceutical or pharmaceutical properties. However, the broad application of S. stipitis is hampered by the lack of stable episomal expression platforms and precise genome-editing tools. Here we report the success in pinpointing the centromeric DNA as the partitioning element to guarantee stable extra-chromosomal DNA segregation. The identified centromeric sequence not only stabilized episomal plasmid, enabled homogeneous gene expression, increased the titer of a commercially relevant compound by 3-fold, and also dramatically increased gene knockout efficiency from <1% to more than 80% with the expression of CRISPR components on the new stable plasmid. This study elucidated that establishment of a stable minichromosome-like expression platform is key to achieving functional modifications of nonconventional yeast species in order to expand the current collection of microbial factories.
许多非传统酵母物种具有模式酵母所没有的非常理想的特性，尽管有效操纵它们存在重大技术障碍。树干毕赤酵母是生物可再生能源行业中最重要的典型非传统酵母之一，具有很高的天然木糖利用能力。最近的研究表明，作为一个适合生产源自莽草酸途径的高价值化合物的微生物生物制造平台，它比酿酒酵母具有更好的潜力，其中许多化合物具有强大的营养保健或药用特性。然而，树干毕赤酵母的广泛应用受到缺乏稳定的附加型表达平台和精确的基因组编辑工具的阻碍。在这里，我们报告成功地确定了着丝粒DNA作为分配元件，以保证稳定的染色体外DNA分离。鉴定出的着丝粒序列不仅稳定了附加体质粒，实现了均匀的基因表达，使一种商业相关化合物的产量提高了3倍，而且在新的稳定质粒上表达CRISPR组件时，还将基因敲除效率从<1%显著提高到80%以上。这项研究阐明，建立一个稳定的类小染色体表达平台是实现非传统酵母物种功能修饰以扩大当前微生物工厂种类的关键。
