CRISPR-Cas9 enabled targeted mutagenesis in the thermotolerant methylotrophic yeast Ogataea thermomethanolica

作者：Chitwadee Phithakrotchanakoon, Aekkachai Puseenam, Sriwan Wongwisansri, Lily Eurwilaichitr, Supawadee Ingsriswang, Sutipa Tanapongpipat, Niran Roongsawang · 发表于：FEMS Microbiology Letters · 年份：2018 · DOI：10.1093/femsle/fny105 · 被引用次数：19 · 研究领域：CRISPR and Genetic Engineering、Fungal and yeast genetics research、RNA and protein synthesis mechanisms

Ogataea thermomethanolica TBRC656 is a thermotolerant methylotrophic yeast suitable for heterologous protein expression at various temperatures. However, the lack of efficient methods for targeted gene mutagenesis limits strain engineering in this yeast. In this study, we applied a CRISPR-Cas9-based tool for targeted gene mutagenesis in O. thermomethanolica. The putative unfolded protein response regulator OtHAC1, and the OtMAL1 (maltase) and OtMAL2 (maltose permease) genes involved with sucrose and maltose utilization were targeted for CRISPR-Cas9 mutagenesis. Plasmids were constructed for integrative and episomal expression of CRISPR-Cas9 elements in O. thermomethanolica in which Cas9 and gRNA are transcribed from the alcohol oxidase (AOX) promoter. The expression of these genome-editing elements is controlled by derepression with glycerol and gRNA are flanked by self-cleaving ribozymes. For integrative system, OtHAC1, OtMAL1 and OtMAL2 were disrupted at 63%, 97% and 93%, respectively. In addition, OtMAL1 was also disrupted with episomal system at 92%. These findings indicate that the CRISPR-Cas9 system described herein is thus applicable for studying gene function and strain engineering in yeast O. thermomethanolica.