CRISPR-Cas9 enabled targeted mutagenesis in the thermotolerant methylotrophic yeast Ogataea thermomethanolica
| Autor: | Sriwan Wongwisansri, Lily Eurwilaichitr, Chitwadee Phithakrotchanakoon, Aekkachai Puseenam, Supawadee Ingsriswang, Sutipa Tanapongpipat, Niran Roongsawang |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Sucrose Monosaccharide Transport Proteins 030106 microbiology Mutagenesis (molecular biology technique) Microbiology 03 medical and health sciences Plasmid CRISPR-Associated Protein 9 Genetics Clustered Regularly Interspaced Short Palindromic Repeats Maltose Molecular Biology Gene Derepression Gene Editing biology Chemistry Cas9 Ribozyme alpha-Glucosidases Yeast Repressor Proteins 030104 developmental biology Biochemistry Metabolic Engineering Mutagenesis Saccharomycetales biology.protein Maltase |
| Zdroj: | FEMS microbiology letters. 365(11) |
| ISSN: | 1574-6968 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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