A reusable method for construction of non-marker large fragment deletion yeast auxotroph strains: A practice in Torulopsis glabrata

作者：Zhou Jingwen; Dong Zhiyao; Liu Liming^*; Du Guocheng; Chen Jian

来源：Journal of Microbiological Methods, 2009, 76(1): 70-74.

DOI：10.1016/j.mimet.2008.09.011

• Fusion PCR
• Nystatin enrichment
• Auxotroph screening
• Gene knockout
• Candida glabrata

A reusable method for construction of yeast auxotrophic mutants with large deletions that do not contain markers was developed and successfully applied to a pyruvate overproducing yeast strain, Torulopsis glabrata CCTCC M202019. A URA3 knockout fragment with a large deletion of the open reading frame and long homologous arms was constructed by fusion PCR and transformed into the parent strain by high-efficiency electroporation. A high concentration of transformed yeast was achieved by subsequently culturing in rich medium for 24 h and in nitrogen-free minimal medium for 4 h. Potential Delta ura3 auxotrophic mutants were enriched by treatment with nystatin and selection on uracil-limited medium. Mutants were confirmed by streaking cultures and colony PCR. Delta arg8 and Delta ura3 Delta arg8 double auxotroph mutants were also successfully constructed using this method.

• 出版日期2009-1
• 单位江南大学