A reusable method for construction of non-marker large fragment deletion yeast auxotroph strains: A practice in Torulopsis 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 Deltaura3 auxotrophic mutants were enriched by treatment with nystatin and selection on uracil-limited medium. Mutants were confirmed by streaking cultures and colony PCR. Deltaarg8 and Deltaura3Deltaarg8 double auxotroph mutants were also successfully constructed using this method.
Zhou
Jingwen
J
The Key Laboratory of Industrial Biotechnology, Ministry of Education; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Dong
Zhiyao
Z
Liu
Liming
L
Du
Guocheng
G
Chen
Jian
J
D052818:Autotrophic Processes; D041221:Candida glabrata / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005821:Genetic Techniques; D009154:Mutation; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014170:Transformation, Genetic
D005656:Fungal Proteins