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.
The Key Laboratory of Industrial Biotechnology, Ministry of Education; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Jingwen
J
Zhou
Zhiyao
Z
Dong
Liming
L
Liu
Guocheng
G
Du
Jian
J
Chen
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