Highly-efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants

摘要

Red yeasts hold great promise in the production of microbial lipids and carotenoids. Genetic study of red yeasts has attracted much attention; however, rapid amplication of genes from red yeast samples remains technically challenging. Here a highly efcient method for the preparation of genomic DNA (gDNA) template, which could be directly used for PCR, was developed. Cells from colonies or liquid cultures were collected and sequentially treated by microwave, plMAN5C, proteinase K and boiling (MMPB) in a single tube to give cell lysates that were qualied as PCR templates. Single-copied gDNA fragments o up to 2.8 kb were successfully amplied. We also demonstrated successful application of this method for species in the Ascomycetes and Basidiomycetes and identication of two leucine auxotroph mutants of Rhodotorula glutinis. This method could be widely employed for the screening and genetic engineering of various yeasts.