Institute for Agro-Environmental Sciences (NIAES), National Agriculture and Food Research Organization, 3-1-3 Kannondai, Tsukuba, Ibaraki, Japan.
Institute of Agrobiological Sciences (NIAS), National Agriculture and Food Research Organization, 1-2 Owashi, Tsukuba, Ibaraki, Japan.
Biosci Biotechnol Biochem. 2022 Jul 22;86(8):1031-1040. doi: 10.1093/bbb/zbac075.
The basidiomycetous yeast Pseudozyma antarctica, which has multiple auxotrophic markers, was constructed, without inserting a foreign gene, as the host strain for the introduction of multiple useful genes. P. antarctica was more resistant to ultraviolet (UV) irradiation than the model yeast Saccharomyces cerevisiae, and a Paura3 mutant (C867T) was obtained after 3 min of UV exposure. A uracil-auxotrophic marker (URA3) recycling system developed in ascomycetous yeasts and fungi was applied to the P. antarctica Paura3 strain. The PaLYS12 and PaADE2 loci were disrupted via site-directed homologous recombination of PaURA3 (pop-in), followed by the removal of PaURA3 (pop-out). In the obtained double auxotrophic strain (Palys12Δ, Paura3), PaADE2 was further disrupted, and PaURA3 was removed to obtain the triple auxotrophic strain PGB800 (Paura3, Palys12Δ, Paade2Δ). The whole-genome sequence of the PGB800 strain did not contain foreign genes used for genetic manipulation and disrupted PaADE2 and PaLYS12, and removed PaURA3, as planned.
极地假丝酵母（Pseudozyma antarctica）是一种担子菌酵母，具有多种营养缺陷型标记，我们在不插入外源基因的情况下，将其构建为宿主菌株，用于引入多个有用基因。与模式酵母酿酒酵母（Saccharomyces cerevisiae）相比，极地假丝酵母对紫外线（UV）辐射的抗性更强，经过 3 分钟的 UV 照射后，得到了一个 Paura3 突变体（C867T）。我们将在子囊菌酵母和真菌中开发的尿嘧啶营养缺陷型标记（URA3）回收系统应用于极地假丝酵母 Paura3 菌株。通过 PaURA3（pop-in）的定点同源重组，破坏了 PaLYS12 和 PaADE2 基因座，随后通过 PaURA3（pop-out）去除了 PaURA3。在获得的双营养缺陷型菌株（Palys12Δ，Paura3）中，进一步破坏了 PaADE2 基因，并去除了 PaURA3，从而获得了三营养缺陷型菌株 PGB800（Paura3，Palys12Δ，Paade2Δ）。PGB800 菌株的全基因组序列中不包含用于遗传操作的外源基因，并且按计划破坏了 PaADE2 和 PaLYS12，并去除了 PaURA3。
