Enhancement of the gene targeting efficiency of non-conventional yeasts by increasing genetic redundancy.
Enhancement of the gene targeting efficiency of non-conventional yeasts by increasing genetic redundancy.
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通过增加遗传冗余提高非常规酵母的基因打靶效率
DOI：
10.1371/journal.pone.0057952
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Li Y
中科院分区：
文献类型：
--
作者：
Chen Z;Sun H;Li P;He N;Zhu T;Li Y
与模式酵母相比，非传统酵母的基因打靶效率通常较低，这极大地限制了这些生物的研究和应用。在这项研究中，我们的目的是提高非常规酵母的基因打靶效率，通过提高突变株的适应性，特别是通过增加宿主细胞的遗传冗余。为了证明这一过程，在巴斯德毕赤酵母中进行OCH 1基因缺失。在敲除基因组DNA中的天然OCH 1基因之前，为巴斯德毕赤酵母GS115菌株提供辅助质粒上的OCH 1基因的额外拷贝。OCH 1基因的冗余显著消除了och1突变体的生长缺陷，并在同源侧翼长度相同的情况下将OCH 1基因的缺失效率提高了两个数量级。使用相同的策略来删除KU70和SGS1基因。KU70和SGS1的靶向效率分别提高了1倍和23倍。因此，本研究为非传统酵母中“顽固”基因的删除提供了一种有效的策略。这项研究进一步表明，细胞适应性可能是限制基因靶向效率的重要因素。
In contrast to model yeasts, gene targeting efficiencies of non-conventional yeasts are usually low, which greatly limits the research and applications of these organisms. In this study, we aimed to enhance the gene targeting efficiency of non-conventional yeasts by improving the fitness of mutant strains, particularly by increasing the genetic redundancy of host cells. To demonstrate this process, OCH1 gene deletion in Pichia pastoris was performed. Extra copies of the OCH1 gene on a helper plasmid were provided for the P. pastoris GS115 strain before the native OCH1 gene in the genomic DNA was knocked out. The redundancy in OCH1 gene significantly eliminated the growth defects of the och1 mutant and increased the deletion efficiency of the OCH1 gene by two orders of magnitude with the same length of homologous flanks. The same strategy was used to delete the KU70 and SGS1 genes. The targeting efficiencies of KU70 and SGS1 were increased by 1- and 23-fold, respectively. Therefore, this study provided an efficient strategy for the deletion of “stubborn” genes in non-conventional yeasts. This study further showed that cellular fitness is potentially an important factor that can limit the efficiency of gene targeting.
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影响因子：
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