Development of flow cytometric procedures for the efficient isolation of improved lipid accumulation mutants in a Chlorella sp microalga
Development of flow cytometric procedures for the efficient isolation of improved lipid accumulation mutants in a Chlorella sp microalga
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DOI：
10.1007/s10811-013-0021-8
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发表时间：
2013-12-01
影响因子：
3.3
通讯作者：
Hildebrand, Mark
中科院分区：
文献类型：
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作者：
Manandhar-Shrestha, Kalpana;Hildebrand, Mark
微藻生物燃料生产的成功开发将依赖于燃料分子前体积累的数量和速度的改善。诱变和选择是提高燃料分子生产率的有吸引力的方法。以前的筛选方法是费力的，分离的突变体的数量很少，并且突变体的总体性能可能由于存在由随机诱变产生的影响其他细胞过程和生长的有害次级突变而受到损害。我们报道了一种改进的分离高三酰甘油（TAG）积累小球藻突变体的方法，KAS 603，使用基于流式细胞术的选择。除了选择高TAG积累菌株之外，该方法还要求突变体的生长与群体中的其他细胞竞争。不仅生长是竞争性的，而且在重复选择的情况下TAG积累的性能得到改善，表明从有害的次级突变中纯化。该程序导致突变体的分离具有高得多的效率（数千倍），其比以前的方法更好地（1.8-2.5倍）显著优于野生型。这打开了大门，以新的方法来表征涉及TAG积累和其他细胞过程的基因。
The successful development of microalgae-based biofuel production will rely on improvements in the amount and rate of fuel molecule precursor accumulation. Mutagenesis and selection is an attractive approach to improve fuel molecule productivity. Previous screening methods have been laborious, the numbers of mutants isolated have been small, and overall performance of mutants may have been compromised by the presence of deleterious secondary mutations generated by random mutagenesis that affect other cellular processes and growth. We report an improved method of isolating high triacylglycerol (TAG) accumulating mutants of a Chlorella sp., KAS603, using flow cytometric-based selection. In addition to selection for high TAG accumulating strains, the method requires that growth of mutants be competitive with other cells in the population. Not only is growth competitive, but there is improved performance in TAG accumulation with repeated selection, suggesting purification from deleterious secondary mutations. The procedure resulted in the isolation of mutants with far higher efficiency (thousands of fold) that outperformed wild type substantially better (1.8-2.5-fold) than with previous methods. This opens the door to new approaches to the characterization of genes involved in TAG accumulation and other cellular processes.