Duan Guangqian, Li Shuoshuo, Li Xin, Huang Kaiyao
Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Sheng Wu Gong Cheng Xue Bao. 2019 Jul 25;35(7):1335-1347. doi: 10.13345/j.cjb.190086.
Docosahexaenoic acid (DHA) has many unique physiological functions such as promoting the development of brain and retina in infants. Therefore, it is widely applied to food, pharmacy, breeding and other industries. To obtain engineered strains of Aurantiochytrium limacinum SR21 suitable for industrial application with increased lipid and DHA production, we designed a simple, fast, accurate and high-throughput screening method based on Nile red staining of oil droplets. First, ultraviolet C (UVC) mutagenesis was used to generate a random mutant library of A. limacinum. Second, screening conditions were optimized including staining conditions of Nile red and the sorting criterion. Thereby, three putative high-lipid mutants (D03432, D05106 and D01521) were selected from the mutant library containing 3 648 mutated clones. The three mutants grew faster and produced higher amounts of lipids and DHA compared to wild type (WT). In 100 mL cultures, the lipid content of D03432 and D05106 mutants reached 64.74% and 63.13% of dry cell weight respectively, whereas the wild strain exhibited only 43.19%. DHA yield in these two mutants were even 2.26-fold and 2.37-fold higher than that of the wild strain. Experiment with 5 L fermentor further confirmed that D03432 and D05106 mutants had better performance than the wild strain on DHA yield (45.51% and 66.46% more than that of the wild strain, respectively), and demonstrated their high potential for industrial application. This work not only generated several high-DHA content mutants with high potential for industrial use, but also provided vital guidance for high-throughput screening of lipid hyper-accumulating mutants in other oil-producing microorganisms.
二十二碳六烯酸（DHA）具有许多独特的生理功能，如促进婴儿大脑和视网膜的发育。因此，它被广泛应用于食品、制药、养殖等行业。为了获得适合工业应用的、能够提高脂质和DHA产量的金黄藻SR21工程菌株，我们基于尼罗红对油滴的染色设计了一种简单、快速、准确且高通量的筛选方法。首先，利用紫外线C（UVC）诱变产生金黄藻的随机突变文库。其次，优化筛选条件，包括尼罗红的染色条件和分选标准。由此，从包含3648个突变克隆的突变文库中筛选出三个假定的高脂质突变体（D03432、D05106和D01521）。与野生型（WT）相比，这三个突变体生长更快，脂质和DHA产量更高。在100 mL培养物中，D03432和D05106突变体的脂质含量分别达到干细胞重量的64.74%和63.13%，而野生菌株仅为43.19%。这两个突变体的DHA产量甚至比野生菌株高2.26倍和2.37倍。5 L发酵罐实验进一步证实，D03432和D05106突变体在DHA产量方面比野生菌株表现更好（分别比野生菌株高45.51%和66.46%），并证明了它们在工业应用中的巨大潜力。这项工作不仅产生了几个具有高工业应用潜力的高DHA含量突变体，还为其他产油微生物中脂质超积累突变体的高通量筛选提供了重要指导。
