裂殖壶菌高产油突变体的高通量筛选

Screening for hyper-accumulating lipid mutants in Aurantiochytrium limacinum using high-throughput fluorescence-based method

摘要:

二十二碳六烯酸 (DHA) 具有促进婴幼儿大脑和视网膜发育等多种生理功能，被广泛应用于食品、医药和养殖等行业。为了获得适合于工业化生产的高产油、高产DHA的裂殖壶菌工程株，文中建立了一套操作简单、快速准确的基于尼罗红染色的高通量筛选方案。首先利用紫外线 (UVC) 诱变的方式快速构建裂殖壶菌的随机突变体库。然后采用优化后的筛选条件如裂殖壶菌的最佳尼罗红染色条件 (二甲基亚砜浓度为20%，尼罗红终浓度为2.0 μg/mL，孵育时间为10 min，孵育温度为40 ℃) 和更合理的筛选依据 (多功能酶标仪实现高通量测量的单位细胞密度油脂量) 等，对3 648株突变体进行筛选，得到了3株高产油突变体 (D03432、D05106和D01521)。摇瓶发酵实验表明，这3株突变体在生物量、油脂含量和DHA产量上均高于野生型菌株，其中突变体D03432和D05106的油脂量分别达到了干重的64.74%和63.13%，远高于野生型菌株的43.19%。而且这两株突变体的DHA产量分别是野生型菌株的2.26倍和2.37倍。最后，对突变体D03432和D05106进行了5 L发酵罐发酵培养，相较于野生型菌株，这两株突变体不仅生物量和油脂含量有所增加，而且DHA产量更是分别增加了45.51%和66.46%，展现出较好的工业应用潜力。此外，本筛选方案对其他产油微生物高产油突变体的高通量筛选具有借鉴作用。

Abstract:

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.