High-throughput fluorescence-activated cell sorting for cell wall-deficient microalgal mutants screening
High-throughput fluorescence-activated cell sorting for cell wall-deficient microalgal mutants screening
复制标题
高通量荧光激活细胞分选用于细胞壁缺陷型微藻突变体筛选
DOI：
10.1016/j.algal.2020.102011
复制
发表时间：
2020-09-01
影响因子：
5.1
通讯作者：
Han, Danxiang
中科院分区：
文献类型：
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作者：
Lin, Juan;Chen, Yi;Han, Danxiang
单细胞绿色藻类雨生红球藻（Haematococcus pluvialis）中天然抗氧化剂虾青素的生物利用度和提取效率受到藻细胞壁刚性的限制。本研究建立了一种高通量的荧光激活细胞分选（FACS）技术，用于筛选细胞壁缺陷突变体。将荧光素缀合的凝集素-蓖麻凝集素I（RCA（120））用作结合藻类细胞壁的糖部分的标记物，这使得能够区分细胞壁不同的藻类菌株。利用已建立的流式细胞仪（FACS）检测系统，从突变体库中筛选到一株RCA（120）-荧光强度显著降低的细胞壁缺陷型突变体（即突变体-264）。与野生型相比，突变体-264的细胞壁结合糖的总含量降低了43.9%，其中甘露糖、甘露醇和半乳糖的含量显著降低。突变体-264的干重、虾青素含量和提取效率分别比野生型提高了21.1%、13.1%和16.5%。本研究不仅证明了利用基于流式细胞仪的高通量筛选细胞壁突变体的可行性，而且提供了一个新的有前途的H。用于工业应用的具有多种改良性状的雨生菌菌株。
Bioavailability and extraction efficiency of the natural antioxidant astaxanthin from the unicellular green alga Haematococcus pluvialis have been limited by the rigid algal cell walls. A high-throughput fluorescence-activated cell sorting (FACS) pipeline was developed in this study with the aim to screen cell wall-deficient mutants. The fluorescein-conjugated lectin-Ricinus communis Agglutinin I (RCA(120)) was used as a marker for binding the sugar moieties of the algal cell walls, which enabled discriminating the algal strains that differed in the cell walls. By utilizing the established FACS pipeline, a cell wall-deficient mutant (i.e. mutant-264) with the significantly reduced RCA(120)-fluorescein intensity was screened from the mutant library. The total content of the cell wall-associated sugars was reduced by 43.9% in the mutant-264 as compared to that of the wild type, with significant decrease in mannose, mannitol and galactose. The dry weight, astaxanthin content and extraction efficiency were improved by 21.1%, 13.1% and 16.5%, respectively, in mutant-264 as compared to that of the wild type. This study not only demonstrated the feasibility of utilization of the FACS-based high-throughput pipeline for cell wall mutant screening, but also delivered a novel promising H. pluvialis strain with multiple improved traits for industrial applications.