Hu Lina, Feng Shuying, Liang Gaofeng, Du Jingxia, Li Aifang, Niu Chunling
School of Basic Medical Sciences, Henan University of Science and Technology, No. 263 Kaiyuan Avenue, Luoyang, 471023, Henan, China.
Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China.
AMB Express. 2021 Jun 7;11(1):83. doi: 10.1186/s13568-021-01242-4.
Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.
杜氏盐藻（D. salina）已被开发成为基因工程领域的一种新型表达系统。然而，由于目标蛋白表达水平较低或不稳定，其在重组蛋白的实际生产中受到了极大限制。鉴于成簇规律间隔短回文重复序列（CRISPR）/Cas系统具有精确的基因编辑功能，本文以β-胡萝卜素羟化酶基因为例，探索杜氏盐藻的应用，旨在提高外源基因的表达水平。基于pKSE401骨架构建了三种CRISPR/Cas9二元载体，用于靶向杜氏盐藻CCAP19/18（Dschyb）的β-胡萝卜素羟化酶的外显子1和3。通过盐梯度转化法获得了杜氏盐藻突变体，并通过实时荧光定量PCR鉴定了Dschyb基因的表达。此外，在高强度处理后的不同时间点，采用高效液相色谱法分析了类胡萝卜素含量。与野生型菌株相比，突变体的β-胡萝卜素水平显著增加，接近1.4μg/ml，而突变体中的玉米黄质水平则有不同程度的下降。所有结果为杜氏盐藻的靶向基因编辑提供了有力证据。本研究首次成功对杜氏盐藻进行了基因编辑，这对于提高胡萝卜素产量和满足实际工业需求具有非常重要的现实意义。此外，它提供了一种克服当前杜氏盐藻障碍的方法，进而为促进杜氏盐藻系统的开发和应用提供了强有力的工具。
