CRISPR/Cas9-induced β-carotene hydroxylase mutation in Dunaliella salina CCAP19/18

Abstract

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.

Carotenoid metabolism: New insights and synthetic approaches

“…The mutated CrOR His strain contained up to 1.6-fold and 3.2-fold enhanced total carotenoid content, compared to the wild-type CrOR overexpressing and the mock line, respectively. In Dunaliella salina , a CRISPR-Cas technique was used to target the 1 st and 3 rd exons of the β-CAROTENE HYDROXYLASE ( Dschyb ) gene, which significantly enhanced the β-carotene level, reaching about 1.4 g/ml ( Hu et al., 2021 ) ( Figure 2D ).…”

Genetic engineering to enhance microalgal-based produced water treatment with emphasis on CRISPR/Cas9: A review

“…Frontiers in Bioengineering and Biotechnology frontiersin.org degradation could induce increased lipid yields in different microalgal species (Nguyen A. D. et al, 2020;Chang et al, 2020). Several other studies were also successful in implementing CRISPR/Cas9 to increase the production of different carotenoids in Dunaliella salina (Hu et al, 2021); and in Chlamydomonas reinhardtii (Baek et al, 2016), to improve the thermal tolerance of Tetraselmis suecica (Xu J. et al, 2020), and to investigate gene function in Phaeodactylum tricornutum (Hao et al, 2022;Llavero-Pasquina et al, 2022). Overall, CRISPR/ Cas9 has been successfully applied on many other algae species to improve their biomass productivities, tolerances to abiotic stressors, or increase lipid content, or that of other biomolecules and value-added products (Kumar et al, 2020;Jeon et al, 2021;Wang et al, 2021).…”

Converting carbon dioxide to high value‐added products: Microalgae‐based green biomanufacturing

“…In addition, CRISPR technology has been applied in Dunaliella salina and Chlorella , Agrobacterium‐mediated genetic transformation was achieved in Euglena gracilis , and intron‐mediated gene expression was realized in Cyclotella spp. The development of these technologies has laid the foundation for the future construction of transformed microalgal cell factories for the industrial production of HVACs (Becker et al, 2021; Hu et al, 2021; Kim et al, 2021).…”

“…However, further development of C. reinhardtii as a green chassis cell has been hindered by the lack of efficient expression elements and comparative evaluation, as well as low transgene titers. Some scholars have systematically evaluated the existing expression elements of C. reinhardtii and created novel synthetic expression elements by promoter engineering, which significantly improved the efficiency of C. reinhardtii as a chassis cell for synthetic biology (Hu et al, 2021).…”