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Abstract
The marine microalgal species Nannochloropsis gaditana has emerged as a model organism for research into algal-derived biofuels due to its ability to produce large amounts of lipids. In addition, this species has biotechnological potential owing to its ease of transformation and availability of a sequenced and annotated genome. The ability to control expression of transgenes in an inducible manner is an important requirement for many genetic engineering approaches and as such, inducible promoters are an important component of the molecular toolkit for a host organism. We designed an expression vector containing a ~ 1.3 kb region upstream of the endogenous nitrate reductase gene of N. gaditana and demonstrated this is capable of promoting the expression of a heterologous enhanced green fluorescent protein (eGFP) reporter. In the presence of ammonium, expression of the eGFP reporter was undetectable; however, in the presence of nitrate, eGFP expression is induced. In addition, we demonstrated how altering the nitrogen source of the growth media can be used to precisely control expression. We have shown that the nitrate reductase promoter can be used as a powerful molecular tool for heterologous protein expression in N. gaditana, further contributing to the development of this species as a candidate strain for biotechnology.
Highlights
Recent advances have seen the emergence of Nannochloropsis spp. as a platform for biotechnological application of marine microalgae
Using an enhanced green fluorescent protein as a fluorescent reporter and for immunoblot analysis, we investigated the ability of the endogenous N. gaditana nitrate reductase promoter to robustly drive inducible expression of heterologous protein in transgeneic cell lines
The efficiency of gene expression driven by the nitrate reductase promoter sequence (PNR) of N. gaditana was analysed using the enhanced green fluorescent protein expression vector pNR (Fig. 1; see Online Resource 3 for complete annotated vector map)
Summary
Recent advances have seen the emergence of Nannochloropsis spp. as a platform for biotechnological application of marine microalgae. Reverse genetic approaches using random mutagenesis combined with high-throughput phenotypic screening have isolated Nannochloropsis strains with improved light-use efficiency and increased lipid accumulation (Doan and Obbard 2012; Beacham et al 2015; Perin et al 2015). Nannochloropsis spp. are haploids and transformation of the nuclear genome with integration constructs has been established and shown to occur by high-efficiency homologous recombination in Nannochloropsis oceanica, allowing the generation of knockout mutants (Kilian et al 2011; Radakovits et al 2012). Wang et al (2016) reported the first use of a CRISPR/Cas9-based approach in a Nannochloropsis species, generating mutant strains of N. oceanica with precise five-base deletions in the nitrate reductase gene, demonstrating the potential for sophisticated gene editing techniques. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas has
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