Abstract
Plastid engineering offers an important tool to fill the gap between the technical and the enormous potential of microalgal photosynthetic cell factory. However, to date, few reports on plastid engineering in industrial microalgae have been documented. This is largely due to the small cell sizes and complex cell-wall structures which make these species intractable to current plastid transformation methods (i.e., biolistic transformation and polyethylene glycol-mediated transformation). Here, employing the industrial oleaginous microalga Nannochloropsis oceanica as a model, an electroporation-mediated chloroplast transformation approach was established. Fluorescent microscopy and laser confocal scanning microscopy confirmed the expression of the green fluorescence protein, driven by the endogenous plastid promoter and terminator. Zeocin-resistance selection led to an acquisition of homoplasmic strains of which a stable and site-specific recombination within the chloroplast genome was revealed by sequencing and DNA gel blotting. This demonstration of electroporation-mediated chloroplast transformation opens many doors for plastid genome editing in industrial microalgae, particularly species of which the chloroplasts are recalcitrant to chemical and microparticle bombardment transformation.
Highlights
Microalga-based biochemical factory is regarded as an ideal strategy for sequestering greenhouse gas and producing valuable molecules ranging from therapeutic proteins to biofuels (Tran et al, 2013; Moody et al, 2014)
Plastid Transformation for Nannochloropsis oceanica (Kilian et al, 2011; Vieler et al, 2012; Li et al, 2014; Iwai et al, 2015; Kang et al, 2015; Poliner et al, 2017; Xin et al, 2017), which facilitates the manipulation of crucial nodes in oil biosynthesis and the development of the RNA interference (RNAi) (Wei L. et al, 2017) and CRISPR/Cas9 methods (Wang et al, 2016)
During the creation of nuclear mutagenesis library for industrial oleaginous microalga Nannochloropsis oceanica, we found that antibiotic constructs were inserted into the plastid genome by electroporation
Summary
Microalga-based biochemical factory is regarded as an ideal strategy for sequestering greenhouse gas and producing valuable molecules ranging from therapeutic proteins to biofuels (Tran et al, 2013; Moody et al, 2014). Genetic engineering of industrial microalgae provides a viable way to optimize crucial traits for commercial feedstock development (Gimpel et al, 2013; Zhang and Hu, 2014; Wang et al, 2016; Cui et al, 2018). Plastid Transformation for Nannochloropsis oceanica (Kilian et al, 2011; Vieler et al, 2012; Li et al, 2014; Iwai et al, 2015; Kang et al, 2015; Poliner et al, 2017; Xin et al, 2017), which facilitates the manipulation of crucial nodes in oil biosynthesis and the development of the RNA interference (RNAi) (Wei L. et al, 2017) and CRISPR/Cas methods (Wang et al, 2016). Despite of a low possibility of functioning in nuclear expression of these utilized plastid promoter and terminator, we cannot exclude the possibility that GFP expressed in nuclear instead of chloroplast.
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