Toward the Exploitation of Sustainable Green Factory: Biotechnology Use of Nannochloropsis spp

Abstract

Securing food, energy, and raw materials for a growing population is one of the most significant challenges of our century. Algae play a central role as an alternative to plants. Wastewater and flue gas can secure nutrients and CO₂ for carbon fixation. Unfortunately, algae domestication is necessary to enhance biomass production and reduce cultivation costs. Nannochloropsis spp. have increased in popularity among microalgae due to their ability to accumulate high amounts of lipids, including PUFAs. Recently, the interest in the use of Nannochloropsis spp. as a green bio-factory for producing high-value products increased proportionally to the advances of synthetic biology and genetic tools in these species. In this review, we summarized the state of the art of current nuclear genetic manipulation techniques and a few examples of their application. The industrial use of Nannochloropsis spp. has not been feasible yet, but genetic tools can finally lead to exploiting this full-of-potential microalga.

Keywords: Nannochloropsis; genetic engineering; genetic manipulation; synthetic biology.

Figure 1

Nannochloropsis phylogenetic tree. Phylogenetic tree showing the evolution of Nannochloropsis spp. with respect to the other algae. The phylogenetic tree is obtained from the multiple sequence alignment of 18S ribosomal RNA gene (18S rDNA) using Clustal omega and neighbor-joining distance matrix. The following sequences from NCBI were used: Physcomitrella patens (AF126289.1), Arabidopsis thaliana (X16077.1), Chlamydomonas reinhardtii (AB511835.1), Chlorella vulgaris (X13688.1), Cyanidioshyzon merolae (XR_002461616.1), Thalassiosira pseudonana (MH545685.1), Phaeodactylum tricornutum (AJ269501.1), Nannochloropsis CCMP505 (U41050.1), and Ectocarpus siliculosus (L43062.1).

Figure 2

Diagram showing primary and secondary endosymbiosis events at the origin of Nannocchloropsis spp. The left panel shows the primary endosymbiosis at the origin of green algae (Chlorophyta), red algae (Rhodophyta), and Glaucophytes. In this process, a eukaryotic cell engulfs a prokaryotic cell that can undergo photosynthesis. As a footprint, the chloroplast of cells belonging to primary endosymbiosis is surrounded by a double membrane. By contrast, Chromalveolates, which include Cryptophyta, Haptophyta, Stramenopiles (or Heterokontophyta), and Alveolata, were originated by a secondary endosymbiosis. In this process, an eukaryotic cell engulfs another eukaryotic cell which has already engulfed a prokaryotic cell in its past (a Rhodophyte in that case). The Chromalveolate chloroplast, as a result of secondary endosymbiosis, is surrounded by four membranes.

Figure 3

CRISPR/Cas9 mechanism. The main components are an RNA-guided Cas9 endonuclease and a single guide RNA (sgRNA). The genome integration-based approach (left side) requires the construction of a vector carrying three different cassettes: one encoding for the Cas9 protein, the second for the sgRNA, and the third for antibiotic resistance. Once the vector is stably integrated into the host genome, Cas9 protein and sgRNA are synthesized through cell machinery. On the contrary, the in vitro ribonucleoprotein (RNP) assembly approach (right side) requires Cas9 purified protein, usually from E. coli, and in vitro synthesized sgRNA. Both in the integration and in the in vitro RNP assembly, Cas9 protein and sgRNA are assembled to form Cas9 (RNP) with the ability to bind and cleave target DNA [92]. The main difference is that in the former, the RNP complex is assembled by the cell itself, while in the latter, RNP is in vitro assembled and later inserted into the cell. Single guide RNA drives the RNP to the targeted site that must be close to the protospacer adjacent motif (PAM) [93]. The cleavage carried out by Cas9 is a double-strand blunt break (DSB). Once the DSB is introduced in the target gene by Cas9 nuclease activity, the cell has two alternatives for the repair: non-homologous end-joining (NHEJ) or homology-directed repair (HDR) [93]. The first can introduce insertion or deletion at cleavage sites, generating a frameshift into the gene open reading frame. The latter can introduce exogenous DNA at the target site by using a homologous DNA repair template. In the HDR, usually, as donor DNA, the resistant marker cassette is used to allow transformant screening.

Figure 4

Screening pipeline for transformants using the “safe harbor” system. Briefly, genes of interest are in silico designed, optimizing codon usage and adding homologous recombination sequences (1). Nannochloropsis landing pad strain nuclear transformation is performed by means of electroporation, and homologous recombination (HR) occurs in the safe harbor site (2). td Tomato fluorescence can be used to identify lines in which homologous recombination occurred. WT shows no fluorescence, background landing pad strains (BG) show maximum fluorescence, while the transformed lines have or do not have fluorescence according to the occurrence of HR (3). Protein quantification allows quantifying the protein of interest; lines showing no tomato fluorescence, in which HR occurs in the safe harbor site, show the highest protein accumulation; other transformed lines show no or lower protein accumulation due to random insertion into the genome (4).

Figure 5

Nannochloropsis lipid metabolism. The main characterized enzymes are written in dark blue. Abbreviations: ACCase, acetyl-CoA carboxylase; DGAT, diacylglycerol acyltransferase; DGD1, digalactosyldiacylglycerol synthase; FAD, fatty acid desaturase; FAS, fatty acid synthase; FAE, fatty acid elongase; GPAT, glycerol 3-phosphate acyltransferase; LPAT, lysophosphatidic acid acyltransferase; MGD1, monogalactosyldiacylglycerol synthase; PAP, phosphatidic acid phosphatase; PDAT, phospholipid:diacylglycerol acyltransferase; PDHC, pyruvate dehydrogenase complex; SQD1, UDP-sulfoquinovose synthase 1; CoA, coenzyme A; DGDG, digalactosyldiacylglycerol; G3P, glycerol 3-phosphate; MGDG, monogalactosyldiacylglycerol; SQDG, sulfoquinovosyldiacylglycerol; TAG, triacylglycerol; PUFA, polyunsaturated fatty acid.