Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana

Abstract

The potential use of algae in biofuels applications is receiving significant attention. However, none of the current algal model species are competitive production strains. Here we present a draft genome sequence and a genetic transformation method for the marine microalga Nannochloropsis gaditana CCMP526. We show that N. gaditana has highly favourable lipid yields, and is a promising production organism. The genome assembly includes nuclear (~29 Mb) and organellar genomes, and contains 9,052 gene models. We define the genes required for glycerolipid biogenesis and detail the differential regulation of genes during nitrogen-limited lipid biosynthesis. Phylogenomic analysis identifies genetic attributes of this organism, including unique stramenopile photosynthesis genes and gene expansions that may explain the distinguishing photoautotrophic phenotypes observed. The availability of a genome sequence and transformation methods will facilitate investigations into N. gaditana lipid biosynthesis and permit genetic engineering strategies to further improve this naturally productive alga.

Figure 1. Biomass production by N. gaditana.

(a) N. gaditana production of biomass, lipids, protein and sugars quantified during continuous growth over a period of 3 months in 50% salinity seawater medium supplemented with nitrate, phosphate and CO₂ with continuous 1,000 μE light. Every week, half of the culture was collected and replaced with fresh medium. Inset values show the yield in mg l⁻¹ per day. Values are from 12 measurements and error bars show the standard deviation. (b) Chart illustrating collected biomass compositions, the majority of which consists of lipids even under nutrient replete conditions. Inset values show percentage of total biomass. (c) Comparison of N. gaditana lipid production rates with other algae examined in this work. Values are from at least three separate experiments and error bars show the standard deviation. (d) Comparison of N. gaditana large-scale production rates with other biofuel production platforms. Bars in green indicate our estimations; bars in grey indicate estimations by Atsumi et al. The values for N. gaditana have been extrapolated from 1 l cultures and adjusted for our observed productivity in 12 h/12 h light/dark cycles. The S. elongatus production values are for 24 h light and would presumably be lower in 12 h/12 h light/dark cycles.

Figure 2. Phylogenetic analysis of the N. gaditana genome.

(a) Schematic phylogenetic tree of stramenopiles and photosynthetic algae. The tree is adapted from Eisenreich et al., and Tyler et al. Filled green circles on the right indicate photosynthetic species. (b) The tree indicates the relationship between different strains of Nannochloropsis based on 18S ribosomal RNA gene sequences. (c) Venn diagram representation of shared/unique genes of N. gaditana in comparison with brown algae, diatoms, red algae and green algae. (d) N. gaditana gene models were compared with all previously sequenced genomes in the non-redundant protein database using BLASTp. The number of times an organism was the top BLASTp hits (E-value less than 1E-3) of a N. gaditana gene model is indicated.

Figure 3. Genes conserved in photosynthetic stramenopiles.

(a) Euler diagram showing the 363 genes that make up the StramenopilePhotoCut genes common to photosynthetic and absent in non-photosynthetic stramenopiles. Number of genes found in each sector is indicated. The centre yellow sector indicates genes unique for the photosynthetic Stramenopiles (not found in green or red lineages). (b) Chart showing the number of 'StramenopilePhotoCut' genes with select GO terms. 'StramenopilePhotoCut' genes with no GO terms are not indicated.

Figure 4. N. gaditana metabolic pathway map.

Light grey background traces indicate KEGG pathways not encoded by the N. gaditana genome. KEGG pathways in green, magenta or blue are present in the N. gaditana genome. Genes that are up- or down-regulated during nitrogen deprivation are labelled in magenta and blue, respectively.

Figure 5. Comparison of TAG biosynthetic pathway genes.

Number of gene homologues in the TAG biosynthetic pathways in N. gaditana as compared with a brown alga (E. siliculosus), a diatom (P. tricornutum), a red alga (C. merolae) and a green alga (C. reinhardtii). For each reaction, coloured squares denote the number of homologous genes in N. gaditana (pink), E. siliculosus (brown), P. tricornutum (orange), C. merolae (red), C. reinhardtii (green).

Figure 6. Carbon-concentrating mechanisms.

Proposed mechanisms in which inorganic carbon is assimilated by N. gaditana and the proposed C₄-like metabolism based on predicted protein localizations. Metabolites (black): G3P, glyceraldehyde 3-phosphate; MA, malic acid; OAA, oxaloacetate; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate; Pyr, pyruvate; RuBP, ribulose-1,5-bisphosphate; Enzymes (red): BCT, bicarbonate transporter; CA, carbonic anhydrase; MDH, malate dehydrogenase; ME, malic enzyme; PC, pyruvate carboxylase; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate, phosphate dikinase; RuBisCO, Ribulose-1,5-bisphosphate carboxylase oxygenase.