Comparative Proteomics Reveals Evidence of Enhanced EPA Trafficking in a Mutant Strain of Nannochloropsis oculata

Abstract

The marine microalga Nannochloropsis oculata is a bioproducer of eicosapentaenoic acid (EPA), a fatty acid. EPA is incorporated into monogalactosyldiacylglycerol within N. oculata thylakoid membranes, and there is a biotechnological need to remodel EPA synthesis to maximize production and simplify downstream processing. In this study, random mutagenesis and chemical inhibitor-based selection method were devised to increase EPA production and accessibility for improved extraction. Ethyl methanesulfonate was used as the mutagen with selective pressure achieved by using two enzyme inhibitors of lipid metabolism: cerulenin and galvestine-1. Fatty acid methyl ester analysis of a selected fast-growing mutant strain had a higher percentage of EPA (37.5% of total fatty acids) than the wild-type strain (22.2% total fatty acids), with the highest EPA quantity recorded at 68.5 mg/g dry cell weight, while wild-type cells had 48.6 mg/g dry cell weight. Label-free quantitative proteomics for differential protein expression analysis revealed that the wild-type and mutant strains might have alternative channeling pathways for EPA synthesis. The mutant strain showed potentially improved photosynthetic efficiency, thus synthesizing a higher quantity of membrane lipids and EPA. The EPA synthesis pathways could also have deviated in the mutant, where fatty acid desaturase type 2 (13.7-fold upregulated) and lipid droplet surface protein (LDSP) (34.8-fold upregulated) were expressed significantly higher than in the wild-type strain. This study increases the understanding of EPA trafficking in N. oculata, leading to further strategies that can be implemented to enhance EPA synthesis in marine microalgae.

Keywords: Nannochloropsis; cerulenin; eicosapentaenoic acid (EPA); ethyl methanesulfonate (EMS) random mutagenesis; galvestine-1; label-free quantitative (LFQ) proteomic analysis.

FIGURE 1

Schematic diagram of experimental cultivation system for selecting two-time points for LFQ proteomics experiments. All experiments were conducted at room temperature at 20°C and illuminated under 130 μmol μmol m⁻² s⁻¹ under a 12-h/12-h (day/night) cycle. The cultures were subjected to continuous filtered aeration and bubbled at 2 L/min.

FIGURE 2

Biomass density of N. oculata mutants after eight days of growth in f/2 medium containing cerulenin. The initial optical density of 595 nm was 0.15 at day 0 (A) Optical density of 82 N. oculata mutants in 50 µM cerulenin. (B) Optical density of 20 N. oculata mutants containing 60 µM cerulenin. The cultures were incubated at 130 μmol m⁻²s⁻¹, 20°C, under a 12-h/12-h (light/dark) cycle. The green rectangle and blue line represent the biomass density of the wild-type N. oculata.

FIGURE 3

Growth rate per day comparisons of mutants M1, M18, M45, and wild-type N. oculata, incubated at 130 μmol m⁻²s⁻¹, 20°C, under a 12-h/12-h (light/dark) cycle and 160 RPM shaking for ten days. Mean ± standard deviation is shown (n = 3) and t-tests determine the statistical significance (p < 0.05 [*]; p < 0.01 [**]; and p < 0.001 [***]) of the M1 mutant strain compared to the wild-type strain.

FIGURE 4

Growth profiles for wild-type and M1 mutant N. oculata cultivated in 1-L flasks under 150 μmol m⁻² s⁻¹, 20°C, and aerated bubbling for mixing and carbon source for 12 days. (A) Growth curves illustrated by optical density at 595 nm and (B) DCW. Comparison of wild-type and M1 mutant N. oculata over 12 days of culturing for (C) chlorophyll concentration and (D) phosphate (P) and nitrate (N) uptake profiles. Mean ± standard deviation is shown (n = 3) and t-tests determine the statistical significance (p < 0.05 [*]; p < 0.01 [**]; and p < 0.001 [***]) of the M1 mutant strain compared to the wild-type strain.

FIGURE 5

Percentages (%) of fatty acids at day 12. (A) Wild-type and (B) M1 mutant N. oculata. Quantification (mg/g) of fatty acids at day 12. (C) Wild-type and (D) M1 mutant N. oculata. Mean ± standard deviation is shown (n = 3) and t-tests determine the statistical significance (p < 0.05 [*]; p < 0.01 [**]; p < 0.001 [***]) for EPA content in the M1 mutant strain compared to the wild-type strain.

FIGURE 6

PCA plots show 12 samples clustered by biological replicates: (A) Wild-type N. oculata samples day 3 (light blue) and day 12 (pink) and (B) M1 mutant N. oculata samples day 2 (light blue) and day 12 (pink). Volcano plots show the significant protein distributions in wild-type (C) and M1 mutant (D) N. oculata.

FIGURE 7

Diagram of enzyme regulations from day 3 to day 12 for carbon fixation toward TAG biosynthesis pathways for wild-type N. oculata. The diagram shows the pathways and their relation to fatty acid synthesis pathways. Significant quantified proteins are shown in the green and red boxes for upregulated and downregulated proteins, respectively.

FIGURE 8

Diagram of enzyme regulations from day 2 to day 12 for carbon fixation toward TAG biosynthesis pathways for M1 mutant N. oculata. The diagram shows the pathways and their relation to fatty acid synthesis pathways. Significant quantified proteins are shown in the green and red boxes for upregulated and downregulated proteins, respectively.

FIGURE 9

Fatty acid content. (A) EPA percentages (%) of TFA in polar lipids and TAG at day 2 (M1 mutant), day 3 (wild-type), and day 12 (wild-type and M1 mutant) N. oculata. (B) Polar lipid (PL):TAG quantity ratio (mg/g) at day 2 (M1 mutant), day 3 (wild-type), and day 12 (wild-type and M1 mutant) N. oculata. Mean ± standard deviation is shown (n = 3) and t-tests determine the statistical significance (p < 0.05 [*]; p < 0.01 [**]; p < 0.001 [***]) for EPA content in the M1 mutant strain compared to the wild-type strain.