Green Microalgae Strain Improvement for the Production of Sterols and Squalene

Abstract

Sterols and squalene are essential biomolecules required for the homeostasis of eukaryotic membrane permeability and fluidity. Both compounds have beneficial effects on human health. As the current sources of sterols and squalene are plant and shark oils, microalgae are suggested as more sustainable sources. Nonetheless, the high costs of production and processing still hinder the commercialization of algal cultivation. Strain improvement for higher product yield and tolerance to harsh environments is an attractive way to reduce costs. Being an intermediate in sterol synthesis, squalene is converted to squalene epoxide by squalene epoxidase. This step is inhibited by terbinafine, a commonly used antifungal drug. In yeasts, some terbinafine-resistant strains overproduced sterols, but similar microalgae strains have not been reported. Mutants that exhibit greater tolerance to terbinafine might accumulate increased sterols and squalene content, along with the ability to tolerate the drug and other stresses, which are beneficial for outdoor cultivation. To explore this possibility, terbinafine-resistant mutants were isolated in the model green microalga Chlamydomonas reinhardtii using UV mutagenesis. Three mutants were identified and all of them exhibited approximately 50 percent overproduction of sterols. Under terbinafine treatment, one of the mutants also accumulated around 50 percent higher levels of squalene. The higher accumulation of pigments and triacylglycerol were also observed. Along with resistance to terbinafine, this mutant also exhibited higher resistance to oxidative stress. Altogether, resistance to terbinafine can be used to screen for strains with increased levels of sterols or squalene in green microalgae without growth compromise.

Keywords: Chlamydomonas; biodiesel; squalene; sterol; terbinafine.

Figure 1

Simplified pathways for sterol, pigment, and triacylglycerol synthesis (G3P; glycerol-3-phosphate; GGPP: geranylgeranyl pyrophosphate; IPP: isopentenyl diphosphate; LPA: lysophosphatidic acid; MEP: 2-C-methyl-d-erythritol 4-phosphate; TAG: triacylglycerol).

Figure 2

Growth phenotype of wild type (WT) and tfr mutants of C. reinhardtii. Serial dilutions of cells were spotted onto the agar medium without terbinafine (left) and with 1 mM terbinafine (right) and cultivated for 1 week.

Figure 3

(A) Sterols and (B) squalene content. Total lipid was extracted from log-phase cultures. All data are means ± SD of three biological replicates. Significant differences between the wild type (WT) and the mutants within the same condition are indicated by asterisks (*) (p < 0.05).

Figure 4

(A) Carotenoid and (B) chlorophyll content under N-replete medium in the presence and the absence of terbinafine. All data are means ± SD of three biological replicates. Significant differences between the wild type (WT) and the mutants within the same condition are indicated by asterisks (*) (p < 0.05). Significant differences between different treatments of the same strain are indicated by plus sign (+) (p < 0.05).

Figure 5

Growth phenotype wild type (WT) and tfr1 under oxidative stress. Serial dilutions of cells were spotted onto the agar medium NaCl and Rose Bengal (RB) and cultivated for 1 week.

Figure 6

Quantification of triacylglycerol (A), chlorophyll (B), carotenoid (C) and squalene (D) from cultures under N-deprived condition with and without terbinafine. All data are means ± SD of three biological replicates. Significant differences between the wild type (WT) and the mutants within the same condition are indicated by asterisks (*) (p < 0.05). Significant differences between different treatments of the same strain are indicated by plus sign (+) (p < 0.05).

Figure 7

Growth and maximum quantum yield (F_v/F_m) parameter of wild type (WT) and tfr1 mutant in nitrogen-replete medium (A,C) and nitrogen-replete medium with terbinafine (B,D). All data are means ± SD of three biological replicates. Significant differences between the wild type (WT) and the mutants within the same condition are indicated by asterisks (*) (p < 0.05).

Figure 8

Growth and maximum quantum yield (F_v/F_m) parameter of wild type (WT) and tfr1 mutant in nitrogen-depleted medium (A,C) and nitrogen-depleted medium with terbinafine (B,D). All data are means ± SD of three biological replicates. Significant differences between the wild type (WT) and the mutants within the same condition are indicated by asterisks (*) (p < 0.05).