Sterol Biosynthesis in Four Green Algae: A Bioinformatic Analysis of the Ergosterol Versus Phytosterol Decision Point

Abstract

Animals and fungi produce cholesterol and ergosterol, respectively, while plants produce the phytosterols stigmasterol, campesterol, and β-sitosterol in various combinations. The recent sequencing of many algal genomes allows the detailed reconstruction of the sterol metabolic pathways. Here, we characterized sterol synthesis in two sequenced Chlorella spp., the free-living C. sorokiniana, and symbiotic C. variabilis NC64A. Chlamydomonas reinhardtii was included as an internal control and Coccomyxa subellipsoidea as a plant-like outlier. We found that ergosterol was the major sterol produced by Chlorella spp. and C. reinhardtii, while C. subellipsoidea produced the three phytosterols found in plants. In silico analysis of the C. variabilis NC64A, C. sorokiniana, and C. subellipsoidea genomes identified 22 homologs of sterol biosynthetic genes from Arabidopsis thaliana, Saccharomyces cerevisiae, and C. reinhardtii. The presence of CAS1, CPI1, and HYD1 in the four algal genomes suggests the higher plant cycloartenol branch for sterol biosynthesis, confirming that algae and fungi use different pathways for ergosterol synthesis. Phylogenetic analysis for 40 oxidosqualene cyclases (OSCs) showed that the nine algal OSCs clustered with the cycloartenol cyclases, rather than the lanosterol cyclases, with the OSC for C. subellipsoidea positioned in between the higher plants and the eight other algae. With regard to why C. subellipsoidea produced phytosterols instead of ergosterol, we identified 22 differentially conserved positions where C. subellipsoidea CAS and A. thaliana CAS1 have one amino acid while the three ergosterol producing algae have another. Together, these results emphasize the position of the unicellular algae as an evolutionary transition point for sterols.

Keywords: Chlorella sorokiniana; Chlorella variabilis NC64A; Clotrimazole; Coccomyxa subellipsoidea; Ketoconazole; algal sterol composition; oxidosqualene cyclase; terbinafine.

Fig. 1

Putative sterol pathway in Chlorella variabilis and Chlorella sorokiniana showing structures of the biosynthetic intermediates and the enzymes which catalyze each step, starting for convenience with cycloartenol, the product of Cas1p. 1: SMT1‐sterol C‐24 methyltransferase; 2: SMO2‐C‐4 sterol methyloxidase; 3: CPI1‐cycloeucanol cycloisomerase; 4: CYP51‐sterol C‐14 demethylase; 5: ERG4/24‐C‐24(28) sterol reductase; 6: HYD1‐C (8, 7) sterol isomerase; 7: STE1‐C‐5 sterol desatirase; 8: CYP710‐C‐22 sterol desaturase. The biosynthetic pathway follows that suggested by Brumfield et al (2017) for C. reinhardtii, overlaid with our data showing the sterol intermediates which accumulate following treatment with ketoconazole or clotrimazole, drugs thought to inhibit the two cytochrome P₄₅₀ enzymes, CYP51 and CYP710, here labeled 4 and 8, respectively.

Fig. 2

Phylogenetic tree for 40 oxidosqualene cyclase (OSC) proteins showing the position of genes from 9 algal species relative to their plant, animal, fungal, and diatom equivalents. Acetobacter tropicallis (not shown) was used as an out‐group to derive the root.

Fig. 3

Multiple sequence alignment of OSC proteins presented in Tables 3 and S2. The three residues identified by Brumfield et al. (2017) as conserved in CAS proteins are colored in orange. The catalytic aspartic acid (D483 for Arabidopsis thaliana) is colored in blue. Residues that are conserved across Chlamydomonas reinhardtii, Chlorella sorokiniana, and C. variabilis but are differently conserved in A. thaliana CAS1 and Coccomyxa subellipsoidea are highlighted in yellow.

Fig. 4

Four views of the tertiary structure prediction of Coccomyxa subellipsoidea CAS. Positions are given for the residues on the C. subellipsoidea sequence (Fig. 3). Yellow residues are conserved across Chlamydomonas reinhardtii, Chlorella sorokiniana, and C. variabilis, but are differentially conserved in Arabidopsis thaliana CAS1 and C. subellipsoidea. The catalytic D480 residue is colored in blue. Residues identified by Brumfield et al. (2017) as conserved in CAS proteins are colored in orange. 15 of the 22 differently conserved residues are shown. These residues cluster on the outer surface of the protein and around the catalytic region and could alter the protein‐protein binding of the CAS proteins and/or reaction dynamics to explain the shift from ergosterol to phytosterol production in C. subellipsoidea.