Lipid extract derived from newly isolated Rhodotorula toruloides LAB-07 for cosmetic applications

Abstract

Rhodotorula toruloides is a non-conventional yeast with a natural carotenoid pathway. In particular, R. toruloides is an oleaginous yeast that can accumulate lipids in high content, thereby gaining interest as a promising industrial host. In this study, we isolated and taxonomically identified a new R. toruloides LAB-07 strain. De novo genome assembly using PacBio and Illumina hybrid platforms yielded 27 contigs with a 20.78 Mb genome size. Subsequent genome annotation analysis based on RNA-seq predicted 5296 protein-coding genes, including the fatty acid production pathway. We compared lipid production under different media; it was highest in the yeast extract salt medium with glycerol as a carbon source. Polyunsaturated α-linolenic acid was detected among the fatty acids, and docking phosphatidylcholine as a substrate to modeled Fad2, which annotated as Δ12-fatty acid desaturase showed bifunctional Δ12, 15-desaturation is structurally possible in that the distances between the diiron center and the carbon-carbon bond in which desaturation occurs were similar to those of structurally identified mouse stearoyl-CoA desaturase. Finally, the applicability of the extracted total lipid fraction of R. toruloides was investigated, demonstrating an increase in filaggrin expression and suppression of heat-induced MMP-1 expression when applied to keratinocytes, along with the additional antioxidant activity. This work presents a new R. toruloides LAB-07 strain with genomic and lipidomic data, which would help understand the physiology of R. toruloides. Also, the various skin-related effect of R. toruloides lipid extract indicates its potential usage as a promising cosmetic ingredient.

Keywords: Desaturase; Fatty acid; Genome sequencing; Keratinocytes; Lipid extract; Rhodotorula toruloides.

Graphical abstract

Fig. 1

Isolation and taxonomical identification of LAB-07 strain. (A) Brief scheme of LAB-07 isolation. The LAB-07 strain was isolated from the soil of Hongcheon, Republic of Korea. After a few rounds of characterization, LAB-07 was isolated, which showed a reddish-pink phenotype when streaked on YM agar. (B) Location of ITS and 26S rRNA D1∼D3 expansion segment domain used for taxonomic identification. Primers used for amplification are displayed. (C) Phylogenetic tree with type strains based on the BLAST result of LAB-07 ITS. Maximum likelihood method with 1000 bootstrap replicates was used for analysis, with values indicated below the branch. Bootstrap values under 50 are collapsed. LAB-07 strain is in red, marked with an asterisk. The evolutionary distances are demonstrated with branch length, marked as a scale indicating the number of base substitutions per site.

Fig. 2

Functional category distribution of predicted R. toruloides coding sequence based on the EggNOG database. Among 6151 total proteins, 5441 were matched with 5318 and 123 single and multiple matches. From the matched proteins, 2104 functions unknown (37.63%) are not illustrated in this figure.

Fig. 3

Fatty acid synthesis in R. toruloides. (A) Brief synthesis pathway of predicted major fatty acid components with corresponding elongase (blue) and desaturases (red). Among the synthesis of linoleic acid by the phosphatidylcholine pathway (dotted line), the synthesis of phosphatidylcholine and hydrolysis of the linoleoyl chain from phosphatidylcholine are not illustrated. The activity of thioesterase removing CoA from each acyl-CoA chain is not illustrated. (B) Lipid production under different media. Lipid titer (red, left axis) and content (black, right axis) were calculated. (C) The relative fraction of each fatty acid under different media. YS+Glu: YS with glucose as carbon source; YS+Gly: YS with glycerol as carbon source.

Fig. 4

Structural analysis of Fad2. (A) Modeled structure of Fad2. The Diiron center is zoomed, and coordinating histidine residues are shown. (B) Predicted docking conformation of 1-palmitoyl-2-linoleoylphosphatidylcholine (PC; purple) with Fad2, displayed with a hydrophobic surface. The choline head and palmitoyl chain are located outside, and the linoleoyl chain is located in a tunnel inside the protein that leads to the diiron catalytic center. (C) Docking result of Fad2. Distance between Fe²⁺ ion and Δ15 (orange), formed through this reaction, is displayed with the location of a Δ9, 12 double bonds (magenta) in the linoleoyl chain (cyan).

Fig. 5

Assay results of R. toruloides lipid extract. (A) Change in the relative expression level of filaggrin in keratinocytes by treatment. (B) Change in the relative expression level of MMP-1 in keratinocytes by treatment. Control without IR treatment is indicated as a hyphen. Asterisks represent the significance between samples and control without IR exposure. Hash represents the significance between samples and control with IR exposure. (C) Relative antioxidant activity by treatment. All values indicate the mean of independent experiments± SD. (*p < 0.05, ** p < 0.01, ***p < 0.001 from Student’s t-test) RA: retinoic acid; AA: ascorbic acid.