Diversity, astaxanthin production, and genomic analysis of Rhodotorula paludigena SP9-15

Abstract

Astaxanthin is a carotenoid known for its powerful antioxidant properties. This study focused on isolating yeast strains capable of producing astaxanthin from flower and fruit samples collected in Thailand. Out of 115 isolates, 11 strains were identified that produced astaxanthin. Molecular identification techniques revealed that these isolates belonged to two species: Rhodotorula paludigena (5 isolates) and Rhodosporidiobolus ruineniae (6 isolates). Whole-genome analysis of one representative strain, R. paludigena SP9-15, identified putative candidate astaxanthin synthesis-associated genes, such as CrtE, CrtYB, CrtI, CrtS, CrtR, CrtW, CrtO, and CrtZ. High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) confirmed astaxanthin production. Further optimization of astaxanthin production was carried out by investigating the effects of various factors on the growth rate and astaxanthin production. The optimal conditions were 40 g/L glucose as a carbon source, pH 7.5, and cultivation at 25 °C with 200 rpm for 3 days. Under these conditions, R. paludigena SP9-15 synthesized biomass of 11.771 ± 0.003 g/L, resulting in astaxanthin with a content of 0.558 ± 0.018 mg/g DCW (dry cell weight), an astaxanthin yield of 6.565 ± 0.238 mg/L, and astaxanthin productivity of 2.188 ± 0.069 g/L/day. These findings provide insights into astaxanthin production using red yeast strains from Thailand and highlight the potential of R. paludigena SP9-15 for further application.

Keywords: Astaxanthin; Biosynthetic pathway; Genome; Putative gene; Rhodotorula paludigena; Yeast.

Fig. 1

The phylogenetic tree of gene sequences of 26S rRNA was generated by Maximum Likelihood method. Bootstrap values (>50%) based on 1000 replications are given at branch nodes. The GenBank accession number of each organism used is given in parentheses. Evolutionary analyses were conducted in MEGA X software.

Fig. 2

The circular genomic map of Rhodotorula paludigena SP9-15 displays the following information: open reading frames (ORFs) in blue, GC content in pink, GC skew (+) in yellow, and GC skew (−) in green. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Venn diagram illustrating the shared and unique protein in Rhodotorula paludigena SP9-15 and comparison with proteins found in R.paludigena P4R5, R. kratochvilovae CBS 7436^T, and R. glutinis CBS20^T, respectively.

Fig. 4

Thin layer chromatography (TLC) of an acetone extract of astaxanthin-producing isolated yeasts and astaxanthin standard. The presence of astaxanthin band of several Rhodosporidiobolus ruineniae isolates (a), and Rhodotorula paludigena isolates (b).

Fig. 5

The HPLC chromatogram of the extracted astaxanthin from R. paludigena SP9-15 in DMSO, non-spiked and spiked with a 10 ppm astaxanthin standard (a). The LC-MS chromatograms in MRM mode (negative scan) of the extracted astaxanthin from R. paludigena SP9-15 in DMSO (b).

Fig. 6

Effect of carbon sources (a), glucose concentration (b), nitrogen supplementation (c), pH (d), temperature (e) and cultivation time (f) on the astaxanthin content (mg/g DCW), astaxanthin yield (mg/L), biomass (g/L), and productivity (mg/L/day) of R. paludigena SP9-15. The experiments were performed in triplicate, and the results were presented as the mean ± standard deviation (SD). The Tukey’s test with a significance level of ⍺ 0.05 was applied, and the levels that are not linked by the same letter indicate significant differences.