F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.164600.2 Research Article Articles Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1

[version 2; peer review: 2 approved, 1 approved with reservations]

 Hoondee Patcharaporn Conceptualization Data Curation Formal Analysis Investigation Methodology Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing 1 2 Tedsree Nisachon Formal Analysis Methodology 3 Phuengjayaem Sukanya Conceptualization Data Curation Formal Analysis Methodology Writing – Original Draft Preparation 4 Kingkaew Engkarat Conceptualization Formal Analysis Methodology 5 Sritularak Boonchoo Formal Analysis Funding Acquisition Writing – Review & Editing 6 7 Rojsitthisak Pornchai Formal Analysis Funding Acquisition Writing – Review & Editing 6 8 Nakashima Takuji Writing – Review & Editing 9 10 Thitikornpong Worathat Conceptualization Data Curation Formal Analysis Funding Acquisition Methodology Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing a 6 8 Tanasupawat Somboon Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-7149-5341 b 6 11 Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology Krungthep, Sathon, Bangkok, 10120, Thailand Biodiversity and Sustainable Utilization Research Unit, Rajamangala University of Technology Krungthep, Sathon, Bangkok, 10120, Thailand Faculty of Science and Arts, Chanthaburi Campus, Burapha University, Tha Mai, Chanthaburi, 22170, Thailand Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Thung Khru, Bangkok, 10140, Thailand Department of Biology, School of Science, King Mongkut's Institute of Technology Ladkrabang, Lat Krabang, Bangkok, 10520, Thailand Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand Department of Field Sciences, University of Human Environments, Mutsuyama, Ehime, 790-0825, Japan Research Organization for Nano and Life Innovation, Waseda University, Shinjuku, Tokyo, 162-0041, Japan Department of Biochemistry and Microbiology, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand worathat.t@pharm.chula.ac.th somboon.t@chula.ac.th

No competing interests were disclosed.

 2 1 2026 2025 14 717 30 12 2025 Copyright: © 2026 Hoondee P et al. 2026 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

Astaxanthin, a red xanthophyll carotenoid, is a powerful antioxidant, anticancer, and glucose and lipid homeostasis regulator. Some pigmented yeasts belonging to the genus Rhodotorula, the well-known yeast for beta-carotene production, have been reported as natural astaxanthin producers. However, the lack of genomic data on astaxanthin-producing strains within these species hinders the identification of biosynthetic routes, molecular characterization of these pathways, and gene editing applications.

 Methods

This study explored the diversity and astaxanthin production capability of cultivable pigmented yeast in flower samples. The astaxanthin production ability was inspected by three consecutive methods, including thin-layer chromatography (TLC) for the preliminary step, followed by quantitative spectrophotometry and high-performance liquid chromatography (HPLC) for qualitative validation. The draft genome sequence and astaxanthin-producing genes of astaxanthin-producing yeasts were examined.

 Results

Twelve of 23 yeasts from floral samples exhibited natural pigmentation, with colors ranging from pinkish-orange to red, and exhibited the potential for astaxanthin synthesis. These yeasts were Rhodotorula paludigena (three strains) and Rhodotorula mucilaginosa (nine strains). Among R. mucilaginosa strains, HL26-1 had the greatest astaxanthin content (104.98 ± 0.13 μg/g DCW) and yield (0.9280 ± 0.0012 mg/L). Strain LL69-1 has the greatest astaxanthin content (251.78 ± 0.27 μg/g DCW) and yield (1.8632 ± 0.0023 mg/L) among R. paludigena strains. The 18.78 Mbp R. mucilaginosa HL26-1 genome includes 5,711 protein-coding genes. Conversely, the R. paludigena LL69-1 genome was 20.99 Mbp, encompassing 6,782 predicted genes. A comprehensive investigation of draft genome sequences of these two strains identified CrtE, CrtYB, CrtI, CrtS, and CrtR as potential astaxanthin transcription genes.

 Conclusion

Here, our results highlight the outstanding potential of two naturally pigmented yeasts, R. mucilaginosa HL26-1 and R. paludigena LL69-1, for astaxanthin production. Furthermore, our findings provide information on the whole genome and protein-encoded genes associated with astaxanthin production, which serve as valuable biological resources for various biotechnological applications.

 astaxanthin pigmented yeast Rhodotorula mucilaginosa Rhodotorula paludigena astaxanthin synthase Thailand Science Research and Innovation Fund Chulalongkorn University CU_FRB65_hea(51)060_33_04 This research was funded by the Thailand Science Research and Innovation Fund Chulalongkorn University (CU_FRB65_hea (51) 060_33_04). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 1

Dear Readers, This revised version of the article has been improved in response to reviewer comments to enhance clarity, transparency, and scientific rigor. The introduction has been reorganized to improve clarity and coherence, with a clearer presentation of the research background, objectives, and research gap. The Materials and Methods section has been substantially expanded to provide comprehensive experimental details, including clearly described statistical analyses and appropriate reporting of variation and significance. The manuscript has been revised to consistently distinguish between total carotenoids and astaxanthin. Additional methodological details, including HPLC and TLC conditions and astaxanthin standard specifications, have been added. Statements regarding astaxanthin yield have been refined to ensure accuracy, and inconsistencies in genome assembly statistics have been corrected throughout the manuscript. Formatting issues, unit consistency, abbreviations, and typographical errors have also been addressed. We believe that these revisions have strengthened the manuscript and hope that the revised version will be suitable for publication. Sincerely, The Authors

 Introduction

Astaxanthin (3,3′-dihydroxy-β, β′-carotene-4,4′-dione) is a red pigment compound belonging to the xanthophyll carotenoid group and is widely recognized as a powerful antioxidant. The effective free radical–scavenging activity of astaxanthin contributes to its anticancer properties by reducing oxidative stress and singlet oxygen–mediated cellular damage. 1 Its antioxidant capacity has been reported to surpass that of vitamin E (α-tocopherol), β-carotene, canthaxanthin, and other natural carotenoids by up to 100-fold. 2 Beyond its antioxidant prowess, astaxanthin serves as a potent metabolic regulator of glucose and lipid homeostasis by increasing insulin sensitivity, augmenting glucose uptake, and modulating lipid turnover in the liver. In dermatology, it has garnered substantial attention for its ability to counteract oxidative stress, facilitate cellular rejuvenation, repair DNA damage, and protect against UV-induced photoaging and skin malignancies. 3, 4 Given these multifaceted biological activities, astaxanthin is extensively utilized across the pharmaceutical, nutraceutical, cosmetic, and animal feed industries. 5

Presently, astaxanthin is obtained either through chemical synthesis or natural extraction. 6 Despite its cost-effectiveness compared to natural sources, synthetic production often generates hazardous byproducts and poses environmental risks. 7 Consequently, the demand for natural sources such as shrimp, krill, fish, and microorganisms has surged. 8 Among microbes, the microalga Haematococcus pluvialis is the primary industrial source due to its high astaxanthin content. However, algal cultivation faces significant logistical and economic hurdles, including slow growth rate, susceptibility to contamination, and complex downstream processing. To overcome these limitations, recent research has shifted toward pigmented yeasts as alternative bio-factories. Compared to microalgae, yeasts offer several biotechnological advantages: they can be cultured in closed bioreactors, possess faster growth cycles, and can utilize various carbon sources, including lignocellulosic biomass and industrial by-products. 6, 9– 11

While Phaffia rhodozyma ( Xanthophyllomyces dendrorhous) has been the traditional yeast source for several decades, 6, 12 certain Rhodotorula species, such as R. toruloides 13, 14 R. paludigena, 15, 16 R. mucilaginosa 17, 18 and Rhodotorula sp., 19 have recently emerged as promising candidates for astaxanthin biosynthesis. Despite this potential, a critical research gap remains. The pathway of astaxanthin biosynthetic in Phaffia rhodozyma has been reported. 20– 22 The regulation starts with beta-carotene formation through mevalonate pathway. Acetyl-CoA was converted to isopentenyl-pyrophosphate (IPP), the precursor of all isoprenoids, which were further condensed to produce phytoene, the colorless carotenoid. Subsequently, phytoene was transformed into β-carotene, which was catalyzed by phytoene synthase/lycopene beta-cyclase and phytoene desaturase (encoded by CrtYB and CrtI genes, respectively). 20– 22 Finally, a single gene called CrtS, which could act as both a ketolase and a hydroxylase, was responsible for converting β-carotene into astaxanthin. 21, 22 However, the genomic information and metabolic pathways of astaxanthin-producing Rhodotorula species remain incompletely understood. Only a handful of studies have employed effective techniques, such as whole-genome sequencing and gene analysis, to elucidate genome-related astaxanthin production in Rhodotorula yeast. 13, 15– 17 As a result, the scarcity of comprehensive genomic data hampers the discovery of essential biosynthetic genes, molecular characterization, and the use of gene-editing technologies to increase production yields.

Flower tissues and nectar are recognized as important reservoirs for diverse yeast communities. Previous studies have reported that pigmented yeasts can function as growth-promoting microorganisms, 23, 24 have shown great potential as biosynthetic fungicides for the control of postharvest fruit decay, 25, 26 and may influence plant–pollinator dynamics through the modification of nectar chemistry. 27 Moreover, pigmented yeasts isolated from flowers are often stress-tolerant, with carotenoid pigments contributing to their stress resistance by providing photoprotection against UV-B radiation, thereby supporting yeast growth and enhancing survivorship in UV-exposed environments. 28 These yeasts are also capable of producing valuable carotenoid pigments, such as β-carotene and astaxanthin, making them promising candidates for a wide range of biotechnological applications. 29

This study aimed to isolate and characterize novel astaxanthin-producing yeast strains obtained from flowers. Furthermore, we investigated the genomic profiles of two chosen astaxanthin-producing yeast strains, namely, Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1. Through analysis of their whole-genome sequences and identification of genes associated with astaxanthin synthesis, our objective was to provide comprehensive insights into the repertoire of proteins encoded within their genomes. This endeavor holds promise for elucidating their functional capabilities and igniting excitement about their potential integration as valuable biological resources across a spectrum of biotechnological applications, from the food and feed industries to the pharmaceutical and cosmetic sectors.

 Methods Isolation and phenotypic characterization

We collected eleven flower samples from the residential areas in Lampang Province, Thailand, in April 2022. Samples were randomly cut and collected into a sterile plastic bag through a scissor flame sterilized. After sampling, the flowers were carefully transported as soon as possible to the laboratory at low temperature (4°C) to maintain integrity during transit. Flower identification was conducted on-site by interviewing the owner of the plant and was then identified by one of the co-authors (B.S.). Specimens have been deposited at the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand. Detailed information regarding the samples, specimen number, and their respective collection locations is provided in Table S1. The enrichment method has been used for yeast isolation. One gram of each sample was inoculated into 15 mL of yeast malt (YM) broth medium (10 g/L glucose, 5 g/L peptone, 3 g/L yeast extract, 3 g/L malt extract, pH 5.5). The inoculated media were incubated at 25°C for 3 days. Subsequently, the resulting cultures were streaked onto YM agar medium (pH 5.5) and incubated at 30°C for 5 days. Following incubation, pigmented yeast colonies displaying the desired pink to red hue were meticulously selected and purified on YM agar media using the streak plate method. Each isolate culture was carefully preserved on a YM agar slant at 4°C to facilitate further analysis and experimentation. Carbon assimilation of the strain was performed using the API ® ID 32 C kit (BioMérieux, France) according to the manufacturer’s instructions. Reactions were visually examined at 72 hours, and then the results were interpreted to be positive or negative based on the presence or absence of turbidity in the carbohydrate wells.

 Molecular identification of yeast strains

Genomic DNA extraction

Genomic DNA was extracted from the pure yeast cultures using the glass bead extraction method 30 with some modification. Briefly, yeast cells were washed twice with sterilized distilled water and then lysed by vortexing with 0.3 g of 0.45-0.52 mm diameter acid washed glass beads in 200 μL of extraction buffer (comprising 2% (v/v) Triton X-100, 1% (w/v) SDS, 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA (pH 8.0)) for 5 minutes. The resulting cell-free supernatant was transferred to a new tube and gently mixed with a 2X volume of phenol (Sigma–Aldrich ®, USA, Cat. No. 242322), chloroform (RCI Labscan ®, Ireland, Cat. No. AR 1027E), and isoamyl alcohol (Sigma–Aldrich ®, USA, Cat. No. 8.22255) at a ratio of 25:24:1. After centrifugation, the supernatant was transferred to a new tube containing 1 mL of absolute ethanol (RCI Labscan ®, Ireland, Cat. No. AR1069) and stored at -20°C for 20 minutes. The DNA pellet obtained after centrifugation was washed with 500 μL of 70% (v/v) ethanol, dried at 37°C, dissolved in 50 μL of TE buffer, and stored at -20°C until further use.

Sequencing of the 26S rRNA gene (D1/D2 domain)

The 26S rRNA gene within the D1/D2 domain of the large subunit (LSU D1/D2 domain) was amplified by PCR using the primers NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′). 31 PCR was conducted in a 20 μL volume comprising 2 μL of DNA template, 0.4 μL of each primer (10 pmol/μL), 10 μL of 2X Go Tag Green, and 7.2 μL of distilled water. The amplification process involved an initial denaturation step at 94°C for 3 minutes, followed by 36 cycles of denaturation at 94°C, annealing at 52°C, and extension at 72°C, each for 30 seconds, and a final extension step at 72°C for 5 minutes. Subsequently, the PCR products were purified using a gel/PCR DNA fragment extraction kit (GenepHlow™, Geneaid Biotech Ltd., Taiwan). Purified PCR products were sequenced bidirectionally using BT sequencing technology (Celemics Inc., Republic of Korea).

Analysis of phylogenetic placement

The LSU D1/D2 sequences were aligned with those of related species using MUSCLE, 32 and any gaps were removed. MEGA11 software 33 was used to construct a neighbor-joining (NJ) tree using Kimura’s two-parameter model. 34 The reliability of the branches was assessed using a bootstrap test with 1000 replicates. 35

Yeast cultivation for astaxanthin production

A single loopful of yeast cultured on YM agar was transferred to YM broth (50 mL) in a 250 mL flask and then incubated at 30°C with agitation at 200 rpm for 24 hours. Subsequently, a 5 mL aliquot of this culture was inoculated into fresh YM broth (45 mL) in a 250 mL flask and incubated under the same conditions for 72 hours. Following incubation, the cells were harvested by centrifugation at 4°C and 6,500 × g, for 10 minutes, washed twice with distilled water, and then subjected to lyophilization for drying. Experiments were performed in triplicate. The resultant dry cell weight (DCW) was determined to quantify the cell biomass. Lyophilized cells were used for further analysis of astaxanthin.

 Astaxanthin analysis

Qualitative analysis of astaxanthin production by thin layer chromatography

The extraction and qualitative analysis of astaxanthin were performed using a slightly modified method described by Ushakumari and Ramanujan. 36 Lyophilized cells (0.01 g DCW) were suspended in 1 mL of acetone (RCI Labscan ®, Ireland, Cat. No. AR1003) and homogenized using a pestle at room temperature for 3 minutes. The supernatant was collected by centrifugation at 12,300 × g, for 10 minutes. Subsequently, an aliquot (20 μL) of the extract was applied onto a silica gel TLC plate using a capillary tube, alongside an astaxanthin standard. Analytical-grade astaxanthin with purity ≥ 98% was purchased from Dayang Chem Co. Ltd. (China) and utilized as a reference standard. Plate development was carried out using acetone:hexane (1:3, v/v) as the mobile phase. Acetone and hexane were purchased from RCI Labscan ® (Ireland) with Cat. No. of AR1003 and AR1085, respectively. After development, colored bands were directly visualized under visible light and compared with the astaxanthin standard. The R f value was calculated to evaluate the migration of pigments on the TLC plate.

Quantification of astaxanthin production

The extraction and measurement of astaxanthin were conducted using previously described methods with slight modifications. According to Li and others 37 and Casella, 38 absorbance at 530 nm shows a strong linear correlation with astaxanthin content while minimizing interference from other carotenoids; therefore, this wavelength was used for astaxanthin quantification in this study. To extract the intracellular carotenoid content, 50 mg of lyophilized cells were suspended in 5 mL of dimethyl sulfoxide (DMSO; Sigma–Aldrich ®, USA, Cat. No. 34869) and ultrasonicated at 37 kHz and 50°C for 30 minutes using an ultrasonic bath (Elmasonic, E60H model, Germany). The extracts were centrifuged at 12,300 × g for 5 minutes. The extraction process was repeated until the supernatant became colorless. Astaxanthin concentration was determined using a Cary 60 UV–Vis spectrophotometer (Agilent) at 530 nm against a DMSO blank. A standard calibration curve was generated using astaxanthin concentration of 0, 0.25, 0.5, 1, 2, 4, 6, and 8 μg/mL prepared in DMSO. Astaxanthin concentrations were calculated based on the standard calibration curve, and the results are presented as the mean of triplicate measurements. Statistical analyses were performed using SPSS software for Windows (version 22). One-way analysis of variance (ANOVA) was applied to evaluate differences among individual factors, and Tukey’s multiple range test was used for post hoc pairwise comparisons at a significance level of α = 0.05. Astaxanthin production efficiency among the different strain was compared. The strains, which showed the highest astaxanthin production among their species, were selected for further study.

Confirmative analysis of astaxanthin synthesis

The astaxanthin was extracted and analyzed based on the described method. 15 In brief, lyophilized cells (0.05 g dry cell weight) were suspended in 5 mL of DMSO (Sigma–Aldrich ®, USA, Cat. No. 34869), mixed thoroughly, and sonicated at 37 kHz (Elmasonic E60H, Germany) at 55°C for 5 minutes. The mixture was centrifuged at 6,500 × g for 10 minutes and the resultanting supernatant was filtered through a 0.22 μm membrane filter prior to high performance liquid chromatography (HPLC) analysis. HPLC analysis was performed using a UHPLC Nexera X2 system (Shimadzu, Japan) equipped with an LC-30AD binary pump, SIL-30AC autosampler, CTO-20AC column oven and SPD-M30A detector, controlled by LabSolutions chromatography software. Seperation was achieved on a C18 column (GL Science InterSustain, 4.6 mm × 150 mm, 5 μm). The flow rate was set at 0.5 mL/minutes from 0.00 to 3.00 minutes and increased to 1.0 mL/minutes from 3.01 to 20.00 minutes. The detection wavelength was 480 nm, the column temperature was maintained at 30°C, and the injection volume was 5 μL. The mobile phase consisted of methanol (RCI Labscan ®, Ireland, Cat. No. LC1115)/acetonitrile (RCI Labscan ®, Ireland, Cat. No. LC1005)/ethyl acetate (RCI Labscan ®, Ireland, Cat. No. LC1070)/formic acid (Sigma–Aldrich ®, USA, Cat. No. 5.43804) (75.9:12:12:0.1, v/v) (solvent A) and methanol (solvent B).

 Whole-genome sequencing and analysis

The whole genome was sequenced using the paired-end (PE) 150 method on the Illumina HiSeq Xten/Novaseq/MGI2000 platform at Vishuo Biomedical Pte. Ltd., Beijing, China. The single-end reads were processed to eliminate adapters and low-quality bases using Fastp (v0.23.0). The resulting data were then assembled into contigs using Velvet de Novo assembler version 1.2.10. 39, 40 Subsequently, the contigs were assembled into scaffolds using SSPACE (version 3.0), 41 and the gaps were filled using GapFiller (versions 1–10). 42 Gene prediction was performed using Augustus version 3.3. 43 The coding genes were annotated using the National Center for Biotechnology Information (NCBI) NR database via BLAST. Subsequently, gene function prediction was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) 44 and KofamKOALA tools, employing default settings and considering all hits ( https://www.genome.jp/kegg/). 45 Proksee ( https://proksee.ca/) was used to generate the circular genome map, and OrthoVenn ( https://orthovenn3.bioinfotoolkits.net/ start/db) was used to create the Venn diagram. The Kostas Lab web-based tool ( http://enve-omics.ce.gatech.edu/) was used to analyze the average nucleotide identity (ANI) values. 46

Results Isolation and identification of yeast strains

Twenty-three yeasts were isolated from the 11 flower samples collected (Table S1). Among them, 12 isolates exhibited pigmented colonies with the desired coloration ranging from pink to orange. All isolates were subjected to identification at the molecular operational taxonomic unit (MOTU) level by sequencing of the 26S rRNA gene (LSU D1/D2 domain), followed by species assignment via comparison with entries in the NCBI GenBank database using the BLASTn program. Consequently, all 12 pigment yeast strains were identified as basidiomycetous yeasts (Table S1), with two species belonging to the Rhodotorula genus: Rhodotorula paludigena (9 strains) and Rhodotorula mucilaginosa (3 strains). The remaining 11 strains were classified into four species of Basidiomycota— Cryptococcus heveanensis (1 strain), Pseudozyma aphidis (2 strains), Pseudozyma hubeiensis (1 strain), and Pseudozyma siamensis (1 strain)—and five species of Ascomycota— Candida parapsilosis (1 strain), Metschnikowia koreensis (1 strain), Saccharomyces cerevisiae (2 strains), Wickerhamiella infanticola (1 strain), and Debaryomyces nepalensis (1 strain).

 Qualitative analysis of astaxanthin production by thin layer chromatography

A total of twelve pigmented strains were selected for qualitative analysis of astaxanthin production using thin-layer chromatography (TLC) technique. Astaxanthin, a red carotenoid, was directly visualized on the TLC plates without derivatization. Comparison with the astaxanthin standard revealed a corresponding band in all extracts. TLC analysis showed the presence of several pigment bands in the extracts. Astaxanthin was identified by comparing the R f value and coloration of the sample band with those of the astaxanthin standard analyzed under identical chromatographic conditions. The astaxanthin bands from all strains migrated within narrow R f range centered around 0.28, corresponding to the R f value of the astaxanthin standard. Minor variations in R f values were observed; however, all astaxanthin bands matched the standard, confirming the presence of astaxanthin in all tested yeast strains ( Figure 1). Based on these results, all yeast strains were selected for further quantitative analysis of astaxanthin content.

Thin layer chromatography (TLC) of an acetone extract of pigment isolated yeasts and astaxanthin standard on Silica gel G plate using a mixture of acetone and hexane (ratio of 1:3 (v/v)) as the mobile phase.

The presence of astaxanthin band of R. paludigena strain TL35-3, HL55-2, TL35-4, and TL35-6 (a), strain LL69-1, IL45-1, IL45-2-1, HL55-1, HL55 (b), and R. mucilaginosa strain TL35-2, AL44-2, and HL26-1 (c).

 Quantification of astaxanthin production

Twelve yeast strains exhibiting positive results in TLC analysis for astaxanthin were subjected to quantitative analysis using spectrophotometry. Intracellular astaxanthin was extracted from powdery cells using the conventional DMSO extraction method 37 coupled with sound energy via an ultrasonic bath to enhance cell disruption efficiency. 47 In the R. mucilaginosa strains, the astaxanthin content ranged from 23.99 ± 0.22 to 104.98 ± 0.13 μg/g DCW, with yields ranging from 0.1991 ± 0.0021 to 0.9280 ± 0.0012 mg/L ( Figure 2). Moreover, several R. paludigena strains exhibited astaxanthin contents and yields ranging from 47.99 ± 0.11 to 251.78 ± 0.27 μg/g DCW and from 0.3589 ± 0.0009 to 1.8632 ± 0.0023 mg/L, respectively ( Figure 2). Strain LL69-1 demonstrated the highest astaxanthin production of R. paludigena, with an astaxanthin content of 251.78 ± 0.27 μg/g DCW and a yield of 1.8632 ± 0.0023 mg/L. Meanwhile, R. mucilaginosa strain HL26-1, the highest producer of its species, which yielded 104.98 ± 0.13 μg/g DCW and 0.9280 ± 0.0012 mg/L.

Quantitative analysis of astaxanthin production by 3 <italic toggle="yes">R. mucilaginosa</italic> strains (AL44-2, TL35-2, and HL26-1) and 9 <italic toggle="yes">R. paludigena</italic> strains (TL35-5, TL35-4, TL35-6, HL55, HL55-1, HL55-2, IL45-2-1, IL45-4, and LL69-1).

The results are obtained from three replicate experiments and reported as the average value ± standard deviation (SD) for n = 3, with different letters indicating significant difference ( p < 0.05 one-way analysis of variance (ANOVA)).

 Confirmative analysis of astaxanthin synthesis in selected astaxanthin-producing strains

The ability of two astaxanthin-producing yeasts, R. mucilaginosa HL26-1 and R. paludigena LL69-1, to synthesize astaxanthin was confirmed by high-performance liquid chromatography (HPLC). The HPLC chromatogram of both strains exhibited peaks corresponding to astaxanthin when compared with a 10 ppm astaxanthin standard. The retention times of the predominant peaks were observed at 2.061 minutes for strain HL26-1 and 2.211 minutes for strain LL69-1. Astaxanthin was identified by comparing the retention times of the sample peaks with that of the authentic astaxanthin standard. In addition, spiking the sample extracts with the astaxanthin standard resulted in a proportional increase in the corresponding peak area without the appearance of additional peaks, confirming the identity of astaxanthin. Representative HPLC chromatograms of the standard, samples, and spiked samples are shown in Supplementary Figure S1. These results demonstrate that both HL26-1 and LL69-1 are capable of astaxanthin production.

 General features of the <italic toggle="yes">R. paludigena</italic> LL69-1 and <italic toggle="yes">R. mucilaginosa</italic> HL26-1 genomes

The genome assembly statistics for the two astaxanthin-producing yeasts, R. mucilaginosa HL26-1 and R. paludigena LL69-1, are presented in Table 1. The assembly of R. mucilaginosa HL26-1 resulted in 107 scaffolds, with the giant scaffold spanning 853,611 base pairs (bp). The N50 length of the scaffolds was 193,129 bp, with a GC content of 60.12% and a genome size of 18.78 Mbp. Conversely, R. paludigena LL69-1 had a genome size of 20.99 Mbp, with a GC content of 63.82%. Its assembly comprised 107 scaffolds with an N50 value of 476521 bp and an L50 value of 24 scaffolds. The largest scaffold in LL69-1 was 1,230,191 bp, whereas the shortest was 524 bp. Circular genomics of the HL26-1 and LL69-1 genomes, illustrating the open reading frame (ORF) positions, GC content, and GC skew of each strain, are depicted in Figure 3. The whole-genome sequences of R. mucilaginosa HL26-1 and R. paludigena LL69-1 were deposited in the NCBI/GenBank database ( http://www.ncbi.nlm.nih.gov), associated with the BioProject identities PRJNA1025132 and PRJNA1025134, BioSample numbers SAMN37714267 and SAMN37714269, and GenBank accession numbers JAZBNE000000000 and JAWJBI000000000, respectively.

Assembly statistics of the <italic toggle="yes">R. paludigena</italic> HL26-1 and <italic toggle="yes">R. mucilaginosa</italic> LL69-1 genomes.
Features Strains
R. mucilaginosa HL26-1 R. paludigena LL69-1
Genome assembly size (bp) 18,775,076 20,987,037
Max length (bp) 853,611 1,230,191
Number of scaffolds 107 154
N50 (bp) 193,129 476,521
GC (%) 60.12 63.82
Predicted protein-coding gene 5,711 6,782
Circular genomic map of <italic toggle="yes">R. mucilaginosa</italic> HL26-1 and <italic toggle="yes">R. paludigena</italic> LL69-1 displaying the following information: GC content in orange, GC skew (+) in blue, GC skew (-) in purple, and open reading frames (ORFs) in green. Phylogenetic and average nucleotide identity (ANI) analysis of strains

Figure S2 depicts a phylogenetic tree using the D1/D2 domains of the large subunit sequences obtained in this study and those available in the NCBI nucleotide database. This analysis confirmed the phylogenetic relationship between these pigmented yeasts and other related species. R. mucilaginosa HL26-1 and R. paludigena LL69-1 were clustered within the same clade as their closest-type strains, R. mucilaginosa CBS316 T and R. paludigena CBS6566 T, respectively, with 100% bootstrap support.

Moreover, we conducted an average nucleotide identity (ANI) analysis, a crucial step in assessing the phylogenomic relationships between the two astaxanthin-producing yeasts and other Rhodotorula species. The pairwise ANI values of the whole genomes of R. mucilaginosa HL26-1, R. paludigena LL69-1, and the other Rhodotorula species varied (Supplementary Figure S3).

 Functional genome annotation and comparative analysis of putative gene families associated with astaxanthin biosynthesis

According to annotation results from the National Center for Biotechnology Information (NCBI) database, the draft genome sequence of R. mucilaginosa HL26-1 comprises 5,711 protein-encoding genes, while R. paludigena LL69-1 was predicted to contain approximately 6,782 coding genes. The Venn diagram in Figure 4 illustrates the genetic variations and distinct characteristics that differentiate the genomes of HL26-1 and LL69-1 from those of closely related species. The protein-coding sequences of HL26-1 and LL69-1, along with those of three other Rhodotorula species ( R. kratochvilovae CBS 7436, R. mucilaginosa GDMCC2.30, and R. paludigena P4R5), were compared to examine the similarity of their protein sequences. This comparison revealed that R. mucilaginosa HL26-1, R. kratochvilovae CBS 7436 , R. mucilaginosa GDMCC2.30, and R. paludigena P4R5 possess 15, 84, 13, and 89 proteins, respectively, which are exclusive to their respective species or strains. Additionally, 11, 85, 35, and 36 protein families were identified as being species- or strain-specific for R. paludigena LL69-1, R. kratochvilovae CBS 7436 T , R. mucilaginosa GDMCC2.30, and R. paludigena P4R, respectively.

Venn diagram displaying the presence of conserved and specific proteins among <italic toggle="yes">Rhodotorula mucilaginosa</italic> HL26-1 (a) or <italic toggle="yes">R. paludigena</italic> LL69-1 (b) and <italic toggle="yes">R. kratochvilovae</italic> CBS 7436, <italic toggle="yes">R. mucilaginosa</italic> GDMCC2.30, and <italic toggle="yes">R. paludigena</italic> P4R5.

KEGG metabolic pathway analysis categorized a total of 2,795 (52.1%) protein-coding genes of HL26-1 into four major groups: metabolism (951 genes), genetic information processing (682 genes), cellular activities (225 genes), and environmental information processing (137 genes). Within the LL69-1 dataset, 3,303 protein-coding genes (48.7%) were subjected to KEGG analysis and classified into four major categories: metabolism (1,064 genes), genetic information processing (734 genes), cellular activities (245 genes), and environmental information processing (168 genes).

The putative gene families associated with astaxanthin biosynthesis in strains HL26-1 and LL69-1 were determined ( Table 2). The pathway of astaxanthin synthesis was divided into two main sections: one that creates the basic building blocks called terpenoids, which are important natural substances, and another that specifically produces astaxanthin.

Putative gene families associated with astaxanthin biosynthesis in <italic toggle="yes">R. mucilaginosa</italic> HL26-1 and <italic toggle="yes">R. paludigena</italic> LL69-1.
Pathways Putative genes Enzyme product (KEGG orthologs number, EC number) LL69-1 HL26-1
Scaffold ID E-value Scaffold ID E-value
Terpenoid backbone biosynthesis ACAT Acetyl-CoA acetyltransferase (K00626, EC:2.3.1.9) Scaffold13.g2648 2.1×10 −168 Scaffold38.g4472 5.1×10 −172
HMGCS Hydroxymethylglutaryl-CoA synthase (K01641, EC:2.3.3.10) Scaffold18.g3608 7.1×10 −197 Scaffold33.g3151 6.4×10 −194
HMGCR Hydroxymethylglutaryl-CoA reductase (K00021, EC:1.1.1.34) Scaffole80.g6723 3.0×10 −296 Scaffold61.g2990 9.1×10 −298
MVD Mevalonate kinase (K00869, EC:2.7.1.36) Scaffold13.g2759 2.5×10 −108 Scaffold39.g1654 7.3×10 −103
PMK Phosphomevalonate kinase (K00938, EC:2.7.4.2) Scaffold13.g2880 7.2×10 −122 Scaffold38.g4446 1.0×10 −121
DPMDC Diphosphomevalonate decarboxylase (K01597, EC:4.1.1.33) Scaffold58.g6593 2.1×10 −150 Undetectable -
IDI Isopentenyl-diphosphate delta isomerase (K01823, EC:5.3.3.2) Scaffold13.g2706 1.5×10 −67 Scaffold39.g1606 8.8×10 −68
GGDPS Geranylgeranyl diphosphate synthase (K00804, EC:2.5.1.1 2.5.1.10 2.5.1.29) Scaffold4.g827 1.0×10 −149 Scaffold103.g709 5.6×10 −148
FDPS Farnesyl diphosphate synthase (K00787, EC:2.5.1.1 2.5.1.10) Scaffold14.g3047 1.1×10 −154 Scaffold77.g4986 1.3×10 −155
CrtE Geranylgeranyl pyrophosphate synthase (K05355, EC:2.5.1.82 2.5.1.83) Scaffold1.g227 2.2×10 −168 Scaffold17.g303 7.0×10 −171
Astaxanthin biosynthesis CrtYB Phytoene synthase/lycopene beta-cyclase (K17841, EC:2.5.1.32 5.5.1.19) Scaffold3.g680 7.9×10 −183 Scaffold38.g4383 8.5×10 −170
CrtI Phytoene desaturase (K15745, EC:1.3.99.30) Scaffold3.g682 2.8×10 −234 Scaffold38.g4386 9.8×10 −237
CrtS Beta-carotene 4-ketolase/3-hydroxylase (K23037, EC:1.14.99.63 1.14.15.24 1.14.99.-) Scaffold27.g4592 3.5×10 −81 Scaffold49.g3491 6.2×10 −73
CrtR Cytochrome P450 (K14338, EC:1.14.14.1 1.6.2.4) Scaffold6.g1523 8.4×10 −68 Scaffold42.g2465 2.9×10 −70

The key genes associated with terpenoid biosynthesis discovered in the genomes of both strains were ACAT (acetyl-CoA acetyltransferase), HMGCS (hydroxymethylglutaryl-CoA synthase), HMGCR (hydroxymethylglutaryl-CoA reductase), MVD (mevalonate kinase), PMK (phosphomevalonate kinase), IDI (isopentenyl-diphosphate delta isomerase), GGDPS (geranylgeranyl diphosphate synthase), FDPS (farnesyl diphosphate synthase), and CrtE (geranylgeranyl pyrophosphate synthase). The diphosphomevalonate decarboxylase ( DPMDC) gene has only been found in strain LL69-1. The multiple gene involved in astaxanthin biosynthesis were annotated in the genomes of both strains, including the CrtYB (phytoene synthase/lycopene beta-cyclase), CrtI (phytoene desaturase), CrtS (beta-carotene 4-ketolase/3-hydroxylase), and CrtR (cytochrome P450) genes.

 Discussion

The pigmented yeast in the genus Rhodotorula is well established in yeast biotechnology applications and holds promise in numerous industrial sectors, including biofuels, carotenoids, enzymes, bioremediation, cosmetics, and biocontrol agents. 48– 51 Recently, some Rhodotorula species have exhibited unique abilities to naturally generate astaxanthin, a red pigment with excellent antioxidant activity. These species include R. paludigena, 15, 16 R. sampaioana, 16 Rhodotorula mucilaginosa, 18 and R. toruloides. 13, 14 Several Rhodotorula species have been isolated in this study. The most prevalent was R. paludigena (75%), which was found in the African marigold, pink West Indian jasmine, yellow hibiscus, and lantana flower samples. The remaining three R. mucilaginosa strains were isolated from flower samples of yellow West Indian jasmine, marigold, and hydrangea. The Rhodotorula genus is widespread and commonly found in diverse natural habitats, including nectar and floral tissue. 28, 52, 53 Earlier studies reported several flower-isolated pigmented yeasts had the ability to produce carotenoid pigment. R. paludigena SP9-15 isolated from Zinnia flowers ( Zinnia violacea Cav.), R. paludigena TL35-5 that inhabited Marigold flowers ( Tagetes erecta), Rhodotorula sp. CP72-2 isolated from Calotropis gigantea flowers, R. sampaioana PL61-2 isolated from white desert rose flowers ( Plumeria obtusa), and Rhodosporidiobolus ruineniae SP3-3/4 Red Cat’s Tail ( Acalypha hispida Burm.f.) flower exhibited astaxanthin production. 15, 16, 19 Moreover, the β-carotene-producing strain, R. graminis TY-99, isolated from flowers, was efficient for SCP production from waste milk. 54

Three investigation techniques were implemented to assess the capacity to generate astaxanthin. In the prescreening step, the TLC technique is considered reliable and accurate. Additionally, several studies have widely used TLC to confirm the presence of astaxanthin. 55– 57 For the specific quantification of astaxanthin, spectrophotometry has been used. 37, 38, 58 Meanwhile, high-performance liquid chromatography (HPLC) has been used as a qualitative confirmative analysis technique for astaxanthin synthesis. All twelve Rhodotorula strains identified in this study had the capability to synthesize astaxanthin, a distinctive and uncommon trait among this genus. Nine strains of R. paludigena exhibited astaxanthin contents ranging from 47.99 ± 0.11 to 251.78 ± 0.27 μg/g DCW and yields ranging from 0.3589 ± 0.0009 to 1.8632 ± 0.0023 mg/L, respectively. Meanwhile, the astaxanthin content was between 23.99 ± 0.22 and 104.98 ± 0.13 μg/g DCW, and the yields were between 0.1991 ± 0.0021 and 0.9280 ± 0.0012 mg/L for three R. mucilaginosa strains. Notably , R. paludigena LL69-1 exhibited the highest astaxanthin production, with an astaxanthin content and yield of 251.78 ± 0.27 μg/g DCW and 1.8632 ± 0.0023 mg/L, respectively, surpassing strain HL26-1, the highest producer of the R. mucilaginosa species, which yielded 104.98 ± 0.13 μg/g DCW of astaxanthin and 0.9280 ± 0.0012 mg/L of yield. These findings suggest that the ability to produce astaxanthin is species independent, as varying results have been obtained from diverse strains of the same species.

A comparison of the astaxanthin yield (mg/L) among the strains investigated in this study revealed that R. paludigena LL69-1 exhibited the highest astaxanthin production (1.86 mg/L) under the tested, non-optimized culture conditions. This yield was higher than those reported of other wild-type yeast strains cultivated under comparable or non-optimized conditions, such as Phaffia rhodozyma (0.2-0.4 mg/L) 59 and R. toruloides (0.93 mg/L), 60 indicating the promising potential of strain LL69-1 as a natural astaxanthin-producing yeast. However, the astaxanthin production of LL69-1 remains lower than that of many natural or genetically modified strains cultivated under optimized conditions. 61 , 62 For example, Phaffia rhodozyma 7B12 (originated from P. rhodozyma Past-1) produced 7.71 mg/L astaxanthin when cultivated in an optimal nitrogen medium consisting of 0.28 g/L (NH 4) 2SO 4, 0.49 g/L KNO 3, and 1.19 g/L beef extract. 61 The wild strain Xanthophyllomyces dendrorhous TISTR 5730 grown in mustard waste precipitated hydrolysate (MPH) under optimal conditions gave the astaxanthin yield of 25.8 mg/L, 62 while the X. dendrorhous strain DW6 produced 374.3 mg/L of astaxanthin using cane molasses in a two-stage pH fermentation system. 63 Similarly, higher astaxanthin yields have been reported for other Rhodotorula strains under optimized conditions, including Rhodotorula sp. CP72-2 (4.13 mg/L) 19 and Rhodotorula paludigena SP9-15 (6.67 mg/L). 15 These comparisons highlight that, although LL69-1 demonstrated the highest astaxanthin yield among the strains examined in this study under the tested conditions, further optimization of culture parameters is essential to enhance its production capacity. Moreover, additional genetic and metabolic information related to astaxanthin biosynthesis in R. paludigena and R. mucilaginosa is needed to support future strain improvement and process optimization.

The genomic information of two recently obtained astaxanthin-producing yeasts, R. paludigena LL69-1 and R. mucilaginosa HL26-1, is presented here. The genome sizes of R. paludigena LL69-1 and R. mucilaginosa HL26-1 were 18.78 Mbp with a GC content of 60.12% and 20.99 Mbp with a GC content of 63.82%, respectively. These findings align with the genome sizes of other pigmented yeast species such as R. paludigena SP9-15 (20.92 Mbp), 15 R. paludigena TL35-5 (20.98 Mbp), 16 R. sampaioana PL61-2, 16 R. glutinis ZHK (21.8 Mbp), 64 R. glutinis X-20 (21.85 Mbp), 65 R. toruloides VN1 (20.01 Mbp), 13 and R. mucilaginosa RIT389 (19.66 Mbp). 66 On the whole genome level, R. mucilaginosa HL26-1 has the highest average nucleotide identity of 99.75% to R. mucilaginosa strain JY1105. Similarly, the ANI of R. paludigena LL69-1 was greater at 99.58 than that of the nearest species, R. paludigena CM33. These high ANI values, typically ≥ 95%, underscore a strong correlation with other biological data, suggesting that the two yeasts likely belong to the same species. 67 Comparative genomic analysis revealed that the predicted number of protein-encoding genes of R. mucilaginosa HL26-1 is 5,711. In contrast, R. paludigena LL69-1 was predicted to contain approximately 6,782 coding genes. This result indicates that organisms of different species commonly possess varying quantities of protein-coding genes. In addition, critical genes involved in the terpenoid backbone and astaxanthin biosynthesis of these yeasts were analyzed based on functional genome annotation. All essential genes involved in terpenoid biosynthesis pathways were identified in the genomes of R. paludigena LL69-1 and strain HL26-1, except for the diphosphomevalonate decarboxylase gene ( DPMDC), which was not found in strain HL26-1. This error may have occurred during the genome sequencing procedure. An estimated 0.1–1% of processed bases will be sequenced incorrectly. 68

For the putative candidate astaxanthin synthesis-associated genes, the CrtYB (phytoene synthase/lycopene beta-cyclase), CrtI (phytoene desaturase), CrtS (beta-carotene 4-ketolase/3-hydroxylase), and CrtR (cytochrome P450) genes were identified and annotated in the genomes of both strains. The enzymes phytoene synthase/lycopene beta-cyclase and phytoene desaturase, encoded by CrtYB and CrtI, respectively, play important roles in the biosynthesis of beta-carotene, which is the precursor of astaxanthin synthesis. Rhodotorula species commonly produce β-carotene, torulene, and torularhodin at different ratios. 69 Additionally, we found that CrtY and CrtB were fused to strains HL26-1 and LL69-1 to form CrtYB. This corresponds to several CrtYBs found in various fungal species. 70, 71 These CrtYBs encode a protein with two functions: lycopene cyclase and phytoene synthase activities.

The CrtS gene encodes a specific astaxanthin synthase enzyme responsible for the ketolation and hydroxylation of β-carotene, facilitating the production of astaxanthin. 72 This enzymatic process is further augmented by the cytochrome P450 reductase enzyme CrtR. Originally identified in the pigmented yeast Xanthophyllomyces dendrorhous, 73 CrtS has since been identified in other pigmented yeasts, such as the genus Rhodotorula. 15, 16, 19 Our investigation specifically revealed the presence of all probable genes involved in astaxanthin biosynthesis in R. mucilaginosa HL26-1 and R. paludigena LL69-1. This finding provides compelling evidence for the capability of these strains to produce astaxanthin and offers valuable insights for future genetic engineering efforts aimed at enhancing astaxanthin synthesis. Furthermore, the yeasts HL26-1 and LL69-1 can utilize various carbon sources, including lignocellulosic sugars such as glucose, xylose, and arabinose (see Supplementary Table S2). Genome analysis also revealed that R. mucilaginosa HL26-1 and R. paludigena LL69-1 possess protein-coding genes involved in glucose, xylose, and arabinose utilization. Hence, these unconventional yeasts present great potential for producing astaxanthin, fatty acids, and other valuable products from low-cost sugars.

 Conclusion

In this study, various pigmented yeasts from the genus Rhodotorula, including Rhodotorula mucilaginosa and Rhodotorula paludigena, were isolated from flowers collected in Lampang Province. These yeasts demonstrate the ability to produce astaxanthin. Among these, R. mucilaginosa HL26-1 and R. paludigena LL69-1 exhibited the highest astaxanthin production among their respective species. Analysis of the draft genome sequences revealed the presence of several genes crucial for astaxanthin biosynthesis. These findings offer valuable insights for further advancements in the biotechnological and genomic applications of two promising astaxanthin-producing yeasts, R. mucilaginosa HL26-1 and R. paludigena LL69-1.

 Ethical approval

Not applicable.

 Data availability statement Underlying data

NCBI/GenBank: Whole-genome sequences of R. mucilaginosa HL26-1. GenBank accession numbers JAZBNE000000000; https://www.ncbi.nlm.nih.gov/nuccore/JAZBNE000000000.1, 74 Whole-genome sequences of R. paludigena LL69-1. GenBank accession numbers JAWJBI000000000; https://www.ncbi.nlm.nih.gov/nuccore/JAWJBI000000000.1. 75

Extended data

Figshare: Supplementary information on characterization of red-pigmented yeasts and genes associated with astaxanthin synthesis, https://doi.org/10.6084/m9.figshare.28953866.v3. 76

This project contains the following underlying data:

Supplymentary information_June4.pdf

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgments

We thank the Pharmaceutical Research Instrument Center, Faculty of Pharmaceutical Sciences, Chulalongkorn University, for providing research facilities.

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Biofuels. 2023;16(1):17. 36740699 10.1186/s13068-023-02270-x PMC9899373 Canto A Herrera CM Rodriguez R : Nectar-living yeasts of a tropical host plant community: diversity and effects on community-wide floral nectar traits. PeerJ. 2017;5:e3517. 28717591 10.7717/peerj.3517 PMC5511698 Khalaf AA Ahmed KN : Isolation and diagnosis of Rhodotorula yeast and evaluation of its effectiveness on the production of beta-carotene pigment. IOP Conf. Ser.: Earth Environ. Sci. 2025;1449(1): 012156. 10.1088/1755-1315/1449/1/012156 Myint KT Otsuka M Okubo A : Isolation and identification of flower yeasts for the development of mixed culture to produce single-cell protein from waste milk. Bioresour. Technol. Rep. 2020;10: 100401. 10.1016/j.biteb.2020.100401 Dymek I Żandarek J Starek M : Studies of TLC-chromatographic quantification of astaxanthin in dietary supplements and its antioxidant activity. Processes. 2024;12(8):1680. 10.3390/pr12081680 Minyuk GS Solovchenko AE : Express analysis of microalgal secondary carotenoids by TLC and UV-Vis spectroscopy. Barreiro C Barredo JL , editors. Microbial Carotenoids: Methods and Protocols. New Jersey: Humana;2018; pp.73–95. Du X Dong C Wang K : Separation and purification of astaxanthin from Phaffia rhodozyma by preparative high-speed counter-current chromatography. J. Chromatogr. B. 2016;1029-1030:191–197. 27433984 10.1016/j.jchromb.2016.06.042 Kothari D Lee JH Chon JW : Improved astaxanthin production by Xanthophyllomyces dendrorhous SK984 with oak leaf extract and inorganic phosphate supplementation. Food Sci. Biotechnol. 2019;28:1171–1176. 31275717 10.1007/s10068-019-00604-w PMC6595044 Luna-Flores CH Wang A Hellens J von : Towards commercial levels of astaxanthin production in Phaffia rhodozyma. Aust. J. 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Green Chem. 2024;26(8):4582–4592. 10.1039/D3GC04918C Li CJ Zhao D Cheng P : Genomics and lipidomics analysis of the biotechnologically important oleaginous red yeast Rhodotorula glutinis ZHK provides new insights into its lipid and carotenoid metabolism. BMC Genomics. 2020;21:816–834. 33243144 10.1186/s12864-020-07244-z PMC7690147 Bo S : Carotenoid biosynthesis: Genome-wide profiling, pathway identification in Rhodotorula glutinis X-20, and high-level production. Front. Nutr. 2022;9: 918240. 35782944 10.3389/fnut.2022.918240 PMC9247606 Gan HM Thomas BN Cavanaugh NT : Whole genome sequencing of Rhodotorula mucilaginosa isolated from the chewing stick ( Distemonanthus benthamianus): insights into Rhodotorula phylogeny, mitogenome dynamics and carotenoid biosynthesis. PeerJ. 2017;5:e4030. 29158974 10.7717/peerj.4030 PMC5691792 Lachance MA Lee DK Hsiang T : Delineating yeast species with genome average nucleotide identity: a calibration of ANI with haplontic, heterothallic Metschnikowia species. Antonie Van Leeuwenhoek. 2020;113(12):2097–2106. 33048250 10.1007/s10482-020-01480-9 Mitchell K Brito JJ Mandric I : Benchmarking of computational error-correction methods for next-generation sequencing data. Genome Biol. 2020;21:13–71. 32183840 10.1186/s13059-020-01988-3 PMC7079412 Kot AM Błażejak S Kurcz A : Rhodotorula glutinis—potential source of lipids, carotenoids, and enzymes for use in industries. Appl. Microbiol. Biotechnol. 2016;100:6103–6117. 27209039 10.1007/s00253-016-7611-8 PMC4916194 Arrach N Fernández-Martín R Cerdá-Olmedo E : A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces. Proc. Natl. Acad. Sci. 2001;98(4):1687–1692. 10.1073/pnas.98.4.1687 Ledetzky N Osawa A Iki K : Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous. Arch. Biochem. Biophys. 2014;545:141–147. 24486200 10.1016/j.abb.2014.01.014 Chi S He Y Ren J : Overexpression of a bifunctional enzyme, CrtS, enhances astaxanthin synthesis through two pathways in Phaffia rhodozyma. Microb. Cell Factories. 2015;14:12–90. 26081576 10.1186/s12934-015-0279-4 PMC4470029 Alcaı́no J Fuentealba M Cabrera R : Modeling the interfacial interactions between CrtS and CrtR from Xanthophyllomyces dendrorhous, a P450 system involved in astaxanthin production. J. Agric. Food Chem. 2012;60(35):8640–8647. 22897793 10.1021/jf302287f Hoondee P Tanasupawat S : Diversity and genomic analysis of Rhodotorula sp: Rhodotorula mucilaginosa strain HL26-1, whole genome shotgun sequencing project.Dataset. NCBI/GenBank. 2023. Reference Source Hoondee P Tanasupawat S : Diversity and genomic analysis of Rhodotorula sp: Rhodotorula paludigena strain LL69-1, whole genome shotgun sequencing project.Dataset. NCBI/GenBank. 2023. Reference Source Hoondee P Phuengjayaem S Kingkaew E : Supplementary information on characterization of red-pigmented yeasts and genes associated with astaxanthin synthesis.Dataset. figshare. 2025. 10.6084/m9.figshare.28953866.v3 10.5256/f1000research.194508.r446928 Reviewer response for version 2 Rao Kokati Venkata Bhaskara 1 Referee Vellore Institute of Technology, Vellore, India

Competing interests: No competing interests were disclosed.

 8 1 2026 Copyright: © 2026 Rao KVB 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

I have carefully evaluated the revised manuscript and find that the authors have satisfactorily addressed all the comments. The revisions have significantly improved the clarity, methodological rigor, and consistency of the manuscript. The introduction and Materials and Methods sections are now well structured and comprehensive, and issues related to astaxanthin quantification, genome statistics, and formatting have been appropriately resolved.

I am satisfied with the revisions and recommend the manuscript for indexing.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Microbiology, drug discovery, pharmacology, Biotechnology, Biodegradation

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.181137.r436643 Reviewer response for version 1 Rao Kokati Venkata Bhaskara 1 Referee Vellore Institute of Technology, Vellore, India

Competing interests: No competing interests were disclosed.

 29 12 2025 Copyright: © 2025 Rao KVB 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

This study presents a well-executed investigation into the astaxanthin production capability of novel Rhodotorula strains isolated from flowers and provides valuable draft whole-genome sequences and gene annotations for the two highest-producing strains. The multi-step approach for astaxanthin confirmation (TLC, spectrophotometry, and HPLC) is scientifically sound and robust. The new genomic data and the finding that these yeasts can utilize lignocellulosic sugars are a significant contribution to the field of industrial microbiology and biotechnological astaxanthin production.

1.Is the work clearly and accurately presented and does it cite the current literature? Yes

2.Is the study design appropriate and is the work technically sound? Yes

3. Are sufficient details of methods and analysis provided to allow replication by others? Yes

4. If applicable, is the statistical analysis and its interpretation appropriate? Partly needs revision on adding statistical method used and p values has to be given to ensure reliability of the data

5. Are all the source data underlying the results available to ensure full reproducibility? Yes

6.Are the conclusions drawn adequately supported by the results? yes   

Comments: Major Revision Required

The work is relevant for biotechnology, provides useful genomic insights, and adds value to the limited genomic data available for Rhodotorula paludigena and R. mucilaginosa. The manuscript is generally well-structured,however, several methodological and interpretative issues must be addressed before acceptance.

1.Inconsistent Genome Assembly Statistics (Critical Error): There is a serious and confusing inconsistency in the reporting of the draft genome sizes for the two selected strains. This must be corrected to maintain the authenticity of the data. Abstract states that the R. mucilaginosa HL26-1 genome is 18.78 Mbp, and the R. paludigena LL69-1 genome is 20.99 Mbp. The results section states that R. mucilaginosa HL26-1 assembly resulted in a genome size of 20.99 Mbp, and R. paludigena LL69-1 had a genome size of 18.78 Mbp. similar controversy in table 1.

2.Astaxanthin Yield Comparison: The discussion rightly notes that the high astaxanthin yield of R. paludigena LL69-1 (1.186mg/L) is superior to that of "other microorganisms". However, the paper later cites Rhodotorula sp. CP72-2 (4.13mg/L) and R. paludigena SP9-15 (6.67mg/L) ephrase the statement on superiority to be more precise, perhaps by comparing it only to unoptimized strains or stating it is the highest yield in this study under the tested conditions, to avoid contradicting the later discussion. The final conclusion that optimizing conditions is necessary is appropriate.

3.Clarity on Strain Selection: In the Abstract, it is stated that HL26-1 had the greatest content and yield among R. mucilaginosa strains, and LL69-1 had the greatest content and yield among R. paludigena strains. While clear, you may consider clarifying in the Methods why only two strains ( R. mucilaginosa HL26-1 and R. paludigena LL69-1) were chosen for the full whole-genome sequencing and analysis (presumably because they were the best producers of their respective species).

4.Distinction between Carotenoids and Astaxanthin: The paper must consistently and clearly differentiate between Total Carotenoids and Astaxanthin. In the Results and Discussion, ensure that when values are presented, it is clear whether they refer to the total pool of pigments (carotenes, torulene, etc.) or specifically to the astaxanthin fraction, which was isolated and quantified by HPLC. The difference is vital for biotechnological assessment.

5.HPLC: In the methods section, add chromatogram specifications, wavelength, flow rate, and injection volume.

6.TLC: TLC Rf value identical for all strains is suspicious—discuss variability .

7.Astaxanthin standard: Specify manufacturer purity in Methods .

8.Statistical Analysis Missing: Spectrophotometric data show mean ± SD, but no statistical tests are described. Mention statistical methods used to compare strains and add p-values where necessary.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Microbiology, drug discovery, pharmacology, Biotechnology, Biodegradation

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Tanasupawat Somboon Biochemistry and Microbiology, Chulalongkorn University, Bangkok, Thailand

Competing interests: No competing interests were disclosed.

 30 12 2025

Dear reviewer,

We sincerely thank the reviewer for these valuable comments. The suggestions provided have been carefully considered and will be incorporated into the revised version of the manuscript. A detailed point-by-point response addressing each of the reviewer’s comments is provided below.

1.Inconsistent Genome Assembly Statistics (Critical Error): There is a serious and confusing inconsistency in the reporting of the draft genome sizes for the two selected strains. This must be corrected to maintain the authenticity of the data. The abstract states that the R. mucilaginosa HL26-1 genome is 18.78 Mbp, and the R. paludigena LL69-1 genome is 20.99 Mbp. The results section states that R. mucilaginosa HL26-1 assembly resulted in a genome size of 20.99 Mbp, and R. paludigena LL69-1 had a genome size of 18.78 Mbp. similar controversy in table 1.

Answer: Thank you very much for pointing out this critical inconsistency. We sincerely apologize for this major error in reporting the genome assembly data, which occurred during manuscript preparation. We have now carefully reviewed the data and corrected the genome sizes to ensure consistency across the Abstract, Results section, and Table 1. We are deeply grateful for your careful reading and for bringing this mistake to our attention, which has significantly improved the accuracy and reliability of the manuscript.

2.Astaxanthin Yield Comparison: The discussion rightly notes that the high astaxanthin yield of R. paludigena LL69-1 (1.186mg/L) is superior to that of "other microorganisms". However, the paper later cites Rhodotorula sp. CP72-2 (4.13mg/L) and R. paludigena SP9-15 (6.67mg/L) rephrase the statement on superiority to be more precise, perhaps by comparing it only to unoptimized strains or stating it is the highest yield in this study under the tested conditions, to avoid contradicting the later discussion. The final conclusion that optimizing conditions is necessary is appropriate.

Answer: Thank you for your comment. We have revised the Discussion to clarify that R. paludigena LL69-1 showed the highest astaxanthin yield among the strains examined in this study under non-optimized conditions, rather than claiming overall superiority over other microorganisms. The text has been rephrased to clearly distinguish our results from reports of higher yields obtained under optimized or genetically modified conditions.

3.Clarity on Strain Selection: In the Abstract, it is stated that HL26-1 had the greatest content and yield among R. mucilaginosa strains, and LL69-1 had the greatest content and yield among R. paludigena strains. While clear, you may consider clarifying in the Methods why only two strains ( R. mucilaginosa HL26-1 and R. paludigena LL69-1) were chosen for the full whole-genome sequencing and analysis (presumably because they were the best producers of their respective species).

Answer: We thank the reviewer for this valuable comment. The criteria employed for selecting the strain for subsequent investigation have been described in the materials and methods section.

4.Distinction between Carotenoids and Astaxanthin: The paper must consistently and clearly differentiate between Total Carotenoids and Astaxanthin. In the Results and Discussion, ensure that when values are presented, it is clear whether they refer to the total pool of pigments (carotenes, torulene, etc.) or specifically to the astaxanthin fraction, which was isolated and quantified by HPLC. The difference is vital for biotechnological assessment.

Answer: We thank the reviewer for this important comment, and we revised as your suggestion. We would like to clarify that astaxanthin was quantified spectrophotometrically at a specific wavelength selected to minimize interference from other carotenoids, following the method reported by Li et al. (2012). In that study, absorbance at 530 nm showed a strong linear correlation with astaxanthin content.

Accordingly, in this study, spectrophotometric measurements performed at the specified wavelength were used for astaxanthin quantification rather than total carotenoid determination. In addition, astaxanthin was independently identified and confirmed by HPLC based on retention time comparison and spiking experiments. We have revised the Results and Discussion sections to more clearly distinguish between total carotenoids and astaxanthin content wherever values are reported.

Reference:

       - Li, Y., Miao, F., Geng, Y., Lu, D., Zhang, C., & Zeng, M. (2012). Accurate quantification of astaxanthin from Haematococcus crude extract spectrophotometrically. Chinese Journal of Oceanology and Limnology, 30(4), 627-637.

5.HPLC: In the methods section, add chromatogram specifications, wavelength, flow rate, and injection volume.

Answer: We thank the reviewer for this comment. Detailed HPLC chromatographic conditions, including column specifications, detection wavelength, flow rate, and injection volume, have now been added to the Methods section.

6.TLC: TLC Rf value identical for all strains is suspicious—discuss variability.

Answer: We thank the reviewer for this insightful comment. TLC was employed as a qualitative screening tool to indicate the presence of astaxanthin rather than for quantitative comparison. Although minor experimental variability in Rf values can occur due to factors such as solvent composition, plate condition, and development distance, the astaxanthin bands from all strains migrated within the same narrow R f range and corresponded to the astaxanthin standard under identical TLC conditions. For clarity, the manuscript has been revised to emphasize that TLC was used for qualitative identification, spectrophotometric analysis was used for astaxanthin quantification, and HPLC was employed to confirm the identity of astaxanthin based on retention time comparison and spiking experiments.

7.Astaxanthin standard: Specify manufacturer purity in Methods.

Answer: Specify manufacturer purity was added in the Methods section as your suggestion.

8.Statistical Analysis Missing: Spectrophotometric data show mean ± SD, but no statistical tests are described. Mention statistical methods used to compare strains and add p-values where necessary.

Answer: We thank the reviewer for this valuable comment. Statistical analysis using one-way ANOVA with a significance level set at p < 0.05 was conducted and mentioned in the Methods section and Fig. 2 description.

Sincerely,

The Authors

 10.5256/f1000research.181137.r434447 Reviewer response for version 1 NANJUNDASWAMY ANANDA 1 Referee South Dakota State University, Brookings, South Dakota, USA

Competing interests: No competing interests were disclosed.

 24 12 2025 Copyright: © 2025 NANJUNDASWAMY A 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

This is an excellent and timely study. With the growing global emphasis on developing natural sources of carotenoids, yeast-based production systems remain highly promising. The authors have evaluated strains of Rhodotorula spp. for the expression of astaxanthin biosynthetic genes, and the molecular tools employed are appropriate for the study. The biochemical analyses used to confirm astaxanthin production—such as TLC and HPLC—are well-suited and strengthen the conclusions.

However, a few minor but important details need to be addressed before publication. The manuscript should clearly specify the type of statistical analyses used to evaluate the biochemical data. Additionally, the number of replicates used in the fermentation experiments must be provided. As currently presented, it appears that only a single replicate was used, which is not statistically acceptable. If biological or technical replicates were indeed included, please ensure that this information is explicitly stated.

Overall, the manuscript contains valuable scientific information and, with these clarifications, would be suitable for publication.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Carotenoids, Bioprocessing, Pigmented yeasts, genetic engineering

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Tanasupawat Somboon Biochemistry and Microbiology, Chulalongkorn University, Bangkok, Thailand

Competing interests: No competing interests were disclosed.

 30 12 2025

Dear reviewer,

We sincerely thank the reviewer for these valuable comments. The suggestions provided have been carefully considered and will be incorporated into the revised version of the manuscript.

We have revised the manuscript to clearly specify the statistical analyses and experimental details used for evaluating the biochemical data. Astaxanthin content was quantified spectrophotometrically by measuring absorbance at the characteristic wavelength of astaxanthin (λ = 530 nm) using a calibration curve constructed from standard astaxanthin. The astaxanthin content and yield are now expressed as µg/g DCW and mg/L, respectively.

We have also clarified the number of replicates used in the fermentation experiments. All experiments were performed in triplicate, and the results are reported as mean ± standard deviation. Statistical analysis was conducted using one-way ANOVA, with the significance level set at p < 0.05. These details have been explicitly added to the Materials and Methods section.

Sincerely,

The Authors

 10.5256/f1000research.181137.r436642 Reviewer response for version 1 Nimsi Kizhakkeyveetil Abdul Saleem 1 Referee https://orcid.org/0000-0002-0347-0716 BIOSCIENCES, MES College Marampally (Ringgold ID: 210878), Aluva, Kerala, India

Competing interests: No competing interests were disclosed.

 23 12 2025 Copyright: © 2025 Nimsi KAS 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Comments to the editor

The study has good potential and demonstrates substantial effort, but several important weaknesses need to be addressed before it can be considered for Indexing. Although the introduction is scientifically rich and relevant, it requires better organization, clearer presentation of the research gap, and improved readability to help readers fully grasp the study’s significance.

The manuscript is structured into four sections, beginning with the isolation of pigmented yeasts; however, the source of samples, details of sample collection, and specific isolation methods are not described. In the screening phase, the procedures for purification steps, statistical validation, and criteria for significance, are absent. While the study focuses on two yeast strains ( Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1, the basis for selecting these strains is not explained in the Materials and Methods section.

Overall, the Methods section is too brief, lacking essential details such as experimental design, number of replicates, appropriate controls, and how measurements were collected. Additional concerns include inconsistent use of units, typographical and formatting errors, repeated media compositions, and inconsistent use of abbreviations. The term “clonality” is inappropriately used in the preliminary selection stage and should be revised.

Addressing these issues will significantly strengthen the manuscript by improving transparency, reproducibility, and scientific clarity.

Comment 1: In the introduction author shall also discuss briefly about the pigmented yeasts in flower, their role, and importance.

Comment 2: Author shall also write about the genes of pigmented yeast discovered so far and their importance.

Comment 3: Media composition can write in bracket. Also, it is repeating in text.

Comment 4: go through the manuscript, avoid typological error (e.g. Axs, Ø).

Comment 5: uniform units throughout the manuscript (e.g. H, hour)

Comment 6: sample collection, and processing are missing from materials and methods.

Comment 7: Quantify the pigment and statistically validate the result.

Comment 7: how can you identify the pigment is pure?

Comment 8: is only one pigment is identified by TLC?

Comment 9: describe the mobile phase in TLC.

Comment10: how the astaxanthin purify for HPLC?

comment: 11: include more reference after 2020. regarding pigmented yeasts from flowers.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Diversity yeasts, pigments, plant microbe interaction, metagenomics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Tanasupawat Somboon Biochemistry and Microbiology, Chulalongkorn University, Bangkok, Thailand

Competing interests: No competing interests were disclosed.

 30 12 2025

Dear reviewer

We sincerely thank the reviewer for these valuable comments. The suggestions provided have been carefully considered and will be incorporated into the revised version of the manuscript.

The introduction has been revised to improve organization, readability, and logical flow. The research gap and the significance of the study are now stated more clearly to highlight the novelty of the work.

The Materials and Methods section has been greatly expanded. The source and details of sample collection were described. The specific method for yeast isolation was added. In addition, the measurement methods, and data analysis procedures have been clarified to enhance transparency and reproducibility. Inconsistencies in units, typographical and formatting errors, repeated media descriptions, and inconsistent use of abbreviations have been corrected. The inappropriate use of the term “clonality” has been revised.

A detailed point-by-point response addressing each of the reviewer’s comments is provided below.

Comment 1: In the introduction author shall also discuss briefly about the pigmented yeasts in flower, their role, and importance.

Answer: We thank the reviewer for this comment. A brief background about role and importance of the pigmented yeasts in flowers has been described in the last paragraph of the introduction part.

Comment 2: Author shall also write about the genes of pigmented yeast discovered so far and their importance.

Answer: We thank the reviewer for this comment. We would like to clarify that we have indeed discussed in the introduction part about the genes associated with pigmented yeasts, particularly those in the genus Phaffia, highlighting their significance mechanism in astaxanthin synthesis. However, concerning the genes of pigmented yeasts in the genus Rhodotorula, we regret to inform you that we cannot provide a detailed report on their expression at this time. Currently, there is a lack of clear studies documenting the expression of these genes in Rhodotorula species. This limitation has prevented us from including specific information in our manuscript.

Comment 3: Media composition can write in bracket. Also, it is repeating in text.

Answer: Media composition was written in bracket and the repeating text was edited.

Comment 4: go through the manuscript, avoid typological error (e.g. Axs, Ø).

Answer: The typographical errors were edited.

Comment 5: uniform units throughout the manuscript (e.g. H, hour)

Answer: The unit “h” throughout the manuscript was edited to be hours.

Comment 6: sample collection, and processing are missing from materials and methods.

Answer: The specifics of sample collection and processing were described.

Comment 7: Quantify the pigment and statistically validate the result.

Answer: The pigment content was quantified spectrophotometrically by measuring absorbance at the characteristic wavelength of astaxanthin (λ = 530 nm) using a calibration curve constructed from standard astaxanthin. The pigment concentration was expressed as mg/L (or µg/g DCW). All experiments were performed in triplicate, and results were reported as mean ± standard deviation. Statistical analysis was conducted using one-way ANOVA with a significance level set at p < 0.05.

Comment 7: how can you identify the pigment is pure?

Answer: We thank the reviewer for this valuable comment. We would like to clarify that the pigment was not subjected to additional purification prior to analysis. Astaxanthin was identified by HPLC based on comparison of its retention time with that of an authentic astaxanthin standard. In addition, a spiking experiment was performed by adding the astaxanthin standard to the sample extract, which resulted in a proportional increase in the target peak without the appearance of additional peaks. The HPLC chromatograms of the standard, sample, and spiked sample are provided in Supplementary Figure 1, which together confirm the identity of astaxanthin in the extract.

Comment 8: is only one pigment is identified by TLC?

Answer: We thank the reviewer for this important comment. TLC analysis revealed the presence of multiple pigment bands, indicating the existence of several carotenoid components in the extract. However, astaxanthin was identified based on the comparison of its R f value with that of the astaxanthin standard analyzed under the same chromatographic conditions. The band corresponding to astaxanthin showed a matching R f value and similar coloration to the standard, confirming its identity.

Comment 9: describe the mobile phase in TLC.

Answer: We thank the reviewer for this comment. The mobile phase used for TLC analysis was a mixture of acetone and hexane at a ratio of 1:3 (v/v). This solvent system enabled effective separation of carotenoid pigments, and astaxanthin was identified by comparing the R f value and coloration of the sample band with those of the astaxanthin standard analyzed under identical conditions. The description of the TLC mobile phase has been added to the Methods section.

Comment10: how the astaxanthin purify for HPLC?

Answer: We thank the reviewer for this comment. We would like to clarify that astaxanthin was not subjected to a separate purification step prior to HPLC analysis. Instead, astaxanthin was extracted from yeast cells using DMSO, followed by centrifugation and filtration to remove cell debris. The clarified extract was directly analyzed by HPLC. Astaxanthin was identified based on comparison of its retention time with an authentic astaxanthin standard, and further confirmed by spiking experiments. This clarification has been added to the revised manuscript.

comment: 11: include more reference after 2020. regarding pigmented yeasts from flowers.

Answer: An update report on pigmented yeast isolated from flowers was presented as your suggestion.

Sincerely,

The Authors