Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1

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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 (275.94 ± 0.16 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. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/14-717/v1", "name": "Characterization of red pigmented yeasts and genes associated with..." } } ] } Home Browse Characterization of red pigmented yeasts and genes associated with... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Hoondee P, Tedsree N, Phuengjayaem S et al. Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.12688/f1000research.164600.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] Patcharaporn Hoondee 1,2 , Nisachon Tedsree 3 , Sukanya Phuengjayaem 4 , [...] Engkarat Kingkaew 5 , Boonchoo Sritularak 6,7 , Pornchai Rojsitthisak 6,8 , Takuji Nakashima 9,10 , Worathat Thitikornpong 6,8 , Somboon Tanasupawat https://orcid.org/0000-0002-7149-5341 6,11 Patcharaporn Hoondee 1,2 , Nisachon Tedsree 3 , [...] Sukanya Phuengjayaem 4 , Engkarat Kingkaew 5 , Boonchoo Sritularak 6,7 , Pornchai Rojsitthisak 6,8 , Takuji Nakashima 9,10 , Worathat Thitikornpong 6,8 , Somboon Tanasupawat https://orcid.org/0000-0002-7149-5341 6,11 PUBLISHED 21 Jul 2025 Author details Author details 1 Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology Krungthep, Sathon, Bangkok, 10120, Thailand 2 Biodiversity and Sustainable Utilization Research Unit, Rajamangala University of Technology Krungthep, Sathon, Bangkok, 10120, Thailand 3 Faculty of Science and Arts, Chanthaburi Campus, Burapha University, Tha Mai, Chanthaburi, 22170, Thailand 4 Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Thung Khru, Bangkok, 10140, Thailand 5 Department of Biology, School of Science, King Mongkut's Institute of Technology Ladkrabang, Lat Krabang, Bangkok, 10520, Thailand 6 Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand 7 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand 8 Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand 9 Department of Field Sciences, University of Human Environments, Mutsuyama, Ehime, 790-0825, Japan 10 Research Organization for Nano and Life Innovation, Waseda University, Shinjuku, Tokyo, 162-0041, Japan 11 Department of Biochemistry and Microbiology, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand Patcharaporn Hoondee Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Nisachon Tedsree Roles: Formal Analysis, Methodology Sukanya Phuengjayaem Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation Engkarat Kingkaew Roles: Conceptualization, Formal Analysis, Methodology Boonchoo Sritularak Roles: Formal Analysis, Funding Acquisition, Writing – Review & Editing Pornchai Rojsitthisak Roles: Formal Analysis, Funding Acquisition, Writing – Review & Editing Takuji Nakashima Roles: Writing – Review & Editing Worathat Thitikornpong Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Somboon Tanasupawat Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Genomics and Genetics gateway. This article is included in the From genes to genomes: Investigating the population species boundary in non-model Fungi collection. Abstract 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 (275.94 ± 0.16 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. READ ALL READ LESS Keywords astaxanthin, pigmented yeast, Rhodotorula mucilaginosa, Rhodotorula paludigena, astaxanthin synthase Corresponding Author(s) Worathat Thitikornpong ( [email protected] ) Somboon Tanasupawat ( [email protected] ) Close Corresponding authors: Worathat Thitikornpong, Somboon Tanasupawat Competing interests: No competing interests were disclosed. Grant information: 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. Copyright: © 2025 Hoondee P et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Hoondee P, Tedsree N, Phuengjayaem S et al. Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.12688/f1000research.164600.1 ) First published: 21 Jul 2025, 14 :717 ( https://doi.org/10.12688/f1000research.164600.1 ) Latest published: 02 Jan 2026, 14 :717 ( https://doi.org/10.12688/f1000research.164600.2 )  There is a newer version of this article available. Suppress this message for one day. Introduction Astaxanthin (3,3′-dihydroxy-β, β′-carotene-4,4′-dione), a red compound belonging to the xanthophyll carotenoid group, exhibits formidable antioxidant capabilities, surpassing those of vitamin E (alpha-tocopherol), β-carotene, canthaxanthin, and other natural carotenoids by a factor of 100. 1 Astaxanthin is widely used in various industries, including beauty, food, animal feed, health supplements, and pharmaceuticals. 2 Astaxanthin serves as a potent metabolic regulator of glucose and lipid homeostasis. Their functions include increased insulin sensitivity, augmented glucose uptake, enhanced lipid turnover, and diminished lipid synthesis in the liver. Moreover, astaxanthin has garnered substantial attention in dermatology because of its ability to counteract oxidative stress, facilitate cellular rejuvenation, repair DNA damage, and protect against UV-induced photoaging and skin malignancies. 3 , 4 Furthermore, astaxanthin shows its anticancer properties, attributable to its ability to scavenge free radicals and quell singlet oxygen. 5 The acquisition of astaxanthin and other pigments involves two primary methods: synthetic production utilizing chemicals and natural extraction. 6 Despite its cost-effectiveness compared to natural sources, chemical pigment manufacturing can generate hazardous byproducts and pose environmental risks. 7 Natural astaxanthin has been identified in diverse organisms, including shrimp, lobsters, fish, crustaceans, salmon, trout, red sea breams, and microbes. 8 Among microorganisms, Haematococcus pluvialis has stood out for decades because of its superior bioactivity compared to that of synthetically produced astaxanthin. However, challenges in both upstream and downstream processes have made the production of algal astaxanthin economically and logistically challenging. 2 Therefore, several studies focused on evaluating the natural astaxanthin-producing ability of other potential microorganisms, particularly the pigmented yeast, in order to realize commercial astaxanthin production. Compared to microalgae, yeast cultivation can be cultured in bioreactors, utilize various carbon sources from lignocellulosic biomass and industrial by-products, and reduce land management and environmental concerns. 6 , 9 – 11 Phaffia rhodozyma ( Xanthophyllomyces dendrorhous ) is the rare yeast species with current biotechnological use in the production of astaxanthin for several decades. 6 , 12 Recently, it has been shown that some Rhodotorula species, which were previously known for their ability to synthesize beta-carotene, might be used as an alternative for astaxanthin such as Rhodotorula toruloides, 13 , 14 Rhodotorula paludigena, 15 , 16 Rhodotorula mucilaginosa, 17 and Rhodotorula sp. 18 The pathway of astaxanthin biosynthetic in Phaffia rhodozyma has been reported. 19 – 21 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). 19 – 21 Finally, a single gene called CrtS , which could act as both a ketolase and a hydroxylase, was responsible for converting β-carotene into astaxanthin. 20 , 21 Nevertheless, the astaxanthin biosynthesis pathway in Rhodotorula species remain limited. Unfortunately, the lack of genomic information on astaxanthin-producing within these genera hampers the discovery of biosynthetic pathways and their molecular characterization and gene editing applications. 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 , 16 , 18 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. 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.). The samples were carefully transported to the laboratory in sterile plastic bags with ice packs to maintain integrity during transit. 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. Each sample was inoculated into 15 mL of yeast malt (YM) medium containing 10 g/L glucose, 5 g/L peptone, 3 g/L yeast extract, and 3 g/L malt extract at pH 5.5. The inoculated media were incubated at 25°C for 3 days. Subsequently, the resulting cultures were streaked onto YM agar medium containing 20 g/L agar, 10 g/L glucose, 5 g/L peptone, 3 g/L yeast extract, and 3 g/L malt extract (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 for clonality on YM agar media using the streak plate method. Each clonal culture was carefully preserved on a YM agar slant at 4°C to facilitate further analysis and experimentation. Carbon assimilation of strain was performed using the API ® ID 32 C kit (BioMérieux, France) according to the manufacturer’s instructions. Reactions were visually examined at 72 h, 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 22 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 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′). 23 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, 24 and any gaps were removed. MEGA11 software 25 was used to construct a neighbor-joining (NJ) tree using Kimura’s two-parameter model. 26 The reliability of the branches was assessed using a bootstrap test with 1000 replicates. 27 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. 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. 28 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 min. The supernatant was collected by centrifugation at 12,300 × g, for 10 minutes. Subsequently, a 20 μL aliquot of the extracted sample was applied to a TLC silica gel G plate using a capillary along with standard astaxanthin (DYCN ® , Dayang Chem Co. Ltd., China). A mixture of acetone (RCI Labscan ® , Ireland, Cat. No. AR1003) and hexane (RCI Labscan ® , Ireland, Cat. No. AR1085) was used as the mobile phase for spot development at a ratio of 1:3 (v/v). The colored bands were directly observed after development and compared with the standard astaxanthin band under visible light. In addition, the retention factor (R f ) value was calculated to quantify the movement of the materials along the plate. Quantification of astaxanthin production The extraction and measurement of astaxanthin were conducted using previously described methods with slight modifications. 29 To extract the intracellular carotenoid content, 50 mg of lyophilized cells were suspended in 5 mL of DMSO (Sigma–Aldrich ® , USA, Cat. No. 34869), ultrasonicated at 37 kHz and 50°C for 30 min (Elmasonic, E60H model, Germany). The resulting cell extracts were centrifuged at 12,300 × g, for 5 minutes. The extraction process was repeated until the supernatant became colorless. The astaxanthin concentration was determined using a Cary 60 UV–Vis spectrophotometer (Agilent) at 530 nm against a pure DMSO blank. 29 , 30 A standard curve of absorbance versus astaxanthin concentration was generated using the following concentrations: 0, 0.25, 0.5, 1, 2, 4, 6, and 8 μg/mL in DMSO. Astaxanthin concentrations were calculated using the standard calibration curve of AXs. The results are presented as the mean of triplicate measurements. Confirmative analysis of astaxanthin synthesis The astaxanthin was extracted and analyzed based on the described method. 15 In brief, 0.05 g of dried yeast cell and 5 mL of DMSO (Sigma–Aldrich ® , USA, Cat. No. 34869) were mixed and sonicated at 55°C, 5 min with 37 kHz of sonication (Elmasonic E60H, Germany). After centrifugation at 6,500 × g for 10 min, the resultant supernatant was filtered (0.22 μm) and then was transferred to chromatography column. Astaxanthin were analyzed by high performance liquid chromatography (HPLC) using UHPLC Nexera X2 system (Shimadzu, Japan) consisting of LC-30AD binary pump with a CTO-20AC column oven, an SPD-M30A detector and an SIL-30AC autosampler. Astaxanthin were separated using C18 column (GL Science InterSustain, 4.6 mm × 150 mm, 5 μm) using mobile phases consisting of mixture 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) and methanol. Analytical-grade astaxanthin was purchased from Dayang Chem Co. Ltd. (China) and utilized as a reference standard. 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. 31 , 32 Subsequently, the contigs were assembled into scaffolds using SSPACE (version 3.0), 33 and the gaps were filled using GapFiller (versions 1–10). 34 Gene prediction was performed using Augustus version 3.3. 35 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) 36 and KofamKOALA tools, employing default settings and considering all hits ( https://www.genome.jp/kegg/ ). 37 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. 38 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 in order to conduct a qualitative examination of their astaxanthin synthesis by using the thin-layer chromatography (TLC) technique. Astaxanthin, which is red, requires no additional processing for spot visualization. Compared with that of the astaxanthin standard, an astaxanthin band was observed in all extracts, with a R f value of 0.28. The experimental results demonstrated the presence of an astaxanthin band in all tested yeast strains ( Figure 1 ). All yeast strains were selected for further quantitative analysis of astaxanthin content. Figure 1. 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 29 coupled with sound energy via an ultrasonic bath to enhance cell disruption efficiency. 39 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 254.78 ± 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 254.78 ± 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. Figure 2. Quantitative analysis of astaxanthin production by 3 R. mucilaginosa strains (AL44-2, TL35-2, and HL26-1) and 9 R. paludigena strains (TL35-5, TL35-4, TL35-6, HL55, HL55-1, HL55-2, IL45-2-1, IL45-4, and LL69-1). The results are reported as the average value ± standard deviation (SD) calculated from three repeated measurements. 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 displayed astaxanthin peaks from strains HL26-1 and LL69-1, along with a 10 ppm astaxanthin standard. The retention time of the standard peak was used as a reference, confirming the presence of astaxanthin in the strain HL26-1 and LL69-1 with accuracy. The highest points within the time intervals during which HL26-1 and LL69-1 were retained were observed at 2.061 and 2.211 minutes, respectively. These findings provided conclusive evidence that both strains, HL26-1 and LL69-1, effectively synthesized astaxanthin (Supplementary Figure S1). General features of the R. paludigena LL69-1 and R. mucilaginosa 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 154 scaffolds, with the giant scaffold spanning 1,230,191 base pairs (bp). The N50 length of the scaffolds was 476,521 bp, with a GC content of 63.82% and a genome size of 20.99 Mbp. Conversely, R. paludigena LL69-1 had a genome size of 18.78 Mbp, with a GC content of 60.12%. Its assembly comprised 107 scaffolds with an N50 value of 254,092 bp and an L50 value of 24 scaffolds. The largest scaffold in LL69-1 was 853,611 bp, whereas the shortest was 329 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. Table 1. Assembly statistics of the R. paludigena HL26-1 and R. mucilaginosa 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 Figure 3. Circular genomic map of R. mucilaginosa HL26-1 and R. paludigena 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. Figure 4. Venn diagram displaying the presence of conserved and specific proteins among Rhodotorula mucilaginosa HL26-1 (a) or R. paludigena LL69-1 (b) and R. kratochvilovae CBS 7436, R. mucilaginosa GDMCC2.30, and R. paludigena 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. Table 2. Putative gene families associated with astaxanthin biosynthesis in R. mucilaginosa HL26-1 and R. paludigena 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. 40 – 43 Recently, some Rhodotorula species have exhibited unique abilities to naturally generate astaxanthin, a red pigment with excellent antioxidant activity. These species include Rhodotorula paludigena SP9-15, 15 R. paludigena TL35-5, 16 Rhodotorula sampaioana PL61-2, 16 Rhodotorula toruloides VN1, 13 , 14 and Rhodotorula sp. CP72-2. 18 Several Rhodotorula species has 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, such as air, soil, lake water, seawater, plants, decomposing plant matter, and various food products and fruit juices. 44 – 49 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. 50 – 52 For the specific quantification of astaxanthin, spectrophotometry has been used. 29 , 30 , 53 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 254.78 ± 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 254.78 ± 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 our strains, particularly R. paludigena LL69-1, which produced the highest astaxanthin yield (1.86 mg/L, compared with that of other microorganisms), revealed the superior performance of LL69-1. Its astaxanthin yield surpassed that of other wild yeast strains, such as Phaffia rhodozyma , which produces 0.2-0.4 mg/L 54 and R. toruloides , producing 0.93 mg/L. 55 However, the astaxanthin production of LL69-1 remains lower than that of many genetically modified strains grown under optimized conditions. 56 , 57 Likewise, the astaxanthin yields generated by LL 69-1 were significantly lower compared to those produced by the other natural yeast strain and modified strain under optimal conditions. The highest astaxanthin yield of Phaffia rhodozyma 7B12 (originated from P. rhodozyma Past-1) was 7.71 mg/L when cultivated in optimal nitrogen medium consisting of 0.28 g/L (NH 4 ) 2 SO 4 , 0.49 g/L KNO 3 , and 1.19 g/L beef extract. 56 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. 57 The X. dendrorhous strain DW6 produced 374.3 mg/L of astaxanthin, which is the highest amount produced from cane molasses using a two-stage pH method. 58 Under optimal conditions, Rhodotorula sp. CP72-2 had the highest astaxanthin yield of 4.13 mg/L. 18 Meanwhile, the highest yield of Rhodotorula paludigena SP9-15 astaxanthin grown in optimized medium and environmental factors was 6.67 mg/L. 15 Hence, optimizing culture conditions is crucial for enhancing astaxanthin production. Additionally, there is a need for more valuable genetic data and information on the metabolic pathways associated with astaxanthin synthesis in R. paludigena and R. mucilaginosa. 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), 59 R. glutinis X-20 (21.85 Mbp), 60 R. toruloides VN1 (20.01 Mbp), 13 and R. mucilaginosa RIT389 (19.66 Mbp). 47 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. 61 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. 62 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. 63 Additionally, we found that CrtY and CrtB were fused to strains HL26-1 and LL69-1 to form CrtYB. This corresponds to several CrtYB s found in various fungal species. 64 , 65 These CrtYB s 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. 66 This enzymatic process is further augmented by the cytochrome P450 reductase enzyme CrtR. Originally identified in the pigmented yeast Xanthophyllomyces dendrorhous, 67 CrtS has since been identified in other pigmented yeasts, such as the genus Rhodotorula. 15 , 16 , 18 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 , 68 Whole-genome sequences of R. paludigena LL69-1. GenBank accession numbers JAWJBI000000000; https://www.ncbi.nlm.nih.gov/nuccore/JAWJBI000000000.1 . 69 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 . 70 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. References 1. Park SY, Binkley RM, Kim WJ, et al. : Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity. Metab. Eng. 2018; 49 : 105–115. PubMed Abstract | Publisher Full Text 2. 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Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 21 Jul 2025 ADD YOUR COMMENT Comment Author details Author details 1 Division of Biology, Faculty of Science and Technology, Rajamangala University of Technology Krungthep, Sathon, Bangkok, 10120, Thailand 2 Biodiversity and Sustainable Utilization Research Unit, Rajamangala University of Technology Krungthep, Sathon, Bangkok, 10120, Thailand 3 Faculty of Science and Arts, Chanthaburi Campus, Burapha University, Tha Mai, Chanthaburi, 22170, Thailand 4 Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Thung Khru, Bangkok, 10140, Thailand 5 Department of Biology, School of Science, King Mongkut's Institute of Technology Ladkrabang, Lat Krabang, Bangkok, 10520, Thailand 6 Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand 7 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand 8 Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand 9 Department of Field Sciences, University of Human Environments, Mutsuyama, Ehime, 790-0825, Japan 10 Research Organization for Nano and Life Innovation, Waseda University, Shinjuku, Tokyo, 162-0041, Japan 11 Department of Biochemistry and Microbiology, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand Patcharaporn Hoondee Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Nisachon Tedsree Roles: Formal Analysis, Methodology Sukanya Phuengjayaem Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation Engkarat Kingkaew Roles: Conceptualization, Formal Analysis, Methodology Boonchoo Sritularak Roles: Formal Analysis, Funding Acquisition, Writing – Review & Editing Pornchai Rojsitthisak Roles: Formal Analysis, Funding Acquisition, Writing – Review & Editing Takuji Nakashima Roles: Writing – Review & Editing Worathat Thitikornpong Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Somboon Tanasupawat Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information 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. Article Versions (2) version 2 Revised Published: 02 Jan 2026, 14:717 https://doi.org/10.12688/f1000research.164600.2 version 1 Published: 21 Jul 2025, 14:717 https://doi.org/10.12688/f1000research.164600.1 Copyright © 2025 Hoondee P et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Hoondee P, Tedsree N, Phuengjayaem S et al. Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.12688/f1000research.164600.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 21 Jul 2025 Views 0 Cite How to cite this report: Rao KVB. Reviewer Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.181137.r436643 ) The direct URL for this report is: https://f1000research.com/articles/14-717/v1#referee-response-436643 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 29 Dec 2025 Kokati Venkata Bhaskara Rao , Vellore Institute of Technology, Vellore, India Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.181137.r436643 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, ... Continue reading READ ALL 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 Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? 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 Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. 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. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Rao KVB. Reviewer Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.181137.r436643 ) The direct URL for this report is: https://f1000research.com/articles/14-717/v1#referee-response-436643 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 07 Jan 2026 Somboon Tanasupawat , Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, 10330, Thailand 07 Jan 2026 Author Response 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. ... Continue reading 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 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 Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 07 Jan 2026 Somboon Tanasupawat , Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, 10330, Thailand 07 Jan 2026 Author Response 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. ... Continue reading 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 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 Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: NANJUNDASWAMY A. Reviewer Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.181137.r434447 ) The direct URL for this report is: https://f1000research.com/articles/14-717/v1#referee-response-434447 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 24 Dec 2025 ANANDA NANJUNDASWAMY , South Dakota State University, Brookings, South Dakota, USA Approved VIEWS 0 https://doi.org/10.5256/f1000research.181137.r434447 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 ... Continue reading READ ALL 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 Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? 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 Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. 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. Close READ LESS CITE CITE HOW TO CITE THIS REPORT NANJUNDASWAMY A. Reviewer Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.181137.r434447 ) The direct URL for this report is: https://f1000research.com/articles/14-717/v1#referee-response-434447 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 10 Jan 2026 Somboon Tanasupawat , Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, 10330, Thailand 10 Jan 2026 Author Response 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 ... Continue reading 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 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 Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 10 Jan 2026 Somboon Tanasupawat , Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, 10330, Thailand 10 Jan 2026 Author Response 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 ... Continue reading 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 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 Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Nimsi KAS. Reviewer Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.181137.r436642 ) The direct URL for this report is: https://f1000research.com/articles/14-717/v1#referee-response-436642 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 23 Dec 2025 Kizhakkeyveetil Abdul Saleem Nimsi , BIOSCIENCES, MES College Marampally (Ringgold ID: 210878), Aluva, Kerala, India Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.181137.r436642 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 ... Continue reading READ ALL 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 Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? 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 Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. 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. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Nimsi KAS. Reviewer Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.181137.r436642 ) The direct URL for this report is: https://f1000research.com/articles/14-717/v1#referee-response-436642 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 10 Jan 2026 Somboon Tanasupawat , Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, 10330, Thailand 10 Jan 2026 Author Response 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 ... Continue reading 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 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 Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 10 Jan 2026 Somboon Tanasupawat , Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, 10330, Thailand 10 Jan 2026 Author Response 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 ... Continue reading 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 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 Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 21 Jul 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 2 (revision) 02 Jan 26 read Version 1 21 Jul 25 read read read Kizhakkeyveetil Abdul Saleem Nimsi , MES College Marampally (Ringgold ID: 210878), Aluva, India ANANDA NANJUNDASWAMY , South Dakota State University, Brookings, USA Kokati Venkata Bhaskara Rao , Vellore Institute of Technology, Vellore, India Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Rao K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 08 Jan 2026 | for Version 2 Kokati Venkata Bhaskara Rao , Vellore Institute of Technology, Vellore, India 0 Views copyright © 2026 Rao K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions 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. Competing Interests No competing interests were disclosed. 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. reply Respond to this report Responses (0) Rao KVB. Peer Review Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.194508.r446928) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-717/v2#referee-response-446928 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2026 Rao K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 29 Dec 2025 | for Version 1 Kokati Venkata Bhaskara Rao , Vellore Institute of Technology, Vellore, India 0 Views copyright © 2026 Rao K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions 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 Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? 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 Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. 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. reply Respond to this report Responses (1) Author Response 07 Jan 2026 Somboon Tanasupawat, Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, 10330, Thailand 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 View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Rao KVB. Peer Review Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.181137.r436643) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-717/v1#referee-response-436643 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 NANJUNDASWAMY A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 24 Dec 2025 | for Version 1 ANANDA NANJUNDASWAMY , South Dakota State University, Brookings, South Dakota, USA 0 Views copyright © 2025 NANJUNDASWAMY A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions 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 Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? 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 Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. 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. reply Respond to this report Responses (1) Author Response 10 Jan 2026 Somboon Tanasupawat, Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, 10330, Thailand 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 View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern NANJUNDASWAMY A. Peer Review Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.181137.r434447) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-717/v1#referee-response-434447 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Nimsi K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 23 Dec 2025 | for Version 1 Kizhakkeyveetil Abdul Saleem Nimsi , BIOSCIENCES, MES College Marampally (Ringgold ID: 210878), Aluva, Kerala, India 0 Views copyright © 2025 Nimsi K. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions 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 Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? 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 Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. 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. reply Respond to this report Responses (1) Author Response 10 Jan 2026 Somboon Tanasupawat, Center of Excellence in Natural Products for Ageing and Chronic Diseases, Chulalongkorn University, Pathumwan, 10330, Thailand 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 View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Nimsi KAS. Peer Review Report For: Characterization of red pigmented yeasts and genes associated with astaxanthin synthesis in Rhodotorula mucilaginosa HL26-1 and Rhodotorula paludigena LL69-1 [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :717 ( https://doi.org/10.5256/f1000research.181137.r436642) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-717/v1#referee-response-436642 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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