Research Article |
Corresponding author: Fengli Hui ( fenglihui@yeah.net ) Academic editor: Thorsten Lumbsch
© 2024 Chunyue Chai, Dan Lu, Jinli Liu, Eentao Wang, Xuemei Han, Fengli Hui.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Chai C, Lu D, Liu J, Wang E, Han X, Hui F (2024) Three novel Ascomycota (Saccharomycetes, Saccharomycetales) yeast species derived from the traditional Mexican alcoholic beverage Pulque. MycoKeys 109: 187-206. https://doi.org/10.3897/mycokeys.109.123870
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The abundant variety of yeasts and their diverse applications have essential roles in traditional Mexican alcoholic beverage fermentation processes. During our investigation of yeast diversity associated with Pulque, 41 yeast strains were characterized. Among them, 31 strains were eight known species belonging to seven genera. According to morphological and phylogenetic analyses (ITS and LSU rDNA), ten unidentified yeast strains were identified to be three novel species and proposed: Starmerella elongatum sp. nov., Kazachstania paragamospora sp. nov. and Pichia teotihuacanensis sp. nov. Our study has resulted in the isolation of yeast species not previously detected in Pulque, expanding our knowledge of the diversity of its associated yeast communities.
Morphology, phylogenetic analysis, Pulque, taxonomy, yeast
Alcoholic beverages arose across various geographical regions over the course of human civilization (
Pulque is a traditional Mexican alcoholic beverage produced by fermenting fresh sap collected from the hearts of mature, uncooked agave plants, including Agave americana, A. atrovirens, A. ferox, A. mapisaga, and A. salmiana (
Studies delving into Pulque-associated microbiota identified an array of associated bacterial and yeast species. These microorganisms generate three unique metabolic products during Pulque fermentation: lactic acid, ethanol, and extracellular polysaccharides (EPS). Regarding the yeast diversity in Pulque, Saccharomyces and non-Saccharomyces species have both been found and proposed as necessary for the production of ethanol, amino acids, vitamins, and volatile flavor compounds contributing to the sensory properties of the beverage (
To investigate the diversity of yeast in Pulque, we collected six Pulque samples in the autumn of 2015 from Mexico, from which different yeasts were isolated. Based on DNA sequence comparisons and phenetic characteristics, the majority of isolates were identified as characterized species. Among them, ten unidentified yeast strains were identified. To examine their taxonomy further, phylogenetic analyses based on integrated the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) rRNA gene sequences were performed. Both morphological characteristics and molecular evidence suggest that these yeasts represent three separate species of Kazachstania, Pichia, and Starmerella genera, which are described in detail in this study.
Six homogenized Pulque samples were obtained from two “pulquerías” (Pulque stores) in 2015 in two different Mexico regions: Teotihuacan in the State of Mexico (19°41'N, 98°50'W) with an altitude of 2377 m, and Mexico City in the State of Distrito Federal (19°28'N, 99°09'W) with an altitude of 2400. Pulque samples were fermented for approximately 36 to 48 hours. We removed approximately 10 mL of each Pulque sample into different sterile plastic bags using long-handled spoons. Samples were then transported on ice to the laboratory. Yeasts were isolated from Pulque samples using yeast extract-malt agar (YMA, composed of 1% glucose, 0.3% yeast extract, 0.3% malt extract, 0.5% peptone, and 2% agar, w/v) supplemented with 0.02% chloramphenicol. Each Pulque sample was serially diluted into a sterilized saline solution (0.9% w/v; NaCl). Subsamples of 100 μL were spread on YM plates and incubated at 25 °C for 3~7 days. The yeast colonies were purified through repeated streaking on YM plates followed by incubations at 25 °C. The isolated strains were maintained at 4 °C for short-term preservation and at −80 °C as 20% glycerol (w/v) stocks for long-term preservation.
Morphological observations and metabolic examinations adhering to the standard yeast description profile were performed as previously reported (
Genomic DNA was extracted using an Ezup Column Yeast Genomic DNA Purification Kit according to the manufacturer’s instructions. The ITS region and the D1/D2 domain of the LSU rRNA gene were amplified via PCR and sequenced using the ITS1 and ITS4 (
Phylogenetic relationships between the novel species and their close relatives were characterized using a combined ITS and LSU sequence dataset. Saccharomyces cerevisiae CBS 1171T, Wickerhamiella sorbophila NRRL Y-7921T and Schizosaccharomyces pombe CBS 356T were utilized as respective outgroups. Except for the sequences acquired in this study, the reference sequences were downloaded from GenBank (Suppl. materials
From the six samples of pulque aguamiel, forty-one phenotypically distinct colonies were chosen, and all were confirmed to be yeasts via microscopic analysis and uniform yeast colonies grew when plated on YM plates. The D1/D2 domain of the LSU rRNA gene were sequenced to characterize each yeast isolate. The nearest relative of each strain was recognized according to a BLAST search against the GenBank database using the BLASTn tool. The strains were classified to the species level based on a threshold of > 99% sequence identity with the type strain of a described species within the ITS region or D1/D2 domain (
Strains isolated from samples of the traditional Mexican alcoholic beverage Pulque.
Location | Sample | Strain (NYNU) | Species |
---|---|---|---|
I | 1 | 16111 | Apiotrichum mycotoxinivorans |
2 | 16113 | ||
II | 4 | 161140 | |
I | 1 | 16116 | Candida boidinii |
3 | 161115 | ||
II | 4 | 161139 | |
5 | 161147 | ||
I | 1 | 16118 | Debaryomyces nepalensis |
3 | 161127 | ||
I | 1 | 161111T | Kazachstania sp. |
2 | 161114 | ||
3 | 161129 | ||
I | 1 | 16114 | Kluyveromyces marxianus |
2 | 161112 | ||
3 | 161113 | ||
II | 4 | 161141 | |
5 | 161145 | ||
6 | 161146 | ||
I | 2 | 161118 | Papiliotrema laurentii |
3 | 161131 | ||
II | 5 | 161143 | |
6 | 161149 | ||
I | 1 | 161110 | Pichia manshurica |
2 | 161116 | ||
3 | 161130 | ||
II | 4 | 161138 | |
I | 2 | 161119T | Pichia sp. |
3 | 161117 | ||
II | 5 | 161142 | |
6 | 161153 | ||
II | 4 | 161136 | Piskurozyma taiwanensis |
6 | 161150 | ||
I | 1 | 16112 | Rhodosporidiobolus ruineniae |
3 | 161125 | ||
II | 4 | 161135 | Saccharomyces paradoxus |
5 | 161137 | ||
6 | 161158 | ||
I | 1 | 161162 | |
I | 1 | 16115T | Starmerella sp. |
2 | 161124 | ||
3 | 161128 |
Among the yeast strains isolated, ten were unidentified as any species because of their significant difference from any described yeast species in the D1/D2 domain sequence. The ITS sequences of these strains were then determined to validate their novelty and phylogenetic positions.
Three strains, NYNU 161124, NYNU 161128, and NYNU 16115T, isolated from two samples in Teotihuacan in the State of Mexico (Table
Phylogenetic trees derived from maximum-likelihood analysis based on the concatenated sequences of the ITS and D1/D2 domain of LSU rRNA, illustrating the positions of the novel yeast species in the genera Starmerella (A), Kazachstania (B) and Pichia (C). The tree backbones were developed using MEGA 7.0. The bootstrap support values (BS) ≥ 50% from ML analysis and Bayesian posterior probabilities (BPP) ≥ 0.90 are depicted on the branches, and a dash (“-”) indicates a value < 0.90 (BPP). Newly described species are indicated in bold. Scale bar, 2% sequence difference. T, type strains.
Three strains, NYNU 161111T, NYNU 161114, and NYNU 161129, with identical ITS and D1/D2 sequences, were located in the Kazachstania clade and closely associated with K. gamospora NBRC 11056T, K. zonata CBS 10326T, and K. hellenica CBS 10706T in the tree derived from the combined ITS and D1/D2 sequences (Fig.
Four strains, NYNU 161119, NYNU 161117, NYNU 161142, NYNU 161153, isolated from four samples (Table
The specific epithet elongatum refers to the elongate vegetative cells of this yeast.
Mexico • State of Mexico, Teotihuacan, in the traditional Mexican alcoholic beverage Pulque, autumn of 2015, F.L. Hui, NYNU 16115 (holotype CICC 33262T preserved in a metabolically inactive state, culture ex-type CBS 15224).
After 3 days growth in YM broth at 25 °C, the cells are mostly ellipsoidal to elongate (2.2–3.4 × 3.9–7.3 μm) and occurred singly or in pairs. Budding is multilateral (Fig.
Morphological characteristics of Starmerella elongatum sp. nov. NYNU 161115T A budding yeast cells, three days, YM broth, 25 °C B pseudohyphae grown on CM agar after 14 d at 25 °C. Kazachstania paragamospora sp. nov. NYNU 161111T C budding yeast cells in YM broth after 3 d, 25 °C. Pichia teotihuacanensis sp. nov. NYNU 161119T D budding yeast cells, three days, YM broth, 25 °C E simple pseudohyphae and F pseudohyphae grown on CM agar over 14 days at 25 °C. Scale bars: 10 μm.
Mexico • Teotihuacan in the State of Mexico (19°41'N, 98°50'W), in the traditional Mexican alcoholic beverage Pulque samples, autumn of 2015, F.L. Hui, NYNU 161124, and NYNU 161128.
Starmerella elongatum sp. nov. can be physiologically differentiated from their nearest phylogenetic neighbor, S. stellata (
Physiological characteristics differentiating three novel species from closely related species.
Fermentation | Assimilation reactions and other characteristics | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Species | D-Glucose | D-Galactose | Raffinose | Inulin | D-Glucose | Inulin | L-Sorbose | Soluble starch | Cadaverine | D-Galactose | L-Arabinose | Trehalose | L-Arabinitol | DL-Lactate | Succinate | Ethanol | Methanol | D-Glucosamine | Citrate | Ethylamine | Glucosamine | 10%Nacl/5%glucose | Acetic acid 1% | Growth in vitamin-free edium | Growth at 30 °C |
S. stellata [a] | + | - | + | + | + | w | v | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | + |
S. elongatum sp. nov. | + | - | + | w | + | - | - | w | w | - | - | - | - | - | - | - | - | - | - | - | - | w | + | + | + |
K. gamospora [b] | + | - | + | - | + | + | - | - | - | - | - | + | - | + | - | + | w | - | - | + | - | + | - | - | + |
K. zonata [c] | + | - | + | - | + | + | - | - | + | - | - | - | + | + | - | + | + | - | - | + | - | - | - | + | + |
K. hellenica [d] | + | + | - | + | + | + | - | + | + | + | + | d | + | - | + | - | w | - | - | - | - | - | - | - | - |
K. paragamospora sp. nov. | + | - | + | + | + | + | - | w | w | - | - | + | w | - | w | - | w | - | - | - | - | - | - | + | + |
P. ethanolica [e] | s | - | - | - | - | - | - | - | + | - | - | - | - | w | w | + | - | - | - | + | - | - | - | + | + |
P. deserticola [f] | - | - | - | - | - | - | - | - | - | - | - | - | - | + | + | + | - | - | - | - | - | - | + | - | + |
P. teotihuacanensis sp. nov. | - | - | - | - | - | w | - | w | - | - | - | - | - | w | w | - | w | + | w | - | w | w | + | + | + |
the specific epithet paragamospora, like gamospora, referring to its phylogenetic closeness to Kazachstania gamospora.
Mexico • State of Mexico, Teotihuacan, in the traditional Mexican alcoholic beverage Pulque, autumn of 2015, F.L. Hui, NYNU 161111 (holotype CICC 33274T preserved in a metabolically inactive state, culture ex-type CBS 15233).
After 3 days culture in YM broth at 25 °C, the cells are spherical or ovoid (3–6.2 × 3.3–7.5 μm) and occurred singly or in pairs (Fig.
Mexico • Teotihuacan in the State of Mexico (19°41'N, 98°50'W), in the traditional Mexican alcoholic beverage Pulque samples, autumn of 2015, F.L. Hui, NYNU 161114 and NYNU 161129.
Kazachstania paragamospora sp. nov. can be differed from the related three species K. gamospora, K. zonata and K. hellenica by its inability to assimilate trehalose (Table
The specific epithet teotihuacan of or belonging to the State of Mexico, the geographical origin of the type strain of the isolated species.
Mexico • State of Mexico, Teotihuacan, in the traditional Mexican alcoholic beverage Pulque sample, autumn of 2015, F.L. Hui, NYNU 161119 (holotype CICC 33275T preserved in a metabolically inactive state, culture ex-type CBS 15277).
After 3 days culture in YM broth at 25 °C, the cells are ovoid (2.5–7.0 × 3.7–9.5 µm) and occurred singly or in pairs. Budding is multilateral (Fig.
Mexico • Teotihuacan in the State of Mexico (19°41'N, 98°50'W) and Mexico City in the State of Distrito Federal (19°28'N, 99°09'W), in the traditional Mexican alcoholic beverage Pulque samples, autumn of 2015, F.L. Hui, NYNU 161117, NYNU 161142 and NYNU 161153.
Pichia teotihuacanensis sp. nov. can be physiologically differentiated from P. ethanolica in terms of positive assimilation of inulin, D-glucosamine, soluble starch, citrate, methanol, and glucosamine, and an inability to assimilate ethanol, ethylamine, and cadaverine. P. teotihuacanensis differed from P. deserticola, in terms of their ability to assimilate D-glucosamine, inulin, and soluble starch, and grow in vitamin-free medium (Table
As previously described, Pulque is a critically important, traditional, non-distilled alcoholic beverage produced in the central states of Mexico. Pulque is the focus of research across numerous laboratories, not only due to its nutritional properties but also to the complex microbial diversity responsible for fermentation, a complex procedure that has proven recalcitrant to industrialization. While various microbial studies regarding Pulque have been performed over the last 100 years, the complex yeast found within the beverage has remained uncharacterized. Recently, several yeast species have been identified in Pulque, such as Candida valida, C. zemplinina, C. stellata, C. parapsilosis, Clavispora lusitaniae, Debaryomices carsonii, Geotrichum candidum, Hanseniaspora uvarum, Kazachstania lactis, Kluyveromyces marxianus, Pichia guilliermondii, P. membranifaciens, Rhodotorula mucilaginosa, Saccharomyces cerevisiae subsp. chevalieri, S. cerevisiae subsp. capensis, S. bayanus, S. paradoxus, Torulaspora delbrueckii and so on (
In this study, we collected six Pulque samples from two “pulquerías” (Pulque stores) in two different regions in Mexico. From these samples, we extracted forty-one yeast strains, clustered into 12 yeast species belonging to 11 genera. Of the total isolates, there were nine representatives of previously known species, like Apiotrichum mycotoxinivorans, Candida boidinii, Debaryomyces nepalensis, Kluyveromyces marxianus, Papiliotrema laurentii, Pichia manshurica, Piskurozyma taiwanensis, Rhodosporidiobolus ruineniae and Saccharomyces paradoxus. In addition to known species, we recovered ten strains belonging to 3 yeast species distinct from any previously described, including Kazachstania sp., Pichia sp., and Starmerella sp. Three more comprehensive separate phylogenetic placements of the genera Kazachstania, Pichia, and Starmerella according to the combined ITS and LSU rDNA sequences are provided. Each phylogenetic tree includes almost all GenBank representatives and newly generated sequences, potentially serving as the references for the three fields. Thereafter, we described these novel species as Starmerella elongatum sp. nov., Kazachstania paragamospora sp. nov., and Pichia teotihuacanensis sp. nov. based on molecular phylogenetic and morphological evidence.
According to the details previously reported, yeast plays an important role in the fermentation of Pulque, producing ethanol, amino acids, vitamins, and volatile compounds. Among the total isolates, two known species, Klu. marxianus and S. paradoxus were found to occur in Pulque, Klu. marxianus can excrete enzymes and be found within Pulque, can ferment at high temperatures (45 °C), use complex sugars such as inulin and hemicellulose, and has been used to generate bioethanol with a broader substrate range and higher temperature tolerance (
This study reports on the isolation of yeast species not previously identified in Pulque, extending our understanding of the genetic diversity of its associated yeast communities. To date, alongside the three newly isolated species in this study, more and more yeast species have been isolated from Pulque. Although the yeast taxonomy of Pulque has been a focus of research in the past, the native microbial community is likely related to the traditional non-aseptic conditions used during the collection, transportation, and fermentation; there are still some species that require discovery in Pulque in the future studies.
The authors express deep gratitude to their colleagues at the School of Life Science, Nanyang Normal University, and at Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional. Special thanks to Dr. Entao Wang and Tan Wang for providing specimens and Dr. Ting Lei for assistance with phylogenetic analysis.
The authors have declared that no competing interests exist.
No ethical statement was reported.
This project was supported by Key scientific research project of colleges and universities in Henan Province (grant no. 23A210018); The Program of Fujian Key Laboratory for Monitoring and Management of Crop Pests (grant no. MIMCP-202403); Open-ended project of Key Laboratory of Integrated Pest Management on Crops in Central China, Ministry of Agriculture and Rural Affairs, P. R. China (grant no. 2021ZTSJJ5).
Data curation: CYC. Methodology: DL, JLL. Molecular phylogeny: ETW, XMH. Writing - original draft: CYC. Writing - review and editing: CYC, ETW, FLH. All authors read and approved the final manuscript.
Chunyue Chai https://orcid.org/0000-0003-0284-5560
Fengli Hui https://orcid.org/0000-0001-7928-3055
All of the data that support the findings of this study are available in the main text or Supplementary Information.
DNA sequences used in the molecular phylogenetic analysis of Starmerella genus
Data type: docx
DNA sequences used in the molecular phylogenetic analysis of Kazachstania genus
Data type: docx
DNA sequences used in the molecular phylogenetic analysis of Pichia genus
Data type: docx