Isolation, genetic identification of Amazonian yeasts and analysis of thermotolerance and alcohol tolerance of Saccharomyces cerevisiae from Theobroma grandiflorum and Eugenia stipitata

Flávia da Silva Fernandes; Luan Reis Honorato da Silva; Érica Simplício de Souza; Lívia Melo Carneiro; João Paulo Alves Silva; Steven Zelski; João Vicente Braga de Souza; Jacqueline da Silva Batista; Flávia da Silva Fernandes; Luan Reis Honorato da Silva; Érica Simplício de Souza; Lívia Melo Carneiro; João Paulo Alves Silva; Steven Zelski; João Vicente Braga de Souza; Jacqueline da Silva Batista

doi:10.3934/bioeng.2024003

AIMS Bioengineering

2024, Volume 11, Issue 1: 24-43. doi: 10.3934/bioeng.2024003

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Isolation, genetic identification of Amazonian yeasts and analysis of thermotolerance and alcohol tolerance of Saccharomyces cerevisiae from Theobroma grandiflorum and Eugenia stipitata

1.
Graduate Program in Genetics, Conservation and Evolutionary Biology - GCBEv, National Institute of Amazonian Research (INPA), Manaus, Amazonas, Brazil
2.
Mycology Laboratory, National Institute of Amazonian Research (INPA), Manaus, Amazonas, Brazil
3.
School of Technology, State University of Amazonas (UEA), Manaus, Amazonas, Brazil
4.
Department of Chemical Engineering, School of Engineering of Lorena, University of São Paulo (USP), Lorena, São Paulo, Brazil
5.
Miami University, Department of Biological Sciences, Middletown, OH, USA
6.
Thematic Laboratory of Molecular Biology, National Institute of Amazonian Research (INPA), Manaus, Amazonas, Brazil

Received: 25 September 2023 Revised: 25 December 2023 Accepted: 08 January 2024 Published: 29 January 2024

Although yeasts of the Saccharomyces cerevisiae species are industrially significant, few studies have investigated their presence in environmental samples from the Amazon rainforest. This study aimed to isolate S. cerevisiae yeasts associated with trees of the Amazon Forest and investigate their thermotolerance, alcohol tolerance, and single nucleotide polymorphism (SNP) characteristics, along with those of regional strains from previous research and reference strains from the industry. We collected fruits, bark and decaying plant material from Theobroma grandiflorum, Spondias mombin L., Mangifera indica L., and Eugenia stipitata, and isolated yeasts using the culture media. To identify the yeasts, we conducted morphological and biochemical analyses, including sugar assimilation and fermentation, and sequencing analyses of the rDNA (ITS and LSU (D1 and D2)). We also performed fermentation tests to determine the optimum temperature, thermotolerance and ethanol tolerance. Finally, we subjected the selected strains to SNP analysis to study the reported genes that are important for alcohol tolerance in S. cerevisiae: FPS1 (farnesyl diphosphate synthase1) and ASR1/YPR093 (alcohol sensitive RING/PHD finger1) genes. As a result, we isolated 53 yeasts, and 10 of which exhibited a sugar assimilation and fermentation profile that was similar to that of S. cerevisiae. These ten isolates were identified using sequencing of the ITS and LSU regions, which revealed the species to be Wickerhamomyces anomalus (n = 4), Torulaspora pretoriensis (n = 3), Debaryomyces hansenni (n = 1), and Saccharomyces cerevisiae (n = 2). Through the analysis of the ASR1 and FPS1 regions, we found an SNP at nucleotide 1552 A > G (FPS1), which was associated with ethanol tolerance under our experimental conditions. This work is significant because it is one of the first studies to focus specifically on the isolation of S. cerevisiae from samples in the Amazon region. Furthermore, the SNP analysis allowed us to differentiate isolates that showed greater tolerance to ethanol.
- Amazonian fruits,
- Saccharomyces cerevisiae,
- SNPs,
- ethanol tolerance,
- yeast,
- thermotolerance
Citation: Flávia da Silva Fernandes, Luan Reis Honorato da Silva, Érica Simplício de Souza, Lívia Melo Carneiro, João Paulo Alves Silva, Steven Zelski, João Vicente Braga de Souza, Jacqueline da Silva Batista. Isolation, genetic identification of Amazonian yeasts and analysis of thermotolerance and alcohol tolerance of Saccharomyces cerevisiae from Theobroma grandiflorum and Eugenia stipitata[J]. AIMS Bioengineering, 2024, 11(1): 24-43. doi: 10.3934/bioeng.2024003

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Abstract

Although yeasts of the Saccharomyces cerevisiae species are industrially significant, few studies have investigated their presence in environmental samples from the Amazon rainforest. This study aimed to isolate S. cerevisiae yeasts associated with trees of the Amazon Forest and investigate their thermotolerance, alcohol tolerance, and single nucleotide polymorphism (SNP) characteristics, along with those of regional strains from previous research and reference strains from the industry. We collected fruits, bark and decaying plant material from Theobroma grandiflorum, Spondias mombin L., Mangifera indica L., and Eugenia stipitata, and isolated yeasts using the culture media. To identify the yeasts, we conducted morphological and biochemical analyses, including sugar assimilation and fermentation, and sequencing analyses of the rDNA (ITS and LSU (D1 and D2)). We also performed fermentation tests to determine the optimum temperature, thermotolerance and ethanol tolerance. Finally, we subjected the selected strains to SNP analysis to study the reported genes that are important for alcohol tolerance in S. cerevisiae: FPS1 (farnesyl diphosphate synthase1) and ASR1/YPR093 (alcohol sensitive RING/PHD finger1) genes. As a result, we isolated 53 yeasts, and 10 of which exhibited a sugar assimilation and fermentation profile that was similar to that of S. cerevisiae. These ten isolates were identified using sequencing of the ITS and LSU regions, which revealed the species to be Wickerhamomyces anomalus (n = 4), Torulaspora pretoriensis (n = 3), Debaryomyces hansenni (n = 1), and Saccharomyces cerevisiae (n = 2). Through the analysis of the ASR1 and FPS1 regions, we found an SNP at nucleotide 1552 A > G (FPS1), which was associated with ethanol tolerance under our experimental conditions. This work is significant because it is one of the first studies to focus specifically on the isolation of S. cerevisiae from samples in the Amazon region. Furthermore, the SNP analysis allowed us to differentiate isolates that showed greater tolerance to ethanol.

Acknowledgments

The authors would like to acknowledge the funding received from the from the Fundação de Amparo à Pesquisa do Estado do Amazonas (Call No. 030/2013 UNIVERSAL AMAZONAS), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This work was funded by the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) via the PAPAC and POSGRAD 2022 calls.

Conflict of interest

The authors declare no conflicts of interest.

Author Contributions:

All authors contributed to the article's conception and design. Flávia da Silva Fernandes, Jacqueline da Silva Batista and João Vicente Braga de Souza had the idea for the article. Flávia da Silva Fernandes, Abrames Francisco Ferreira Goes, Matheus Alberto Vasconcelos de Lima and Luan Reis Honorato da Silva performed the literature search and data analysis. Flávia da Silva Fernandes wrote the first draft of the manuscript and Érica Simplício de Souza, Lívia Melo Carneiro and João Paulo Alves Silva critically revised the work. Flávia da Silva Fernandes, Jacqueline da Silva Batista and João Vicente Braga de Souza wrote the posterior and final drafts, and all the authors read and approved the final version of the manuscript.

References

[1]	Renewable Fuel Association, 2023 Ethanol Industry outlook, Annual World Fuel Ethanol Production (2023). Available from: https://d35t1syewk4d42.cloudfront.net/file/2432/2023%20RFA%20Outlook%20FINAL.pdf
[2]	Basso LC, De Amorim HV, De Oliveira AJ, et al. (2008) Yeast selection for fuel ethanol production in Brazil. FEMS Yeast Res 8: 1155-1163. https://doi.org/10.1111/j.1567-1364.2008.00428.x
[3]	Amorim HV, Gryschek M, Lopes ML, et al. (2010) The success and sustainability of the Brazilian sugarcane− fuel ethanol industry. Sustainability of the Sugar and Sugar−Ethanol Industries. USA: ACS Publications 73-82. https://doi.org/10.1021/bk-2010-1058.ch005
[4]	You KM, Rosenfield CL, Knipple DC (2003) Ethanol tolerance in the yeast Saccharomyces cerevisiae is dependent on cellular oleic acid content. Appl Environ Microbiol 69: 1499-1503. https://doi.org/10.1128/AEM.69.3.1499-1503.2003
[5]	Caspeta L, Chen Y, Nielsen J (2016) Thermotolerant yeasts selected by adaptive evolution express heat stress response at 30 C. Sci Rep 6: 1-9. https://doi.org/10.1038/srep27003
[6]	Della Bianca BE, van Maris AJA, Daran JM, et al. (2012) How different are laboratorial and industrial yeast strains under stress conditions. 13th international congress on yeasts .
[7]	Almeida S de F, Silva LRC, Junior GCA, et al. (2019) Diversity of yeasts during fermentation of cocoa from two sites in the Brazilian Amazon. Acta Amazon 49: 64-70. https://doi.org/10.1590/1809-4392201703712
[8]	Marton JM, Felipe MGA, Almeida e Silva JB, et al. (2006) Evaluation of the activated charcoals and adsorption conditions used in the treatment of sugarcane bagasse hydrolysate for xylitol production. Braz J Chem Eng 23: 9-21. https://doi.org/10.1590/S0104-66322006000100002
[9]	Junior GCAC, do Espírito-Santo JCA, Ferreira NR, et al. (2020) Yeast isolation and identification during on-farm cocoa natural fermentation in a highly producer region in northern Brazil. Scientia Plena 16: 1-10. https://doi.org/10.14808/sci.plena.2020.121502
[10]	Matos ÍTSR, de Souza VA, D'Angelo G do R, et al. Yeasts with fermentative potential associated with fruits of camu-camu (myrciaria dubia, kunth) from north of brazilian amazon (2021). https://doi.org/10.21203/rs.3.rs-159931/v1
[11]	Cassa-Barbosa LA, Procópio REL, Matos I, et al. (2015) Isolation and characterization of yeasts capable of efficient utilization of hemicellulosic hydrolyzate as the carbon source. Genet Mol Res 14: 11605-11612. https://doi.org/10.4238/2015.September.28.12
[12]	Ferreira O, Chagas-Junior G, Chisté R, et al. (2022) Saccharomyces cerevisiae and Pichia manshurica from Amazonian biome affect the parameters of quality and aromatic profile of fermented and dried cocoa beans. J Food Sci 87: 4148-4161. https://doi.org/10.1111/1750-3841.16282
[13]	Komatsuzaki N, Okumura R, Sakurai M, et al. (2016) Characteristics of Saccharomyces cerevisiae isolated from fruits and humus: Their suitability for bread making. Prog Biol Sci 6: 55-63.
[14]	Kurtzman CP, Fell JW, Boekhout T, et al. (2011) Methods for isolation, phenotypic characterization and maintenance of yeasts. The yeasts. The Netherlands: Elsevier 87-110. https://doi.org/10.1016/B978-0-444-52149-1.00007-0
[15]	Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. USA: Cold spring harbor laboratory press.
[16]	O'Donnel K (1993) Mitotic, meiotic and pleomorphic speciation in fungal systematics. Fusarium and its near relatives 1: 225-233.
[17]	White TJ, Bruns T, Lee S, et al. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc.: Guide Methods Appl 18: 315-322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
[18]	Tamura K, Peterson D, Peterson N, et al. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731-2739. https://doi.org/10.1093/molbev/msr121
[19]	Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids symposium series. London: Information Retrieval Ltd. 95-98.
[20]	Altschul SF, Gish W, Miller W, et al. (1990) Basic local alignment search tool. J Mol Biol 215: 403-410. https://doi.org/10.1016/S0022-2836(05)80360-2
[21]	Kumar S, Stecher G, Li M, et al. (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35: 1547-1549. https://doi.org/10.1093/molbev/msy096
[22]	Jhariya U, Dafale NA, Srivastava S, et al. (2021) Understanding ethanol tolerance mechanism in saccharomyces cerevisiae to enhance the bioethanol production: current and future prospects. Bioenergy Res 14: 670-688. https://doi.org/10.1007/s12155-020-10228-2
[23]	Betz C, Schlenstedt G, Bailer SM (2004) Asr1p, a novel yeast ring/PHD finger protein, signals alcohol stress to the nucleus. J Biol Chem 279: 28174-28181. https://doi.org/10.1074/jbc.M401595200
[24]	Izawa S, Ikeda K, Kita T, et al. (2006) Asr1, an alcohol-responsive factor of Saccharomyces cerevisiae, is dispensable for alcoholic fermentation. Appl Microbiol Biotechnol 72: 560-565. https://doi.org/10.1007/s00253-005-0294-1
[25]	Ding J, Huang X, Zhao N, et al. (2010) Response of Saccharomyces cerevisiae to ethanol stress involves actions of protein Asr1p. J Microbiol Biotechnol 20: 1630-1636. https://doi.org/10.4014/jmb.1007.07021
[26]	Singh K, Sethi R, Das E, et al. (2022) The role of the glycerol transporter channel Fps1p in cellular proteostasis during enhanced proteotoxic stress. Appl Microbiol Biotechnol 106: 6169-6180. https://doi.org/10.1007/s00253-022-12118-3
[27]	Lourenço AB, Roque FC, Teixeira MC, et al. (2013) Quantitative 1H-NMR-metabolomics reveals extensive metabolic reprogramming and the effect of the aquaglyceroporin FPS1 in ethanol-stressed yeast cells. PLoS One 8: e55439. https://doi.org/10.1371/journal.pone.0055439
[28]	Shashkova S, Andersson M, Hohmann S, et al. (2021) Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells. Methods 193: 46-53. https://doi.org/10.1016/j.ymeth.2020.05.003
[29]	Jubany S, Tomasco I, Ponce de León I, et al. (2008) Toward a global database for the molecular typing of saccharomyces cerevisiae strains. FEMS Yeast Res 8: 472-484. https://doi.org/10.1111/j.1567-1364.2008.00361.x
[30]	Yi S, Zhang X, Li H, et al. (2018) Screening and mutation of saccharomyces cerevisiae UV-20 with a high yield of second generation bioethanol and high tolerance of temperature, glucose and ethanol. Indian J Microbiol 58: 440-447. https://doi.org/10.1007/s12088-018-0741-1
[31]	Udomsaksakul N, Kodama K, Tanasupawat S, et al. (2018) Diversity of ethanol fermenting yeasts in coconut inflorescence sap and their application potential. Sci Asia 44: 371-381. https://doi.org/10.2306/scienceasia1513-1874.2018.44.371
[32]	Techaparin A, Thanonkeo P, Klanrit P (2017) High-temperature ethanol production using thermotolerant yeast newly isolated from greater mekong subregion. Braz J Microbiol 48: 461-475. https://doi.org/10.1016/j.bjm.2017.01.006
[33]	Lee SS, Robinson FM, Wang HY (1981) Rapid determination of yeast viability. Biotechnol. Bioeng. Symp.;(United States). Ann Arbor: Univ. of Michigan.
[34]	Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31: 426-428. https://doi.org/10.1021/ac60147a030
[35]	Zimmermann HW (1963) Studies on the dichromate method of alcohol determination. Am J Enol Vitic 14: 205-213. https://doi.org/10.5344/ajev.1963.14.4.205
[36]	Ozmen Togay S, Capece A, Siesto G, et al. (2020) Molecular characterization of yeasts isolated from traditional turkish cheeses. Food Sci Technol 40: 871-876. https://doi.org/10.1590/fst.24319
[37]	Agarbati A, Canonico L, Marini E, et al. (2020) Potential probiotic yeasts sourced from natural environmental and spontaneous processed foods. Foods 9: 287. https://doi.org/10.3390/foods9030287
[38]	Šuranská H, Vránová D, Omelková J (2016) Isolation, identification and characterization of regional indigenous Saccharomyces cerevisiae strains. Brazi J Microbiol 47: 181-190. https://doi.org/10.1016/j.bjm.2015.11.010
[39]	Nasir A, Rahman SS, Hossain MM, et al. (2017) Isolation of saccharomyces cerevisiae from pineapple and orange and study of metal's effectiveness on ethanol production. Eur J Microbiol Immunol 7: 76-91. https://doi.org/10.1556/1886.2016.00035
[40]	Lee NK, Hong JY, Yi SH, et al. (2019) Bioactive compounds of probiotic saccharomyces cerevisiae strains isolated from cucumber jangajji. J Funct Foods 58: 324-329. https://doi.org/10.1016/j.jff.2019.04.059
[41]	Beato FB, Bergdahl B, Rosa CA, et al. (2016) Physiology of saccharomyces cerevisiae strains isolated from Brazilian biomes: new insights into biodiversity and industrial applications. FEMS Yeast Res 16: fow076. https://doi.org/10.1093/femsyr/fow076
[42]	Tian S, Liang X, Chen J, et al. (2020) Enhancement of 2-phenylethanol production by a wild-type Wickerhamomyces anomalus strain isolated from rice wine. Bioresour Technol 318: 124257. https://doi.org/10.1016/j.biortech.2020.124257
[43]	Visintin S, Alessandria V, Valente A, et al. (2016) Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa. Int J Food Microbiol 216: 69-78. https://doi.org/10.1016/j.ijfoodmicro.2015.09.004
[44]	Delgado-Ospina J, Triboletti S, Alessandria V, et al. (2020) Functional biodiversity of yeasts isolated from Colombian fermented and dry cocoa beans. Microorganisms 8: 1086. https://doi.org/10.3390/microorganisms8071086
[45]	Del Bove M, Lattanzi M, Rellini P, et al. (2009) Comparison of molecular and metabolomic methods as characterization tools of Debaryomyces hansenii cheese isolates. Food Microbiol 26: 453-459. https://doi.org/10.1016/j.fm.2009.03.009
[46]	Mounier J, Irlinger F, Leclercq-Perlat MN, et al. (2006) Growth and colour development of some surface ripening bacteria with debaryomyces hansenii on aseptic cheese curd. J Dairy Res 73: 441-448. https://doi.org/10.1017/S0022029906001919
[47]	Cappelli A, Ulissi U, Valzano M, et al. (2014) A wickerhamomyces anomalus killer strain in the malaria vector anopheles stephensi. PLoS One 9: e95988. https://doi.org/10.1371/journal.pone.0095988
[48]	Oda Y, Iwamoto H, Hiromi K, et al. (1993) Purification and characterization of α-glucosidase from Torulaspora pretoriensis YK-1. Biosci Biotechnol Biochem 57: 1902-1905. https://doi.org/10.1271/bbb.57.1902
[49]	Jiménez J, Benítez T (1986) Characterization of wine yeasts for ethanol production. Appl Microbiol Biotechnol 25: 150-154. https://doi.org/10.1007/BF00938939
[50]	Tadayon RA (1978) Identification of yeasts isolated from bread dough of bakeries in Shiraz, Iran. J Food Prot 41: 717-721. https://doi.org/10.4315/0362-028X-41.9.717
[51]	Oda Y, Tonomura K (1994) Purification and characterization of invertase from torulaspora pretoriensis YK-1. Biosci Biotechnol Biochem 58: 1155-1157. https://doi.org/10.1271/bbb.58.1155
[52]	Petersen KM, Jespersen L (2004) Genetic diversity of the species debaryomyces hansenii and the use of chromosome polymorphism for typing of strains isolated from surface-ripened cheeses. J Appl Microbiol 97: 205-213. https://doi.org/10.1111/j.1365-2672.2004.02293.x
[53]	Choudhary J, Singh S, Nain L (2017) Bioprospecting thermotolerant ethanologenic yeasts for simultaneous saccharification and fermentation from diverse environments. J Biosci Bioeng 123: 342-346. https://doi.org/10.1016/j.jbiosc.2016.10.007
[54]	Kuroda K, Ueda M (2018) Adaptive evolution of yeast under heat stress and genetic reconstruction to generate thermotolerant yeast. Origin and Evolution of Biodiversity. Cham: Springer 23-36. https://doi.org/10.1007/978-3-319-95954-2_2
[55]	Pattanakittivorakul S, Lertwattanasakul N, Yamada M, et al. (2019) Selection of thermotolerant saccharomyces cerevisiae for high temperature ethanol production from molasses and increasing ethanol production by strain improvement. Antonie Van Leeuwenhoek 112: 975-990. https://doi.org/10.1007/s10482-019-01230-6
[56]	Nuanpeng S, Thanonkeo S, Yamada M, et al. (2016) Ethanol production from sweet sorghum juice at high temperatures using a newly isolated thermotolerant yeast saccharomyces cerevisiae DBKKU Y-53. Energies 9: 253. https://doi.org/10.3390/en9040253
[57]	Negi B, Sharma P, Kashyap S, et al. (2013) Screening of yeast strains for vinification of fruits from cold desert regions of North West India. Int Food Res J 20: 975-979.
[58]	Udomsaksakul N, Kodama K, Tanasupawat S, et al. (2018) Indigenous saccharomyces cerevisiae strains from coconut inflorescence sap: characterization and use in coconut wine fermentation. CMU J Nat Sci 17: 219-230. https://doi.org/10.12982/CMUJNS.2018.0016
[59]	Tikka C, Osuru HP, Atluri N, et al. (2013) Isolation and characterization of ethanol tolerant yeast strains. Bioinformation 9: 421-425. https://doi.org/10.6026/97320630009421
[60]	Tesfaw A, Oner ET, Assefa F (2021) Optimization of ethanol production using newly isolated ethanologenic yeasts. Biochem Biophys Rep 25: 100886. https://doi.org/10.1016/j.bbrep.2020.100886
[61]	Dash PK, Jyoti M, Patnaik SC, et al. (2015) Characterization, identification and comparative evaluation of bioethanol tolerance and production capacity of isolated yeast strains from fermented date palm sap (toddy). Malays J Microbiol 11: 223-230. https://doi.org/10.21161/mjm.57213
[62]	Jacobus AP, Stephens TG, Youssef P, et al. (2021) Comparative genomics supports that Brazilian bioethanol saccharomyces cerevisiae comprise a unified group of domesticated strains related to cachaça spirit yeasts. Front Microbiol 12: 644089. https://doi.org/10.3389/fmicb.2021.644089
[63]	Goddard MR, Anfang N, Tang R, et al. (2010) A distinct population of saccharomyces cerevisiae in New Zealand: evidence for local dispersal by insects and human-aided global dispersal in oak barrels. Environ Microbiol 12: 63-73. https://doi.org/10.1111/j.1462-2920.2009.02035.x
[64]	Strope PK, Skelly DA, Kozmin SG, et al. (2015) The 100-genomes strains, an s. cerevisiae resource that illuminates its natural phenotypic and genotypic variation and emergence as an opportunistic pathogen. Genome Res 25: 762-774. https://doi.org/10.1101/gr.185538.114

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