غربالگری مولکولی مخمرهای مولد آستاگزانتین تراوشات درختان توس استان البرز
محورهای موضوعی : میکروب شناسی محیطی
فائزه اجرلو
1
(دانشجوی دکتری، سازمان پژوهش های علمی و صنعتی ایران، پژوهشکده زیست فناوری)
محسن واعظ
2
(استادیار، سازمان پژوهش های علمی و صنعتی ایران، پژوهشکده زیست فناوری)
جعفر همت
3
(استادیار، سازمان پژوهش های علمی و صنعتی ایران، پژوهشکده زیست فناوری)
کلید واژه: مخمر, آستاگزانتین, کاروتنوئید, واکنش زنجیره‌ای پلیمراز چندگانه, درختان توس,
چکیده مقاله :
سابقه و هدف: مخمرهای مولد کاروتنوئید دارای پراکنش وسیعی در مناطق مختلف دنیا می‌باشند. در این بین جداسازی مخمرهای مولد کاروتنوئید با ارزش آستاگزانتین تنها از مناطق محدود و زیستگاه‌های خاص گزارش شده‌اند. این مطالعه با هدف بکارگیری روش واکنش زنجیره‌ای پلیمراز چندگانه برای غربالگری مولکولی سریعتر جدایه‌ها با توان تولید آستاگزانتین از تراوشات درختان توس استان البرز و بررسی کارآیی آن انجام شد.مواد و روش‌ها: این پژوهش بصورت مقطعی با نمونه‌برداری در اردیبهشت ماه 1397 از یکی از رویشگاه‌های طبیعی درختان توس گونه بتولا پندولا در استان البرز صورت پذیرفت. پس از جداسازی مخمرها بر روی محیط‌های انتخابی و خالص‌سازی کلنی‌هایی با رنگ‌های متنوع صورتی تا قرمز، اسیدنوکلئیک ژنومی مخمرها استخراج و غربالگری مولکولی جدایه‌ها از نظر قابلیت تولید آستاگزانتین با واکنش زنجیره‌ای پلیمراز چندگانه بررسی گردید.یافته ها: از مجموع 42 کلنی واجد رنگدانه در هنگام نمونه‌برداری و جداسازی اولیه، تعداد 23 کلنی به رنگ‌های صورتی تا قرمز با روش واکنش زنجیره‌ای پلیمراز چندگانه مورد ارزیابی قرار گرفتند. از بین این جدایه‌ها سه مورد مثبت و بقیه منفی تشخیص داده شدند. صحت نتایج با تعیین ترادف ریبوزومی تایید شد.نتیجه‌گیری: مطالعه حاضر بیانگر قابلیت روش واکنش زنجیره‌ای پلیمراز چندگانه در غربالگری مولکولی سریع و مقرون به صرفه جدایه‌های مخمری مولد آستاگزانتین و امکان بکارگیری این روش برای بررسی زیستگاه‌های طبیعی دیگر این مخمرها می‌باشد.
Background & Objectives: The carotenoid-producing yeasts have a widespread distribiution worldwide. However, isolation of carotenoid-producing yeasts with valuable astaxanthin have been only reported from limited areas and specific habitats. The aim of this study was to apply the multiple polymerase chain reaction method for faster molecular screening of isolates for the yeasts in the exudation sap from birch trees (Betula pendula Roth.) in Alborz Province of Iran with astaxanthin production capacity and toevaluate its efficiency.Materials & Methods: This cross-sectional study was carried out by sampling in May 2018 from one of the natural habitats of birch trees Batula Pendula in Alborz Province, Iran. After isolation of yeasts using selected media and purification of yeast colonies with various pink to red colors, yeasts genomic nucleic acids were extracted and molecular screening for astaxanthin-producing detection of isolates was performed by multiple polymerase chain reaction (Multiplex-PCR).Results: Out of 42 pigmented colonies during sampling and isolation in one cross-sectional study, 23 pink to red colonies were evaluated by Multiplex-PCR method. Of these isolates, three were positive and the rest were negative. The results were validated by determining the ribosomal gene sequencing.Conclusion: The present study demonstrates the specificity of the multiple polymerase chain reaction method in fast and cost-effective molecular screening of astaxanthin-producing yeast isolates and the applicability of this method to study other natural habitats of these yeasts.
2. Stefanini I. Yeast‐insect associations: It takes guts. Yeast. 2018 Apr;35(4):315-30.
3. Fonseca A, Inácio J. Phylloplane yeasts. InBiodiversity and ecophysiology of yeasts 2006 (pp.
263-301). Springer, Berlin, Heidelberg.
4. Phaff HJ. Yeasts associated with plants, insects and soil. Yeasts/edited by Anthony H. Rose, JS
Harrison. 1987.
5. Kurtzman C, Fell JW, Boekhout T, editors. The yeasts: a taxonomic study. Elsevier;2011May9.
6. Boynton PJ. The ecology of killer yeasts: Interference competition in natural habitats. yeast.
2019 Aug;36(8):473-85.
7. Pereira da Costa D, Campos Miranda-Filho K. The use of carotenoid pigments as food additives
for aquatic organisms and their functional roles. Reviews in Aquaculture. 2020 Aug;12
(3):1567-78.
8. Schmidt I, Schewe H, Gassel S, Jin C, Buckingham J, Hümbelin M, Sandmann G, Schrader J.
Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces
dendrorhous. Applied microbiology and biotechnology. 2011 Feb 1;89(3):555-71.
9. Hosseinzadeh CA, Fallah F, Yousefzadeh H. Genetic Diversity and Differentiation of the
Iranian's Betula pendula Populations by DNA Polymorphisms of Three (CD, DT, K1K2)
Chloroplast Genome Regions. Iranian Journal of Biology. 2015; 28(2) 191-201.
10. Weber RW. On the ecology of fungal consortia of spring sap-flows. Mycologist. 2006 Nov
1;20(4):140-3.
11. Grabek-Lejko D, Kasprzyk I, Zaguła G, Puchalski C. The bioactive and mineral compounds in
birch sap collected in different types of habitats. Baltic Forestry. 2017 Jan 1;23(2):394-401.
12. Lõoke M, Kristjuhan K, Kristjuhan A. Extraction of genomic DNA from yeasts for PCR-based
applications. Biotechniques. 2011 May;50(5):325-8.
13. Ajorloo, F., Vaez, M., Hemmat, J. Isolation and molecular identification of the carotenoid
producing yeasts in the exudation sap from birch trees (Betula pendula Roth.) at Marmisho
region of Northwest Iran. Journal of Microbial World, 2019; 12(2): 139-149. [In Persian]
14. Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A. Systematics of basidiomycetous yeasts: a
comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS yeast
research. 2002 Dec 1;2(4):495-517.
15. White TJ, Bruns T, Lee SJ, Taylor JL. Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications.
1990;18(1):315-22.
16. Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A. Biodiversity and
systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain
sequence analysis. International journal of systematic and evolutionary microbiology. 2000
May 1;50(3):1351-71.
17. ROSA CA, LACHANCE MA. The yeast genus Starmerella gen. nov. and Starmerella
bombicola sp. nov., the teleomorph of Candida bombicola (Spencer, Gorin & Tullock) Meyer
& Yarrow. International Journal of Systematic and Evolutionary Microbiology. 1998 Oct 1;48
(4):1413-7.
18. Filatov DA. ProSeq: a software for preparation and evolutionary analysis of DNA sequence
data sets. Molecular Ecology Notes. 2002 Dec;2(4):621-4.
19. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool.
Journal of molecular biology. 1990 Oct 5;215(3):403-10.
20. Woffelman C. DNAMAN for Windows, Version 5.2. 10. Lynon Biosoft. Institute of
Molecular Plant Sciences, Netherlands: Leiden University. 2004.
21. Valduga E, Valério A, Tatsch PO, Treichel H, Furigo A, Luccio M. Assessment of Cell
Disruption and Carotenoids Extraction from Sporidiobolus salmonicolor (CBS 2636). Food
and Bioprocess Technology. 2009;2(2):234-8.
22. Weber RW, Davoli P. Teaching techniques for mycology: 20. Astaxanthin, a carotenoid of
biotechnological importance from yeast and salmonid fish. Mycologist. 2003 Feb1;17(1):30-4.
23. Barredo JL, García-Estrada C, Kosalkova K, Barreiro C. Biosynthesis of astaxanthin as a main
carotenoid in the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous. Journal of
Fungi. 2017 Sep;3(3):44.
24. Sorokina M, Steinbeck C. Review on natural products databases: where to find data in 2020.
Journal of cheminformatics. 2020 Dec;12:1-51.
25. Schroeder WA, Johnson EA. Carotenoids protect Phaffia rhodozyma against singlet oxygen
damage. Journal of industrial microbiology. 1995 Jun 1;14(6):502-7.
26. Golubev WI. Xanthophyllomyces Golubev. InThe Yeasts (Fourth Edition) 1998(pp. 718-719).
27. Nahvi I, Vaez M, Emtiazi G. Evaluation of carotenoid-producing yeasts associated with birch
trees (Betula pendula) in the North of Iran-Shahrestanak village. Pajouhesh-va-Sazandegi.
2000; 13(3):70-74. [In Persian]
28. Weber RW, Davoli P, Anke H. A microbial consortium involving the astaxanthin producer
Xanthophyllomyces dendrorhous on freshly cut birch stumps in Germany. Mycologist. 2006
May 1;20(2):57-61.
29. Libkind D, Moliné M, de García V, Fontenla S, van Broock M. Characterization of a novel
South American population of the astaxanthin producing yeast Xanthophyllomyces
dendrorhous (Phaffia rhodozyma). Journal of industrial microbiology & biotechnology. 2008
Mar 1;35(3):151-8.
30. Colabella F, Libkind D. PCR-based method for the rapid identification of
astaxanthin-accumulating yeasts (Phaffia spp.). Revista Argentina de Microbiología. 2016 Jan
1;48(1):15-20.
31. Muir A, Harrison E, Wheals A. A multiplex set of species-specific primers for rapid
identification of members of the genus Saccharomyces. FEMS yeast research.2011 Nov 1;11
(7):552-63.
32. Glushakova AM, Chernov IY. Seasonal dynamics in a yeast population on leaves of the
common wood sorrel Oxalis acetosella L. Microbiology. 2004 Mar 1;73(2):184-8.
33. Gilbert DG. Dispersal of yeasts and bacteria by Drosophila in a temperate forest. Oecologia.
1980 Jul 1;46(1):135-7.
34. Mittelbach M, Yurkov AM, Nocentini D, Nepi M, Weigend M, Begerow D. Nectar sugars and
bird visitation define a floral niche for basidiomycetous yeast on the Canary Islands. BMC
ecology. 2015 Dec;15(1):2.
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2. Stefanini I. Yeast‐insect associations: It takes guts. Yeast. 2018 Apr;35(4):315-30.
3. Fonseca A, Inácio J. Phylloplane yeasts. InBiodiversity and ecophysiology of yeasts 2006 (pp.
263-301). Springer, Berlin, Heidelberg.
4. Phaff HJ. Yeasts associated with plants, insects and soil. Yeasts/edited by Anthony H. Rose, JS
Harrison. 1987.
5. Kurtzman C, Fell JW, Boekhout T, editors. The yeasts: a taxonomic study. Elsevier;2011May9.
6. Boynton PJ. The ecology of killer yeasts: Interference competition in natural habitats. yeast.
2019 Aug;36(8):473-85.
7. Pereira da Costa D, Campos Miranda-Filho K. The use of carotenoid pigments as food additives
for aquatic organisms and their functional roles. Reviews in Aquaculture. 2020 Aug;12
(3):1567-78.
8. Schmidt I, Schewe H, Gassel S, Jin C, Buckingham J, Hümbelin M, Sandmann G, Schrader J.
Biotechnological production of astaxanthin with Phaffia rhodozyma/Xanthophyllomyces
dendrorhous. Applied microbiology and biotechnology. 2011 Feb 1;89(3):555-71.
9. Hosseinzadeh CA, Fallah F, Yousefzadeh H. Genetic Diversity and Differentiation of the
Iranian's Betula pendula Populations by DNA Polymorphisms of Three (CD, DT, K1K2)
Chloroplast Genome Regions. Iranian Journal of Biology. 2015; 28(2) 191-201.
10. Weber RW. On the ecology of fungal consortia of spring sap-flows. Mycologist. 2006 Nov
1;20(4):140-3.
11. Grabek-Lejko D, Kasprzyk I, Zaguła G, Puchalski C. The bioactive and mineral compounds in
birch sap collected in different types of habitats. Baltic Forestry. 2017 Jan 1;23(2):394-401.
12. Lõoke M, Kristjuhan K, Kristjuhan A. Extraction of genomic DNA from yeasts for PCR-based
applications. Biotechniques. 2011 May;50(5):325-8.
13. Ajorloo, F., Vaez, M., Hemmat, J. Isolation and molecular identification of the carotenoid
producing yeasts in the exudation sap from birch trees (Betula pendula Roth.) at Marmisho
region of Northwest Iran. Journal of Microbial World, 2019; 12(2): 139-149. [In Persian]
14. Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A. Systematics of basidiomycetous yeasts: a
comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS yeast
research. 2002 Dec 1;2(4):495-517.
15. White TJ, Bruns T, Lee SJ, Taylor JL. Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications.
1990;18(1):315-22.
16. Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A. Biodiversity and
systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain
sequence analysis. International journal of systematic and evolutionary microbiology. 2000
May 1;50(3):1351-71.
17. ROSA CA, LACHANCE MA. The yeast genus Starmerella gen. nov. and Starmerella
bombicola sp. nov., the teleomorph of Candida bombicola (Spencer, Gorin & Tullock) Meyer
& Yarrow. International Journal of Systematic and Evolutionary Microbiology. 1998 Oct 1;48
(4):1413-7.
18. Filatov DA. ProSeq: a software for preparation and evolutionary analysis of DNA sequence
data sets. Molecular Ecology Notes. 2002 Dec;2(4):621-4.
19. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool.
Journal of molecular biology. 1990 Oct 5;215(3):403-10.
20. Woffelman C. DNAMAN for Windows, Version 5.2. 10. Lynon Biosoft. Institute of
Molecular Plant Sciences, Netherlands: Leiden University. 2004.
21. Valduga E, Valério A, Tatsch PO, Treichel H, Furigo A, Luccio M. Assessment of Cell
Disruption and Carotenoids Extraction from Sporidiobolus salmonicolor (CBS 2636). Food
and Bioprocess Technology. 2009;2(2):234-8.
22. Weber RW, Davoli P. Teaching techniques for mycology: 20. Astaxanthin, a carotenoid of
biotechnological importance from yeast and salmonid fish. Mycologist. 2003 Feb1;17(1):30-4.
23. Barredo JL, García-Estrada C, Kosalkova K, Barreiro C. Biosynthesis of astaxanthin as a main
carotenoid in the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous. Journal of
Fungi. 2017 Sep;3(3):44.
24. Sorokina M, Steinbeck C. Review on natural products databases: where to find data in 2020.
Journal of cheminformatics. 2020 Dec;12:1-51.
25. Schroeder WA, Johnson EA. Carotenoids protect Phaffia rhodozyma against singlet oxygen
damage. Journal of industrial microbiology. 1995 Jun 1;14(6):502-7.
26. Golubev WI. Xanthophyllomyces Golubev. InThe Yeasts (Fourth Edition) 1998(pp. 718-719).
27. Nahvi I, Vaez M, Emtiazi G. Evaluation of carotenoid-producing yeasts associated with birch
trees (Betula pendula) in the North of Iran-Shahrestanak village. Pajouhesh-va-Sazandegi.
2000; 13(3):70-74. [In Persian]
28. Weber RW, Davoli P, Anke H. A microbial consortium involving the astaxanthin producer
Xanthophyllomyces dendrorhous on freshly cut birch stumps in Germany. Mycologist. 2006
May 1;20(2):57-61.
29. Libkind D, Moliné M, de García V, Fontenla S, van Broock M. Characterization of a novel
South American population of the astaxanthin producing yeast Xanthophyllomyces
dendrorhous (Phaffia rhodozyma). Journal of industrial microbiology & biotechnology. 2008
Mar 1;35(3):151-8.
30. Colabella F, Libkind D. PCR-based method for the rapid identification of
astaxanthin-accumulating yeasts (Phaffia spp.). Revista Argentina de Microbiología. 2016 Jan
1;48(1):15-20.
31. Muir A, Harrison E, Wheals A. A multiplex set of species-specific primers for rapid
identification of members of the genus Saccharomyces. FEMS yeast research.2011 Nov 1;11
(7):552-63.
32. Glushakova AM, Chernov IY. Seasonal dynamics in a yeast population on leaves of the
common wood sorrel Oxalis acetosella L. Microbiology. 2004 Mar 1;73(2):184-8.
33. Gilbert DG. Dispersal of yeasts and bacteria by Drosophila in a temperate forest. Oecologia.
1980 Jul 1;46(1):135-7.
34. Mittelbach M, Yurkov AM, Nocentini D, Nepi M, Weigend M, Begerow D. Nectar sugars and
bird visitation define a floral niche for basidiomycetous yeast on the Canary Islands. BMC
ecology. 2015 Dec;15(1):2.
