PL244546B1 - Unconventional yeast strain Aureobasidium pullulans URC2 capable of efficient linoleic acid production in standard YPD type medium - Google Patents
Unconventional yeast strain Aureobasidium pullulans URC2 capable of efficient linoleic acid production in standard YPD type medium Download PDFInfo
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- PL244546B1 PL244546B1 PL441040A PL44104022A PL244546B1 PL 244546 B1 PL244546 B1 PL 244546B1 PL 441040 A PL441040 A PL 441040A PL 44104022 A PL44104022 A PL 44104022A PL 244546 B1 PL244546 B1 PL 244546B1
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- aureobasidium pullulans
- urc2
- linoleic acid
- yeast strain
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- 241000223678 Aureobasidium pullulans Species 0.000 title claims abstract description 39
- 101150004794 URC2 gene Proteins 0.000 title claims abstract description 17
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 title claims abstract description 15
- 235000020778 linoleic acid Nutrition 0.000 title claims abstract description 14
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 title claims abstract description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims description 33
- 238000004519 manufacturing process Methods 0.000 title abstract description 18
- 244000005700 microbiome Species 0.000 claims abstract description 8
- 239000007222 ypd medium Substances 0.000 claims abstract description 6
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- 108020004463 18S ribosomal RNA Proteins 0.000 claims abstract 2
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- 239000002609 medium Substances 0.000 abstract description 15
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- 239000008103 glucose Substances 0.000 abstract description 12
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- 150000004665 fatty acids Chemical class 0.000 abstract description 6
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 abstract description 2
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- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 description 3
- 235000020664 gamma-linolenic acid Nutrition 0.000 description 3
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- NEDIAPMWNCQWNW-UHFFFAOYSA-N massoia lactone Chemical compound CCCCCC1CC=CC(=O)O1 NEDIAPMWNCQWNW-UHFFFAOYSA-N 0.000 description 2
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Szczep Aureobasidium pullulans URC2 zdeponowany w Kolekcji Kultur Drobnoustrojów Przemysłowych pod numerem KKP 2086p będący przedmiotem zgłoszenia odznacza się zespołem cech stanowiących o jego unikalności, nie ograniczającym jednak jego zakresu ochrony, do których najważniejsze to: unikatowa sekwencja regionu hiperzmiennego ITS1 genu 18S rRNA zgodna z załącznikiem nr 1, zdolność produkcji w standardowej kompletnej pożywce YPD, której składnikami jest pepton (1%), ekstrakt drożdżowy (1%) i glukoza (2%) przy pH pożywki w zakresie od 6,4 do 6,6 oraz w temperaturze od 23°C do 25°C następujących składników: betaglukanów w zakresie od 41,89% do 43,89% oraz trehalozy od 5 do 6 mg na 1 g suchej masy oraz 1,1 g kwasów tłuszczowych na 100 gram suchej masy, w których udział kwasów wielonienasyconych wynosi od 45% do 48%, a udział kwasu linolowego (C18:2n6c) wynosi od 40,44% do 43,84% to jest od 0,44 g do 0,48 g na 100 g biomasy.The Aureobasidium pullulans URC2 strain deposited in the Collection of Industrial Microorganism Cultures under the number KKP 2086p, which is the subject of the application, is characterized by a set of features that make it unique, but do not limit its scope of protection, the most important of which are: a unique sequence of the ITS1 hypervariable region of the 18S rRNA gene in accordance with Annex No. 1, production capacity in standard complete YPD medium, the ingredients of which are peptone (1%), yeast extract (1%) and glucose (2%) at a medium pH ranging from 6.4 to 6.6 and at a temperature of 23° C to 25°C of the following ingredients: beta-glucans ranging from 41.89% to 43.89% and trehalose from 5 to 6 mg per 1 g of dry matter and 1.1 g of fatty acids per 100 grams of dry matter, in which the fatty acids polyunsaturated fatty acids ranges from 45% to 48%, and the share of linoleic acid (C18:2n6c) ranges from 40.44% to 43.84%, i.e. from 0.44 g to 0.48 g per 100 g of biomass.
Description
Przedmiotem wynalazku jest szczep drożdży niekonwencjonalnych z gatunku Aureobasidium pullulans URC2 wydajnie produkujący kwas linolowy w standardowej pożywce YPD, której składnikami jest pepton (1%), ekstrakt drożdżowy (1%) i glukoza (2%) przy pH pożywki w zakresie od 6,4 do 6,6 oraz w temperaturze wzrostu od 23 do 25°C.The subject of the invention is a strain of unconventional yeast of the species Aureobasidium pullulans URC2 that efficiently produces linoleic acid in standard YPD medium, the components of which are peptone (1%), yeast extract (1%) and glucose (2%) at the pH of the medium in the range of 6.4 to 6.6 and at a growth temperature of 23 to 25°C.
Poszukiwanie nowych mikroorganizmów o unikalnych cechach metabolicznych tj. zdolnych do wytwarzania określonych metabolitów jest kluczowe dla rozwoju gospodarki opartej na biotechnologii (Binati RL, Salvetti E, Bzducha-Wróbel A, Basinskiene L, Cizeikiene D, Bolzonella D, Felis GE, Non-conventional yeasts for food and additives production in a circular economy perspective. FEMS Yeast Res. 2021 Oct 2:foab052. doi: 10.1093/femsyr/foab052).The search for new microorganisms with unique metabolic features, i.e. capable of producing specific metabolites, is crucial for the development of an economy based on biotechnology (Binati RL, Salvetti E, Bzducha-Wróbel A, Basinskiene L, Cizeikiene D, Bolzonella D, Felis GE, Non-conventional yeasts for food and additives production in a circular economy perspective. FEMS Yeast Res. 2021 Oct 2:foab052. doi: 10.1093/femsyr/foab052).
Najczęściej wykorzystywanym gatunkiem mikroorganizmów w przeprowadzaniu procesów biotechnologicznych są drożdże konwencjonalne Saccharomyces cerevisiae. Są one powszechnie stosowane w tradycyjnych procesach fermentacji, głównie w przemyśle spożywczym do wytwarzania etanolu, pieczywa, produkcji wina i piwa (Nandy SK, Srivastava RK. A review on sustainable yeast biotechnological processes and applications, Microbiol Res. 2018, 207:83-90. doi: 10.1016/j.micres.2017.11.013), Drożdże te w procesach metabolicznych wykorzystają glukozę jako podstawowe źródła węgla. Optimum temperaturowe dla wzrostu i rozmnażania niezmodyfikowanych genetycznie szczepów drożdży Saccharomyces cerevisiae to 28-30°C. Wykorzystywanie drożdży Saccharomyces cerevisiae jako producentów metabolitów obarczone jest wieloma trudnościami. Jedną z nich jest inhibicja podziałów komórek drożdży będąca konsekwencją wzrostu zakwaszenia środowiska hodowli podczas prowadzenia bioprocesów. Inną wadą stosowania drożdży Saccharomyces cerevisiae jest termowrażliwość, która często stanowi istotną przeszkodę technologiczną ich wykorzystania dla określonych celów biotechnologicznych. Dodatkowo drożdże Saccharomyces cerevisiae nie są także w stanie przeprowadzać wyspecjalizowanych bioprocesów, które prowadzi się z wykorzystaniem innych drożdży określanych mianem niekonwencjonalnych (Sun L, Alper HS. Non-conventional hosts for the production of fuels and chemicals. Curr Opin Chem Biol. 2020, 59:15-22. doi: 10.1016/j.cbpa.2020.03.004.). Innym potencjalnym ograniczeniem technologicznego wykorzystania drożdży Saccharomyces cerevisiae jest także ich brak zdolności syntezy, wielonienasyconych kwasów tłuszczowych (PUFA) (Yazawa H, Iwahashi H, Kamisaka Y, Kimura K, Uemura H, Production of polyunsaturated fatty acids in yeast Saccharomyces cerevisiae and its relation to alkaline pH: tolerance. Yeast. 2009, 26(3):167-84. doi: 10.1002/yea.1659; Uemura H. Synthesis and production of unsaturated and polyunsaturated fatty acids in yeast: current state and perspectives. Appl Microbiol Biotechnol. 2012, 95(1):1-12. doi: 10.1007/800253-012-4105-1.). Biorąc pod uwagę te względy poszukiwanie nowych gatunków drożdży w celu ich przemysłowych zastosowań jest kluczowe dla zaspokojenia potrzeb sektora gospodarczego oraz przezwyciężenia trudności, które są spotykane podczas wykorzystywania szczepów Saccharomyces cerevisiae.The most frequently used species of microorganisms in biotechnological processes is the conventional yeast Saccharomyces cerevisiae. They are widely used in traditional fermentation processes, mainly in the food industry for the production of ethanol, bread, wine and beer production (Nandy SK, Srivastava RK. A review on sustainable yeast biotechnological processes and applications, Microbiol Res. 2018, 207:83-90 . doi: 10.1016/j.micres.2017.11.013), these yeasts will use glucose as the main source of carbon in their metabolic processes. The temperature optimum for the growth and reproduction of non-genetically modified Saccharomyces cerevisiae yeast strains is 28-30°C. The use of the yeast Saccharomyces cerevisiae as producers of metabolites is fraught with many difficulties. One of them is the inhibition of yeast cell division, which is a consequence of the increase in acidification of the culture environment during bioprocesses. Another disadvantage of using Saccharomyces cerevisiae yeast is thermosensitivity, which often constitutes a significant technological obstacle to their use for specific biotechnological purposes. Additionally, Saccharomyces cerevisiae yeast is also unable to carry out specialized bioprocesses that are carried out using other yeasts called unconventional (Sun L, Alper HS. Non-conventional hosts for the production of fuels and chemicals. Curr Opin Chem Biol. 2020, 59 :15-22. doi: 10.1016/j.cbpa.2020.03.004.). Another potential limitation of the technological use of Saccharomyces cerevisiae yeast is its lack of ability to synthesize polyunsaturated fatty acids (PUFA) (Yazawa H, Iwahashi H, Kamisaka Y, Kimura K, Uemura H, Production of polyunsaturated fatty acids in Saccharomyces cerevisiae yeast and its relation to alkaline pH: tolerance. Yeast. 2009, 26(3):167-84. doi: 10.1002/yea.1659; Uemura H. Synthesis and production of unsaturated and polyunsaturated fatty acids in yeast: current state and perspectives. Appl Microbiol Biotechnol. 2012, 95(1):1-12. doi: 10.1007/800253-012-4105-1.). Taking these considerations into account, the search for new yeast species for industrial applications is crucial to meet the needs of the economic sector and to overcome the difficulties encountered when using Saccharomyces cerevisiae strains.
Jednym z takich gatunków jest Aureobasidium pullulans, który jest grzybem drożdżopodobnym (ang. yeast-like fungus) występującym w glebie, roślinach, ściekach, odpadach oraz wodzie morskiej (Prasongsuk S, Lotrakul P, Ali I. et al. The current status of Aureobasidium pullulans in biotechnology. Folia Microbiol. 2018, 63:129-140. doi.org/10.1007/s12223-017-0561-4). Aureobasidium pullulans jest znanym producentem wytwarzającym szeroką gamę produktów naturalnych o znaczeniu biotechnologicznym. Stanowi on źródło: pullulanu będącego biodegradowalnym zewnątrzkomórkowym polisacharydem (poIi-a-1,6-maltotriozy) (Zalar P, Gostincar C, de Hoog GS, Ursic V, Sudhadham M, Gunde-Cimerman N. Redefinition of Aureobasidium pullulans and its varieties. Studies in Mycology 2008, 61:21-38.One of such species is Aureobasidium pullulans, which is a yeast-like fungus found in soil, plants, sewage, waste and seawater (Prasongsuk S, Lotrakul P, Ali I. et al. The current status of Aureobasidium pullulans in biotechnology. Folia Microbiol. 2018, 63:129-140. doi.org/10.1007/s12223-017-0561-4). Aureobasidium pullulans is a well-known producer producing a wide range of natural products of biotechnological importance. It is a source of: pullulan, which is a biodegradable extracellular polysaccharide (poIi-a-1,6-maltotriose) (Zalar P, Gostincar C, de Hoog GS, Ursic V, Sudhadham M, Gunde-Cimerman N. Redefinition of Aureobasidium pullulans and its varieties. Studies in Mycology 2008, 61:21-38.
doi.org/10.3114/sim.2008.61.02); enzymów takich jak β-glukozydaza, amylazy, celulazy, lipazy, proteazy, ksylanazy, mannanazy (Bozoudi D, Tsaltas D. The Multiple and Versatile Roles of Aureobasidium pullulans in the Vitivinicultural Sector. Fermentation 2018, 4:85. doi:10.3390/fermentation4040085); a także esterazy (Meneses DP, Paixao LMN, Fonteies TV, Gudina EJ, Rodrigues LR, Fernandes FAN, Rodrigues S. Esterase production by Aureobasidium pullulans URM 7059 in stirred tank and airlift bioreactors using residual biodiesel glycerol as substrate. Biochemical Engineering Journal 2021, 168:107954]); liamocyny o działaniu antybakteryjnym (Bischoff KM, Leathers TD, Price NPJ, Manitchotpisit P. Liamocin oil from Aureobasidium pullulans has antibacterial activity with specificity for species of Streptococcus. J. Antibiot. 2015, 68:642] i antyproliferacyjnym w stosunku do niektórych linii komórek nowotworowych; a także grzybowych sideroforów (Chi Z, Wang F, Chi Z, Yue L, Liu G, Zhang T, Bioproducts from Aureobasidium pullulans, a Biotechnologically Important Yeast. Appl. Microbiol. Biotechnol . 2009, 82:793-804; Feng J, Yang J, Li X, Guo M, Wang B, Yang ST, Zou X. Reconstruction of a Genome-Scale Metabolic Model and in Silico Analysis of the Polymalic Acid Producer Aureobasidium pullulans CCTCC M2012223. Gene 2017, 607:1-8). Gatunek ten opisano również jako mikroorganizm mający zdolność wytwarzania związków o charakterze przeciwgrzybiczym (Prasongsuk S, Ployngam S, Wacharasindhu S, Lotrakul P, Punnapayak H. Effects of sugar and amino acid supplementation on Aureobasidium pullulans NRRL 58536 antifungal activity against four Aspergillus species. ApplMicrobiol Biotechnol 2013, 97:7821-7830). W patencie PL/EP 25001793. T3 wykazano zastosowanie szczepu Aureobasidium pullulans jako mikroorganizmu wspomagającego proces fermentacji owoców w temperaturze 14°C, który jednocześnie pomaga ograniczyć wzrost patogenów obecnych na owocach użytych jako surowca w tym procesie. Z kolei w patencie WO2015199349A1 opisano wykorzystanie szczepu Aureobasidium pullulans ksy-0516 do produkcji e-1,3/1,6-glukanu w temperaturze 30°C. Patent US5260214A ujawnił wykorzystanie Aureobasidium pullulans jako producenta antybiotyku R106 w temperaturze 15 do 30°C i w zakresie pH od 2 do 8. Patent WO2017030503A1 wskazał wykorzystanie Aureobasidium pullulans w procesie produkcji na drodze fermentacji związków aromatycznych o nazwie lakton massoia. Aureobasidium pullulans był także oceniany w zakresie jego potencjalnej przydatności jako producent kwasów tłuszczowych, w tym wielonienasyconych kwasów tłuszczowych (Ruinen J, Deinema MH. Composition and properties of the extracellular lipids of yeast species from the phyllosphere. Antonie van Leeuwenhoek. 1964, 30:377-384; Kurosawa T, Sakai K, Nakahara T, Oshima Y, Tabuch T. Extracellular Accumulation of the Polyol Lipids, 3,5-Dihydroxydecanoyl and 5-Hydroxy-2-decenoyl Esters of Arabitol and Mannitol, by Aureobasidium sp., Bioscience, Biotechnology, and Biochemistry. 1994 58(11):2057-2060. doi: 10.1271/bbb.58.2057; Wang C-L, Li Y, Xin F-H, Liu Y-Y, Chi Z-M, Evaluation of single cell oil from Aureobasidium pullulans var. melanogenum P10 isolated from mangrove ecosystems for biodiesel production, Process Biochemistry. 2014, 49(5):725-731. doi.Org/10.1016/j.procbio.2014.02.017; Wang G, Bai T, Miao Z, Ning W, Liang W. Simultaneous production of single cell oil and fumaric acid by a newly isolated yeast Aureobasidium pullulans var. aubasidani DH177. Bioprocess Biosyst Eng. 2018, 41(11):1707-1716. doi: 10.1007/s00449-018-1994-0; Certik M, Breierova E, Jurśikova P. Effect of cadmium on lipid composition of Aureobasidium pullulans grown with added extracellular polysaccharides. International Biodeterioration & Biodegradation. 2005, 55:195-202). Jak wykazano jednak w przytoczonych powyżej publikacjach zawartość produkowanego przez Aureobasidium pullulans kwasu linolowego wahała się od 3,7% do 26%. Z kolei według danych zawartych w tabeli 1 w patencie EP0153134B1 dotyczącym metody umożliwiającej otrzymanie kwasu γ-linolenowego w wyniku prowadzenia procesu fermentacji z użyciem m.in. komórek Aureobasidium pullulans w temperaturze od 23°C do 27°C wymagane jest zachowanie następujących specjalnych warunków takich jak: pH podłoża w zakresie od 3,75 do 6,25, wzbogacenie pożywki hodowlanej w NH4Cl w ilości 5,6 g na litr medium, MgSO4 · 7H2O w ilości 1,2 g na litr medium, glukozę w ilości 151,2 g na litr medium, KH2PO4 w ilości 6 g na litr medium oraz dodatek drożdżowego ekstraktu w ilości 0,12 g na litr medium. Według autorów, w takich warunkach Aureobasidium pullulans jest w stanie produkować kwas linolowy C18:2 w ilości od 33% do 54,9%. Należy jednak podkreślić, że zastrzeżenia patentowe przytoczonego dokumentu EP0153134B1 dotyczą metody otrzymywania kwasu γ-linolenowego z hodowli komórek Rhizopus arrhizus. Dodatkowo przedstawiona metoda otrzymywania kwasu γ-linolenowego posiada także liczne ograniczenia technologiczne związane z koniecznością suplementowania medium w składniki takie jak NH4O, MgSO4 · 7H2O, KH2PO4 oraz glukozę w stężeniu 15,12% jak również koniecznością użycia specjalnej linii technologicznej przedstawionej na rycinie 1 i 2 patentu EP0153134B1. W wymienionym wyżej patencie nie ma również informacji o innych cechach szczepu Aureobasidium pullulans, który został użyty jako producent, a także o miejscu zdeponowania tego mikroorganizmu, tym samym dokument ten nie może stanowić jakiejkolwiek przeszkody dla patentowania nowych szczepów gatunku Aureobasidium pullulans.doi.org/10.3114/sim.2008.61.02); enzymes such as β-glucosidase, amylases, cellulases, lipases, proteases, xylanases, mannanase (Bozoudi D, Tsaltas D. The Multiple and Versatile Roles of Aureobasidium pullulans in the Vitivinicultural Sector. Fermentation 2018, 4:85. doi:10.3390/fermentation4040085 ); and esterases (Meneses DP, Paixao LMN, Fonteies TV, Gudina EJ, Rodrigues LR, Fernandes FAN, Rodrigues S. Esterase production by Aureobasidium pullulans URM 7059 in stirred tank and airlift bioreactors using residual biodiesel glycerol as substrate. Biochemical Engineering Journal 2021, 168:107954]); liamocin with antibacterial activity (Bischoff KM, Leathers TD, Price NPJ, Manitchotpisit P. Liamocin oil from Aureobasidium pullulans has antibacterial activity with specificity for species of Streptococcus. J. Antibiot. 2015, 68:642] and antiproliferative activity in relation to some cell lines cancer; as well as fungal siderophores (Chi Z, Wang F, Chi Z, Yue L, Liu G, Zhang T, Bioproducts from Aureobasidium pullulans, a Biotechnologically Important Yeast. Appl. Microbiol. Biotechnol . 2009, 82:793-804; Feng J, Yang J, Li X, Guo M, Wang B, Yang ST, Zou This species has also been described as a microorganism with the ability to produce antifungal compounds (Prasongsuk S, Ployngam S, Wacharasindhu S, Lotrakul P, Punnapayak H. Effects of sugar and amino acid supplementation on Aureobasidium pullulans NRRL 58536 antifungal activity against four Aspergillus species. ApplMicrobiol Biotechnol 2013, 97:7821-7830). The patent PL/EP 25001793. T3 demonstrates the use of the Aureobasidium pullulans strain as a microorganism supporting the fruit fermentation process at a temperature of 14°C, which at the same time helps limit the growth of pathogens present on the fruit used as raw material in this process. In turn, the patent WO2015199349A1 describes the use of the Aureobasidium pullulans ksy-0516 strain for the production of e-1,3/1,6-glucan at a temperature of 30°C. Patent US5260214A disclosed the use of Aureobasidium pullulans as a producer of the antibiotic R106 at a temperature of 15 to 30°C and in the pH range from 2 to 8. Patent WO2017030503A1 indicated the use of Aureobasidium pullulans in the production process by fermentation of aromatic compounds called massoia lactone. Aureobasidium pullulans was also assessed for its potential usefulness as a producer of fatty acids, including polyunsaturated fatty acids (Ruinen J, Deinema MH. Composition and properties of the extracellular lipids of yeast species from the phyllosphere. Antonie van Leeuwenhoek. 1964, 30:377 -384; Kurosawa T, Sakai K, Nakahara T, Oshima Y, Tabuch T. Extracellular Accumulation of the Polyol Lipids, 3,5-Dihydroxydecanoyl and 5-Hydroxy-2-decenoyl Esters of Arabitol and Mannitol, by Aureobasidium sp., Bioscience , Biotechnology, and Biochemistry. 1994 58(11):2057-2060. doi: 10.1271/bbb.58.2057; Wang C-L, Li Y, Xin F-H, Liu Y-Y, Chi Z-M, Evaluation of single cell oil from Aureobasidium pullulans var. melanogenum P10 isolated from mangrove ecosystems for biodiesel production, Process Biochemistry. 2014, 49(5):725-731. doi.Org/10.1016/j.procbio.2014.02.017; Wang G, Bai T, Miao Z, Ning W, Liang W. Simultaneous production of single cell oil and fumaric acid by a newly isolated yeast Aureobasidium pullulans var. aubasidani DH177. Bioprocess Biosyst Eng. 2018, 41(11):1707-1716. doi: 10.1007/s00449-018-1994-0; Certik M, Breierova E, Jurśikova P. Effect of cadmium on lipid composition of Aureobasidium pullulans grown with added extracellular polysaccharides. International Biodeterioration & Biodegradation. 2005, 55:195-202). However, as shown in the publications cited above, the content of linoleic acid produced by Aureobasidium pullulans ranged from 3.7% to 26%. In turn, according to the data contained in Table 1 in the patent EP0153134B1 concerning a method enabling obtaining γ-linolenic acid as a result of the fermentation process using, among others, Aureobasidium pullulans cells at a temperature of 23°C to 27°C require the following special conditions, such as: pH of the medium in the range of 3.75 to 6.25, enrichment of the culture medium with NH4Cl in the amount of 5.6 g per liter of medium, MgSO4 · 7H2O in the amount of 1.2 g per liter of medium, glucose in the amount of 151.2 g per liter of medium, KH2PO4 in the amount of 6 g per liter of medium and the addition of yeast extract in the amount of 0.12 g per liter of medium. According to the authors, under such conditions Aureobasidium pullulans is able to produce C18:2 linoleic acid in amounts ranging from 33% to 54.9%. However, it should be emphasized that the patent claims of the cited document EP0153134B1 concern the method of obtaining γ-linolenic acid from Rhizopus arrhizus cell cultures. Additionally, the presented method of obtaining γ-linolenic acid also has numerous technological limitations related to the need to supplement the medium with ingredients such as NH4O, MgSO4 · 7H2O, KH2PO4 and glucose at a concentration of 15.12%, as well as the need to use a special technological line shown in Figures 1 and 2 patent EP0153134B1. The above-mentioned patent also does not contain information about other features of the Aureobasidium pullulans strain that was used as the producer, as well as about the place of deposit of this microorganism, therefore this document cannot constitute any obstacle to the patenting of new strains of the Aureobasidium pullulans species.
W niniejszym zgłoszeniu zaproponowano nowy szczep Aureobasidium pullulans URC2 pozwalający przezwyciężyć szereg trudności technologicznych opisanych między innymi w patencie EP0153134B1 oraz innych wymienionych we wcześniej wspomnianych dokumentach. Główną zaletą stosowania szczepu Aureobasidium pullulans URC2 jest wydajna produkcja kwasu linolowego w porównaniu do innych znanych szczepów tego gatunku bez konieczności stosowania zmodyfikowanych pożywek hodowlanych. W szczególności zastosowanie szczepu Aureobasidium pullulans URC2 pozwala ograniczyć zużycie glukozy do produkcji kwasu linolowego ponad 7-krotnie, co ma szczególne znaczenie w obniżeniu kosztów ekonomicznych prowadzenia procesów technologicznych z wykorzystaniem wymienionego szczepu.This application proposes a new strain of Aureobasidium pullulans URC2 that allows overcoming a number of technological difficulties described, among others, in the patent EP0153134B1 and others mentioned in the previously mentioned documents. The main advantage of using the Aureobasidium pullulans URC2 strain is the efficient production of linoleic acid compared to other known strains of this species without the need to use modified culture media. In particular, the use of the Aureobasidium pullulans URC2 strain allows to reduce the consumption of glucose for the production of linoleic acid by more than 7 times, which is of particular importance in reducing the economic costs of conducting technological processes using the mentioned strain.
Istotną zaletą szczepu drożdży niekonwencjonalnych Aureobasidium pullulans URC2 jest zdolność do wzrostu i produkcji kwasu linolowego przy wykorzystaniu standardowej kompletnej pożywki YPD zawierającej pepton (1%), ekstrakt drożdżowy (1%) i glukozę (2%).An important advantage of the unconventional yeast strain Aureobasidium pullulans URC2 is the ability to grow and produce linoleic acid using standard complete YPD medium containing peptone (1%), yeast extract (1%) and glucose (2%).
Szczep Aureobasidium pullulans URC2 ma także istotną przewagę technologiczną w odniesieniu do cech metabolicznych innych szczepów opisywanych w przytoczonych powyżej dokumentach. Szczep Aureobasidium pullulans URC2 będący przedmiotem zgłoszenia patentowego w temperaturze 23-25°C jest w stanie produkować 1,6 razy więcej kwasu linolowego w porównaniu do zidentyfikowanego szczepu tego gatunku opisanego w publikacji Ruinen J, Deinema MH. Composition and properties of the extracellular lipids of yeast species from the phyllosphere. Antonie van Leeuwenhoek. 1964, 30:377-384. Według autorów cytowanego artykułu opisany przez nich szczep Aureobasidium pullulans produkuje kwasy tłuszczowe, których 26% stanowi kwasu linolowy; to jest o1,6 razy mniej niż szczep Aureobasidium pullulans URC2.The Aureobasidium pullulans URC2 strain also has a significant technological advantage in relation to the metabolic features of other strains described in the documents cited above. The strain of Aureobasidium pullulans URC2, which is the subject of the patent application, is able to produce 1.6 times more linoleic acid at a temperature of 23-25°C compared to the identified strain of this species described in the publication by Ruinen J, Deinema MH. Composition and properties of the extracellular lipids of yeast species from the phyllosphere. Antoni van Leeuwenhoek. 1964, 30:377-384. According to the authors of the cited article, the Aureobasidium pullulans strain they describe produces fatty acids, 26% of which is linoleic acid; this is 1.6 times less than the Aureobasidium pullulans strain URC2.
Wyżej opisany szczep Aureobasidium pullulans URC2 zdeponowany w Kolekcji Kultur Drobnoustrojów Przemysłowych pod numerem KKP 2086p będący przedmiotem zgłoszenia zgodnie z wynalazkiem charakteryzuje się tym, że posiada zespół cech stanowiących o jego unikalności, nie ograniczającym jednak jego zakresu ochrony, do których należą: sekwencja regionu hiperzmiennego 1TS1 genu 18S rRNA zgodna z załącznikiem nr 1, duże zróżnicowanie morfologiczne, elipsoidalnego kształtu komórek o długości w zakresie od 5 do 15 μm oraz szerokości w zakresie od 3 do 7,5 μm zgodnie ryciną A załącznika nr 2, charakterystyczny profil ilości DNA w jądrze komórkowym według histogramu przedstawionego na rycinie B załącznika nr 2 zgodnie z procedurą podaną w publikacji Potocki L, Baran A, Oklejewicz B, Szpyrka E, Podbielska M, Schwarzbacherova V. Synthetic Pesticides Used in Agricultural Production Promote Genetic Instability and Metabolic Variability in Candida spp. Genes (Basel): 2020, 11(8):848. Published 2020. Jul 24. doi:10.3390/genes11080848, obecność dużego prążka chromosomowego w zakresie od 2,2 Mb do 1,6 Mb uwidocznionego po zakończeniu elektroforezy w zmiennym, homogennym polu elektrycznym -PFGE-CHEF w warunkach rozdziału typowego dla chromosomów Saccharomyces cerevisiae (separację prowadzono przez 24 godziny w buforze 0,5X TBE, w temperaturze 14°C; pierwsze 14 godzin z początkowym czasem pulsu 60 s i końcowym czasem pulsu 120 sekund przy napięciu 6 Volt/cm, a pozostałe 10 godzin z początkowym czasem pulsu 60 s i końcowym czasem pulsu 120 sekund przy napięciu 4,5 V/cm), co zobrazowano na rycinie C załącznika nr 2, a także zdolność wzrostu na źródłach węgla takich jak glukoza, fruktoza, galaktoza, ksyloza oraz glicerol oraz zdolność produkcji etanolu w zakresie od 17 do 53 g/L w czasie alkoholowej fermentacji glukozy w temperaturze 25°C w pożywce minimalnej o składzie 1,7% yeast nitrogen base, 0,5% siarczan amonu, 10% glukoza przy mieszaniu 140 obr./min oraz zdolność produkcji etanolu w zakresie od 5 do 15 g/L w czasie alkoholowej fermentacji fruktozy w temp. 25°C w pożywce minimalnej o składzie 1,7% yeast nitrogen base, 0,5% siarczan amonu, 10% fruktoza przy mieszaniu 140 obr./min oraz zdolność produkcji etanolu od 2 do 9 g/L w czasie alkoholowej fermentacji ksylozy w temp. 25°C w pożywce minimalnej o składzie 1,7% yeast nitrogen base, 0,5% siarczan amonu, 10% ksylozy, przy mieszaniu 140 obr./min oraz zdolność alkalizowania środowiska w 7 dniu hodowli przez kolonie komórkowe oraz zdolność produkcji w standardowej kompletnej pożywce YPD, której składnikami jest 1% pepton, 1% ekstrakt drożdżowy i 2% glukoza przy pH pożywki w zakresie od 6,4 do 6,6 oraz w temperaturze od 23 do 25°C następujących składników: betaglukanów w zakresie od 41,89 do 43,89% oraz trehalozy od 5 do 6 mg na 1 g suchej masy oraz 1,1 g kwasów tłuszczowych na 100 g suchej masy, w których udział kwasów wielonienasyconych wynosi od 45 do 48%, a udział kwasu linolowego (C18:2n6c) wynosi od 40,44 do 43,84% to jest od 0,44 g do 0,48 g na 100 g biomasy.The above-described Aureobasidium pullulans URC2 strain deposited in the Collection of Industrial Microorganism Cultures under the number KKP 2086p, which is the subject of the application in accordance with the invention, is characterized by the fact that it has a set of features that make it unique, but do not limit its scope of protection, which include: the sequence of the 1TS1 hypervariable region 18S rRNA gene in accordance with Annex No. 1, large morphological diversity, ellipsoidal shape of cells with a length ranging from 5 to 15 μm and a width ranging from 3 to 7.5 μm in accordance with Figure A of Annex No. 2, a characteristic profile of the amount of DNA in the cell nucleus according to the histogram presented in Figure B of Appendix 2 in accordance with the procedure given in the publication Potocki L, Baran A, Oklejewicz B, Szpyrka E, Podbielska M, Schwarzbacherova V. Synthetic Pesticides Used in Agricultural Production Promote Genetic Instability and Metabolic Variability in Candida spp. Genes (Basel): 2020, 11(8):848. Published 2020. Jul 24. doi:10.3390/genes11080848, presence of a large chromosome band ranging from 2.2 Mb to 1.6 Mb, visible after electrophoresis in an alternating, homogeneous electric field -PFGE-CHEF under separation conditions typical for Saccharomyces cerevisiae chromosomes (separation was carried out for 24 hours in 0.5X TBE buffer, at 14°C; the first 14 hours with an initial pulse time of 60 s and a final pulse time of 120 seconds at a voltage of 6 Volts/cm, and the remaining 10 hours with an initial pulse time of 60 s and final pulse time of 120 seconds at a voltage of 4.5 V/cm), as shown in Figure C of Appendix 2, as well as the ability to grow on carbon sources such as glucose, fructose, galactose, xylose and glycerol and the ability to produce ethanol in the range of 17 up to 53 g/L during alcoholic fermentation of glucose at 25°C in minimal medium composed of 1.7% yeast nitrogen base, 0.5% ammonium sulfate, 10% glucose with mixing at 140 rpm and the ability to produce ethanol in range from 5 to 15 g/L during alcoholic fermentation of fructose at 25°C in a minimal medium composed of 1.7% yeast nitrogen base, 0.5% ammonium sulfate, 10% fructose with mixing at 140 rpm and ethanol production capacity from 2 to 9 g/L during alcoholic fermentation of xylose at 25°C in minimal medium composed of 1.7% yeast nitrogen base, 0.5% ammonium sulfate, 10% xylose, with mixing at 140 rpm. /min and the ability to alkalize the environment on the 7th day of culture by cell colonies and the ability to produce in the standard complete YPD medium, the ingredients of which are 1% peptone, 1% yeast extract and 2% glucose at the pH of the medium ranging from 6.4 to 6.6 and at a temperature of 23 to 25°C of the following ingredients: beta-glucans ranging from 41.89 to 43.89% and trehalose from 5 to 6 mg per 1 g of dry matter and 1.1 g of fatty acids per 100 g of dry matter, in the share of polyunsaturated acids is from 45 to 48%, and the share of linoleic acid (C18:2n6c) is from 40.44 to 43.84%, i.e. from 0.44 g to 0.48 g per 100 g of biomass.
Przedmiot wynalazku-przedstawiono w niżej zamieszczonym przykładzie jego wykonania, nie ograniczającym zakresu ochrony.The subject of the invention is presented in the example of its embodiment below, which does not limit the scope of protection.
P rzykła d 1 wykorzystania szczepu drożdży niekonwencjonalnych Aureobasidium pullulans URC2 do wydajnej produkcji kwasu linolowego w standardowej pożywce typu YPD.Example 1 of using the unconventional yeast strain Aureobasidium pullulans URC2 for efficient production of linoleic acid in standard YPD type medium.
Hodowlę prowadzono w kolbach Erlenmeyera o pojemności 100 mL w płynnej kompletnej pożywce YPD (pepton 10 g/L; ekstrakt drożdżowy 10 g/L) i glukoza 20 g/L; pH 6,5) poddawanej uprzednio sterylizacji w temperaturze 121°C przez 20 minut. Objętość pożywki do hodowli wynosiła 25 mL, tak przygotowaną pożywkę zaszczepiano kulturą komórek Aureobasidium pullulans URC2. Hodowlę prowadzono przez okres 24 h na wytrząsarce obrotowej w temperaturze 25°C i 150 obr./min, po czym hodowle komórek przenoszono do sterylnych próbówek o pojemności 50 mL i zbierano komórki przez wirowanie przy 7000 RPM przez 5 min w temperaturze 20°C. Supernatant usuwano a komórki w próbówkach przepłukiwano sterylną wodą w objętości 15 ml po czym ponownie je zwirowywano przy tych samych parametrach wirowania. Po zakończonym wirowaniu supernatant usuwano pozostawiając pelet komórek, które zostawały poddane liofilizacji przy następujących parametrach: temp. -40°C, ciśnienie 0,12 mbarów, czas 48 h, Po zakończeniu procesu liofilizacji oceniano wagę suchej biomasy Aureobasidium pullulans URC2. Do dalszej procedury odważano 10 mg liofilizowanych drożdży Aureobasidium pullulans URC2, które umieszczono w 2 mL fiolce chromatograficznej, do której dodatkowo, dodawano 25 μΐ wzorca wewnętrznego C15:0 (1000 μg/mL), 200 μΐ mieszaniny dichlorometan : metanol (2 : 1, v/v] oraz 300 μΐ 0,6 M roztworu HCl w metanolu. Fiolki szczelnie zamykano, a zawartość fiolki wytrząsano do momentu umieszczenia jej w suszarce ogrzanej do temperatury 85°C ±3°C na 1 godzinę. Po tym czasie fiolki wyjmowano z suszarki i studzono przez okres 15 minut do temperatury pokojowej a następnie do fiolki dodawano 1 mL eteru naftowego i zawartość fiolki wytrząsano przez 5 minut, po czym pozostawiano na 1 godzinę do rozdzielenia faz. Z górnej fazy pobierano 100 μL cieczy, którą przenoszono do 2 mL fiolki chromatograficznej i następnie dodawano 400 μΐ eteru naftowego. Tak przygotowane próbki poddawano analizie na zawartość kwasów tłuszczowych za pomocą techniki chromatografii sprzężonej ze spektrometrią mas (chromatograf gazowy, model 7890A, Agilent Technologies, Palo Alto, CA, USA sprzężony z detektorem mas, model 7000).Culture was performed in 100 mL Erlenmeyer flasks in liquid complete YPD medium (peptone 10 g/L; yeast extract 10 g/L) and glucose 20 g/L; pH 6.5) previously sterilized at 121°C for 20 minutes. The volume of the culture medium was 25 mL, the prepared medium was inoculated with a culture of Aureobasidium pullulans URC2 cells. The culture was carried out for 24 h on a rotary shaker at 25°C and 150 rpm, after which the cell cultures were transferred to sterile 50 mL test tubes and the cells were collected by centrifugation at 7000 RPM for 5 min at 20°C. The supernatant was removed and the cells in the tubes were rinsed with sterile water in a volume of 15 ml and then centrifuged again using the same centrifugation parameters. After centrifugation, the supernatant was removed, leaving a cell pellet that was lyophilized at the following parameters: temperature -40°C, pressure 0.12 mbar, time 48 h. After the lyophilization process, the weight of the dry Aureobasidium pullulans URC2 biomass was assessed. For the further procedure, 10 mg of freeze-dried Aureobasidium pullulans URC2 yeast was weighed and placed in a 2 mL chromatography vial, to which were additionally added 25 μΐ of the internal standard C15:0 (1000 μg/mL), 200 μΐ of the dichloromethane: methanol mixture (2: 1, v/v] and 300 μΐ 0.6 M HCl solution in methanol. The vials were tightly closed and the contents of the vial were shaken until they were placed in a dryer heated to 85°C ± 3°C for 1 hour. After that time, the vials were removed from the dryer and cooled for 15 minutes to room temperature, then 1 mL of petroleum ether was added to the vial and the contents of the vial were shaken for 5 minutes and then left for 1 hour for phase separation. 100 μL of liquid was taken from the upper phase and transferred to 2 mL chromatography vial and then 400 μΐ of petroleum ether were added. The samples thus prepared were analyzed for fatty acid content using the chromatography technique coupled with mass spectrometry (gas chromatograph, model 7890A, Agilent Technologies, Palo Alto, CA, USA coupled with a mass detector, model 7000 ).
Do analizy zastosowano następujące parametry pracy aparatu: tryb monitorowania wybranych jonów - SIM (Single lon Monitoring), temperatury dozownika, źródła jonów, linii transferowej i kwadrupoli wynosiły odpowiednio: 250°C, 230°C, 250°C i 150°C, typ jonizacji - elektronowa (El, 70 eV), gaz nośny - hel 1,0 mL/min, program temperaturowy: 40°C utrzymywane przez 2 minuty, wzrost do 220°C (30°C/min), temperatura końcowa 260°C (2°C/min), utrzymywana przez 1 min. Czas analizy 29 minut. Objętość nanoszonej próbki 1 μl. Kolumna HP-5 MS (Ultra Inert / 30 m χ 0.25 mm I.D. χ 0.25-μm). Liniowość została oznaczona na podstawie 6-punktowych krzywych kalibracyjnych (współczynnik R2 0,925-0,999). Do akwizycji i przetwarzania danych zastosowano oprogramowanie Mass Hunter ver. 07.06.The following operating parameters of the apparatus were used for analysis: selected ion monitoring mode - SIM (Single lon Monitoring), temperatures of the dispenser, ion source, transfer line and quadrupole were respectively: 250°C, 230°C, 250°C and 150°C, type ionization - electron (El, 70 eV), carrier gas - helium 1.0 mL/min, temperature program: 40°C maintained for 2 minutes, increase to 220°C (30°C/min), final temperature 260°C (2°C/min), held for 1 min. Analysis time 29 minutes. The volume of sample applied is 1 μl. HP-5 MS column (Ultra Inert/30 m χ 0.25 mm ID χ 0.25-μm). Linearity was determined on the basis of 6-point calibration curves (R 2 coefficient 0.925-0.999). Mass Hunter ver. software was used for data acquisition and processing. 07/06.
Oznaczenie ilościowe wykonano metodą wzorca zewnętrznego z zastosowaniem 37 Supelco component FAME MIX CRM47885 (Merck KGaA, Darmstadt, Germany).Quantification was performed using the external standard method using 37 Supelco component FAME MIX CRM47885 (Merck KGaA, Darmstadt, Germany).
W obliczeniach uwzględniono odzysk metody, który obliczono na podstawie dodanego wzorca wewnętrznego (ISTD tripentadecanoin, Merck KGaA, Darmstadt, Germany).The calculations included the method recovery, which was calculated based on the added internal standard (ISTD tripentadecanoin, Merck KGaA, Darmstadt, Germany).
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