Utilization of α-glucosidic sugars by Ogataea (Hansenula) polymorpha
Date
2018-08-27
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Abstract
Pärmid armastavad suhkruid, eelistades glükoosi – kui keskkonnas on palju glükoosi, siis pidurdatakse teiste suhkrute, nt α-glükosiidsete disahhariidide maltoosi ja sahharoosi kasutamist. Seda mehhanismi nimetatakse glükoosi repressiooniks. Maltoosi ja sahharoosi metabolismi on põhjalikult uuritud pagaripärmil Saccharomyces cerevisiae tema kasutuse tõttu pagari- ja õlletööstuses ning bioetanooli tootmisel. Õllepruulimisel on põhiline kääritatav suhkur linnasesuhkur ehk maltoos. Pagaripärmil paiknevad maltoosi kasutamiseks vajalikud geenid MAL-lookustes, mis asuvad telomeeride lähedal. Ka metülotroofne pärm (kasvab metanoolil) O. polymorpha kasvab maltoosil ja sahharoosil.
Selle töö eesmärgiks oli iseloomustada α-glükosiidsete disahhariidide transporti ja metabolismi pärmil O. polymorpha. DNA sekveneerimisel tuvastati O. polymorpha’l neljageeniline MAL-lookus, mis kodeerib maltaas-isomaltaasi MAL1, α-glükosiidi permeaasi MAL2 ja kahte oletavat MAL-aktivaatorit. MAL1 ja MAL2 geenid on hädavajalikud α-glükosiidide kasutamiseks, kuid oletatavate MAL-aktivaatorgeenide funktsioon vajab veel tõestamist. MAL2 on laia substraadivalikuga prootonsümporter, mis transpordib paljusid α-glükosiidseid di- ja trisahhariide. Sarnase substraadivalikuga maltaas-isomaltaas MAL1 meenutab pagaripärmi maltaaside ja isomaltaaside hüpoteetilist eellast – nn ürgmaltaasi. MAL1 mutantide uurimine näitas, et ensüümi substraadi sidumistaskus võiks olla kaks pluss-sidumiskohta ning substraadieelistuse määramisel on valgus väga oluline Thr200. O. polymorpha MAL1-MAL2 geenide vahel on kahesuunaline promootorala, mis võimaldab mõlema geeni koosreguleerimist kasvusubstraatidega.
O. polymorpha heksoosi kinaaside mutantide uurimisel selgus, et MAL1 promootori vaigistamiseks glükoosiga on vajalik glükoosi fosforüülimine rakus, samas toimib fosforüülimata glükoos promootori aktiveerijana. Püstitati hüpotees, et glükoos-6-fosfaat võiks toimida MAL1 promootorit represseeriva signaalina.
Yeasts grow on many different sugars preferring glucose. If glucose is present, utilization of more complex sugars, including disaccharides will be down-regulated. Genetics, biochemistry and regulation of utilization of α-glucosidic disaccharides maltose and sucrose has been extensively studied in baker’s yeast Saccharomyces cerevisiae as baking, brewing and bioethanol production relies mostly on these sugars. In S. cerevisiae, the genes required for maltose metabolism are clustered in MAL-loci at subtelomeric regions of the chromosomes. A methylotrophic yeast Ogataea polymorpha can also assimilate α-glucosidic disaccharides. The aim of this thesis was to characterize the genes and proteins responsible for metabolism of α-glucosidic disaccharides in O. polymorpha. A four-gene MAL cluster was disclosed in O. polymorpha encoding maltase-isomaltase MAL1, α-glucoside permease MAL2 and two putative MAL-activators. MAL1 and MAL2 proteins were shown indispensable for utilization of a wide range of α-glucosidic sugars by O. polymorpha, functionality of the two putative MAL-activators still has to be proven. Maltase-isomaltase MAL1 of O. polymorpha was shown highly similar to ancMALS – a hypothetical ancestor of Saccharomyces maltases and isomaltases. Study of MAL1 mutants revealed two plus-subsites at the substrate binding pocket. Substitution of Thr200 with Val in MAL1 drastically reduced the hydrolysis of maltose-like substrates (α-1,4-glucosides), making it more similar to isomaltases. The MAL1-MAL2 bidirectional promoter was shown coordinately regulated by carbon sources in both directions. On the basis of study of O. polymorpha sugar kinase mutants a hypothesis was raised according to which monosaccharides glucose and fructose repress the MAL1 promoter only if phosphorylated suggesting that glucose-6-phosphate is a sugar repression signaling metabolite for O. polymorpha.
Yeasts grow on many different sugars preferring glucose. If glucose is present, utilization of more complex sugars, including disaccharides will be down-regulated. Genetics, biochemistry and regulation of utilization of α-glucosidic disaccharides maltose and sucrose has been extensively studied in baker’s yeast Saccharomyces cerevisiae as baking, brewing and bioethanol production relies mostly on these sugars. In S. cerevisiae, the genes required for maltose metabolism are clustered in MAL-loci at subtelomeric regions of the chromosomes. A methylotrophic yeast Ogataea polymorpha can also assimilate α-glucosidic disaccharides. The aim of this thesis was to characterize the genes and proteins responsible for metabolism of α-glucosidic disaccharides in O. polymorpha. A four-gene MAL cluster was disclosed in O. polymorpha encoding maltase-isomaltase MAL1, α-glucoside permease MAL2 and two putative MAL-activators. MAL1 and MAL2 proteins were shown indispensable for utilization of a wide range of α-glucosidic sugars by O. polymorpha, functionality of the two putative MAL-activators still has to be proven. Maltase-isomaltase MAL1 of O. polymorpha was shown highly similar to ancMALS – a hypothetical ancestor of Saccharomyces maltases and isomaltases. Study of MAL1 mutants revealed two plus-subsites at the substrate binding pocket. Substitution of Thr200 with Val in MAL1 drastically reduced the hydrolysis of maltose-like substrates (α-1,4-glucosides), making it more similar to isomaltases. The MAL1-MAL2 bidirectional promoter was shown coordinately regulated by carbon sources in both directions. On the basis of study of O. polymorpha sugar kinase mutants a hypothesis was raised according to which monosaccharides glucose and fructose repress the MAL1 promoter only if phosphorylated suggesting that glucose-6-phosphate is a sugar repression signaling metabolite for O. polymorpha.
Description
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Keywords
methylotrophic yeasts, disaccharides, carbohydrate metabolism, regulation (biol.)