α-glükosiidsete suhkrute kasutamine pärmil Ogataea (Hansenula) polymorpha

Väitekirja elektrooniline versioon ei sisalda publikatsioonePärmid 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

Maltase protein of <i>Ogataea </i>(<i>Hansenula</i>) <i>polymorpha </i>is a counterpart to resurrected ancestor protein ancMALS of yeast maltases and isomaltases

Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α‐methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α‐glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α‐glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose‐like substrates (α‐1,4‐glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate‐binding pocket of MAL1 has three subsites (–1, +1 and +2) and that binding is strongest at the –1 subsite. The DSF assay results were in good accordance with affinity (K (m)) and inhibition (K (i)) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α‐glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd

Genome Mining of Non-Conventional Yeasts: Search and Analysis of MAL Clusters and Proteins

Genomic clustering of functionally related genes is rare in yeasts and other eukaryotes with only few examples available. Here, we summarize our data on a nontelomeric MAL cluster of a non-conventional methylotrophic yeast Ogataea (Hansenula) polymorpha containing genes for &alpha;-glucosidase MAL1, &alpha;-glucoside permease MAL2 and two hypothetical transcriptional activators. Using genome mining, we detected MAL clusters of varied number, position and composition in many other maltose-assimilating non-conventional yeasts from different phylogenetic groups. The highest number of MAL clusters was detected in Lipomyces starkeyi while no MAL clusters were found in Schizosaccharomyces pombe and Blastobotrys adeninivorans. Phylograms of &alpha;-glucosidases and &alpha;-glucoside transporters of yeasts agreed with phylogenesis of the respective yeast species. Substrate specificity of unstudied &alpha;-glucosidases was predicted from protein sequence analysis. Specific activities of Scheffersomycesstipitis &alpha;-glucosidases MAL7, MAL8, and MAL9 heterologously expressed in Escherichia coli confirmed the correctness of the prediction&mdash;these proteins were verified promiscuous maltase-isomaltases. &alpha;-Glucosidases of earlier diverged yeasts L. starkeyi, B. adeninivorans and S. pombe showed sequence relatedness with &alpha;-glucosidases of filamentous fungi and bacilli

Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans

Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two &alpha;-glucosidases of GH13 family. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 4‒7) and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates (palatinose, &alpha;-methylglucoside) were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 &micro;M) and Tris (Ki = 70.5 &micro;M). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product&mdash;glucose. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae

High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan

Bacterial levansucrases polymerize fructose residues of sucrose to β-2,6 linked fructans—fructooligosaccharides (FOS) and levan. While β-2,1-linked FOS are widely recognized as prebiotics, the health-related effects of β-2,6 linked FOS are scarcely studied as they are not commercially available. Levansucrase Lsc3 (Lsc-3) of Pseudomonas syringae pv. tomato has very high catalytic activity and stability making it a promising biotechnological catalyst for FOS and levan synthesis. In this study we evaluate feasibility of several high-throughput methods for screening and preliminary characterization of levansucrases using 36 Lsc3 mutants as a test panel. Heterologously expressed and purified His-tagged levansucrase variants were studied for: (1) sucrose-splitting activity; (2) FOS production; (3) ability and kinetics of levan synthesis; (4) thermostability in a Thermofluor assay. Importantly, we show that sucrose-splitting activity as well as the ability to produce FOS can both be evaluated using permeabilized levansucrase-expressing E. coli transformants as catalysts. For the first time we demonstrate the key importance of Trp109, His113, Glu146 and Glu236 for the catalysis of Lsc3. Cost-effective and high-throughput methods presented here are applicable not only in the levansucrase assay, but have a potential to be adapted for high-throughput (automated) study of other enzymes

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal <i>Bacteroides thetaiotaomicron</i> Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass

<div><p><i>Bacteroides thetaiotaomicron</i>, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by <i>Zymomonas mobilis</i> degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by <i>B</i>. <i>thetaiotaomicron</i>, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of <i>Pseudomonas syringae</i> pv. tomato, its mutant Asp300Asn, levansucrases of <i>Zymomonas mobilis</i>, <i>Erwinia herbicola</i>, <i>Halomonas smyrnensis</i> as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the T_m of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for <i>B</i>. <i>thetaiotaomicron</i> and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of <i>Bacteroides xylanisolvens</i> of human origin a putative levan degradation locus was disclosed.</p></div

Size-distribution of levans used in the study.

<p>Size-distribution of levans used in the study.</p

Initial velocities of reducing sugar release by the endo-levanase BT1760 from various levans added at 5 g/L.

<p>* Synthesized by <i>Pseudomonas syringae</i> pv. tomato levansucrase Lsc3 or its mutant Asp300Asn (D300N) from sucrose or raffinose (Raf). Mean values and standard deviation were calculated from at least three independent experiments. For additional information on levans, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169989#pone.0169989.t001" target="_blank">Table 1</a>.</p