Pseudomonas syringae pv. tomato DC3000 levaansukraasid: ekspressioon, biokeemiline iseloomustamine, mutatsioonanalüüs ja polümerisatsiooniproduktide spekter

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Suhkrud on maakeral äärmiselt levinud molekulid ning neil on väga oluline roll elusorganismides. Fruktoosijääkidest koosnevaid suhkruid nimetatakse fruktaanideks ja nad on organismidele põhiliselt varuaineteks. Mõned taimed, näiteks sigur ja maapirn sisaldavad oma juurtes väga palju fruktaani. Fruktaanidele on leitud ka rakendusi näiteks toidutehnoloogias prebiootikumide, emulgaatorite või magusainetena ja meditsiinis vereplasma asendajana. Prebiootikumidena toimivad eelkõige lühiahelalised fruktaanid ehk fruktooligosahhariidid (FOS). FOS-id stimuleerivad kasulike piimhappebakterite hulka ja aktiivsust jämesooles, parandades sellega organismi tervist, pikaahelalised fruktaanid aga turgutavad immuunsüsteemi ja toimivad vähivastaselt. Fruktaane sünteesivad fruktosüültransferaaside abil paljud taime-, seene- ja bakteriliigid. Oma doktoritöös uurisin taimi nakatava bakteri Pseudomonas syringae levaansukraase. Need on põnevad ensüümid, mis sünteesivad sahharoosist, aga ka veel odavamast toorainest, suhkrupeedi melassist, polümeerset fruktaani – levaani ja FOS-e. Huvitaval kombel on sellel mikroobil levaansukraase kodeerivaid geene kolm (lsc1, lsc2, lsc3), kuigi enamus levaani tootvaid baktereid saab hakkama ühega. Kui panna ükskõik milline neist kolmest mainitud DNA lõigust soolekepikesse (Escherichia coli), siis sünteesitakse täiesti töökorras ensüüm. Kui selline soolekepike kasvab sahharoosiga tardsöötmel, siis kattub tema koloonia ohtra limaga – levaaniga. Näitasime esmakordselt, et lisaks sahharoosile võivad pseudomoonastest pärit levaansukraasid kasutada ka rafinoosi ja stahhüoosi, mida leidub palju näiteks sojaoas. Minu doktoritöö peategelane, valk Lsc3 on väga aktiivne ning äärmiselt stabiilne katalüüsija – tema töövõime säilis kõrgel temperatuuril ja ka pikaaegsel säilitamisel. Sellised omadused teevad Lsc3-st valgu, mida võiks kasutada fruktaanide biotehnoloogilisel tootmisel, sest tööstuslikus protsessis on biokatalüsaatori efektiivsus ja stabiilsus ülioluline. Uudse kiibipõhise molekulide massi määramise meetodiga uurisime, milliseid fruktaane Lsc3 sünteesib. Avastasime, et ensüüm võib fruktoosijääke liita mitmetele erinevatele molekulidele nagu ksüloos, ksülobioos, ksülitool ja galakturoonhape, tekitades segusuhkruid. Segusuhkrud on huvitavad, sest neile ennustatakse uudseid bioloogilisi omadusi ja toimeid, näiteks võiksid nad olla eriti tõhusad prebiootikumid.Levansucrases are bacterial enzymes belonging to family 68 of glycoside hydro¬lases (GHs). They catalyze hydrolysis of their substrate but also have prominent fructosyl transferase activity. The main substrate for levansucrases is sucrose which is major disaccharide in plants. Raffinose, likewisely abundant in some plants, is also used as a substrate. Spectrum of reaction products of levan¬sucrases comprises highly polymeric levan and fructooligosaccharides (FOS) of various degree of polymerization (DP). The entity of levansucrase reaction products depends on the enzyme and its origin, but also on reaction conditions enabling manipulation of the product spectrum. Levansucrases share highly similar five-blade β-propeller fold with other GH68 and 32 enzymes including bacterial inulosucrases, plant and microbial invertases, fructan exohydrolases and fructosyl transferases. In this thesis, levansucrases Lsc2 and Lsc3 from a plant pathogenic bac¬terium Pseudomonas syringae pv. tomato were expressed in a bacterial host Escherichia coli, purified and characterized. As a comparison, levansucrase LscA from P. chlororaphis subsp. aurantiaca was studied. The main results of this thesis are summarized as follows: 1. Lsc2 and Lsc3 of P. syringae pv. tomato were expressed with high yield in a bacterial host Escherichia coli exerting two expression systems. The first system relies on maltase gene promoter PMAL from a methylotrophic yeast Hansenula polymorpha. We verified that functionality of PMAL in E. coli is caused by the presence of σ70-like boxes in the eucaryotic promoter. The PMAL was shown to have suitable strength in E. coli providing a sufficient amount of catalytically active protein of interest. Due to its dual activity, it can be used as a promoter shared by yeasts and bacteria in heterologous protein expression trials. A pURI3 vector-based expression system was ad¬justed to obtain mutant and wild-type N-terminally His-tagged Lsc3 proteins. 2. P. syringae pathovars are exceptional among other bacterial species because they possess up to three levansucrase alleles in their genomes. We showed that all three lsc genes (lsc1, lsc2, lsc3) of P. syringae pv. tomato DC3000 encode functional levansucrase proteins, if expressed from a heterologous promoter in E. coli. 3. Enzymology and biochemistry of Lsc2 and Lsc3 was addressed and com¬pared with that of LscA from P. chlororaphis subsp. aurantiaca. All three proteins were shown to use sucrose, raffinose and stachyose as substrates. Low hydrolytic activity towards levan was also recorded. Affinities for sucrose of Lsc3, Lsc2 and LscA were similar, the Km values being around 20 mM. The maximum reaction velocity and catalytic efficiency of LscA was much lower than that of Lsc2 and Lsc3 proteins. Polymerization properties of the enzymes differed. At low sucrose concentration, Lsc3 polymerized much more effectively than LscA. At high substrate concentration, the difference in transfructosylating activity was evened out, but the FOS spectrum was still different – the LscA produced more high-DP FOS than Lsc3 or Lsc2. 4. As a novel feature for levansucrases of pseudomonads, this study shows the ability of Lsc3 and LscA to produce heterooligofructans (HOF) by trans¬fructosylating nonconventional fructosyl acceptors. For the first time, levansucrases were shown to transfructosylate D-sorbitol, D-galacturonic acid, D-mannitol, xylitol, methyl-α-D-glycopyranoside and a disaccharide xylobiose. Novel high-throughput nanoESI HCT mass spectrometry method was implemented and optimized to specify the HOF and conventional FOS. 5. Lsc2 and Lsc3 were shown to be stabile and catalytically active proteins that preserved their activity at various pH and temperature values. They also tolerated presence of several metal ions and detergents. Those characteristics are essential for extracellular proteins and they are important for enzymes to be used in industry. As we showed that the levansucrases of P. syringae pv. tomato can produce biotechnologically promising products levan, FOS and HOF from a cheap substrate, sucrose, they should certainly be regarded as feasible biocatalysts for technological approaches. 6. Whereas no data on structure-function relationships among levansucrases of Pseudomonas bacteria were available, mutational analysis of Lsc3 was initiated. Asp62, Asp219 and Glu303 were predicted as catalytic triad residues of Lsc3. Mutation analysis of Lsc3 specified Thr302 and His321 as residues implicated in substrate binding and transfructosylation reaction possibly belonging to the +1 subsite of the Lsc3 active centre

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

Single-chamber microbial electrosynthesis reactor for nitrate reduction from waters with a low-electron donors’ concentration : from design and set-up to the optimal operating potential

Funding Information: This research was supported by the Estonian Research Council (grant numbers PSG631, PSG714, PRG352) and by the European Union (EU) through the European Regional Development Fund: Centre of Excellence EcolChange, TK 141 Advanced materials and high-technology devices for energy recuperation systems (grant number 2014-2020.4.01.15-0011), the University of Tartu Feasibility Fund (grant number PLTOMARENG51), and the European Structural and Investment Funds.Peer reviewedPublisher PD

Preparation of onion-like multilayered particles comprising mainly poly(iso-butyl methacrylate)-block-polystyrene by two-step AGET ATRP

The role of dietary fiber in supporting healthy gut microbiota and overall well-being of the host has been revealed in several studies. Here, we show the effect of a bacterial polyfructan levan on the growth dynamics and metabolism of fecal microbiota in vitro by using isothermal microcalorimetry. Eleven fecal samples from healthy donors were incubated in phosphate-buffered defined medium with or without levan supplementation and varying presence of amino acids. The generation of heat, changes in pH and microbiota composition, concentrations of produced and consumed metabolites during the growth were determined. The composition of fecal microbiota and profile of metabolites changed in response to substrate (levan and amino acids) availability. The main products of levan metabolism were acetic, lactic, butyric, propionic and succinic acids and carbon dioxide. Associated growth of levan-degrading (e.g. Bacteroides) and butyric acid-producing (e.g. Faecalibacterium) taxa was observed in levan-supplemented media. The study shows that the capacity of levan and possibly also other dietary fibers/prebiotics to modulate the composition and function of colon microbiota can be predicted by using isothermal microcalorimetry of fecal samples linked to metabolite and consortia analyses

Lipopolysaccharide associated with β-2,6 fructan mediates TLR4-dependent immunomodulatory activity in vitro

Levan, a β-2,6 fructofuranose polymer produced by microbial species, has been reported for its immunomodulatory properties via interaction with toll-like receptor 4 (TLR4) which recognises lipopolysaccharide (LPS). However, the molecular mechanisms underlying these interactions remain elusive. Here, we investigated the immunomodulatory properties of levan using thoroughly-purified and characterised samples from Erwinia herbicola and other sources. E. herbicola levan was purified by gel-permeation chromatography and LPS was removed from the levan following a novel alkali treatment developed in this study. E. herbicola levan was then characterised by gas chromatography–mass spectrometry and NMR. We found that levan containing LPS, but not LPS-depleted levan, induced TLR4-mediated cytokine production by bone marrow-derived dendritic cells and/or activated TLR4 reporter cells. These data indicated that the immunomodulatory properties of the levan toward TLR4-expressing immune cells were mediated by the LPS. This work also demonstrates the importance of LPS removal when assessing the immunomodulatory activity of polysaccharides

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

Fructan Enzymes in Microbes and Plants : Structure, Function, and Product Formation

Fructans—fructose-based oligo- or polysaccharides—are de novo synthesized from sucrose by transfructosylating enzymes of microorganisms and plants. Fructan-producing enzymes belong to glycoside hydrolase families 32 and 68. Levansucrases, inulosucrases, some invertases, sucrose:sucrose 1-fructosyl transferases, fructan:fructan 1-fructosyl transferases, sucrose:fructan 6-fructosyl transferases and β-fructofuranosidases synthesize independently or in cascades a wide variety of fructose-containing saccharides. Fructans from different sources often differ in the linkage between the monosaccharide residues and the degree of polymerization. This chapter reviews the literature on fructan-metabolizing enzymes from bacteria, haloarchaea, yeasts, filamentous fungi, mono- and dicot plants. The focus is mostly on the product spectra of the enzymes, structure-function relationships that determine substrate specificities, and on the enzymatic production of fructans or fructo-oligosaccharides that at later stages may lead to practical applications

Structural insight into a yeast maltase : the BaAG2 from blastobotrys adeninivorans with transglycosylating activity

An early-diverged yeast, Blastobotrys (Arxula) adeninivorans (Ba), has biotechnological potential due to nutritional versatility, temperature tolerance, and production of technologically ap-plicable enzymes. We have biochemically characterized from the Ba type strain (CBS 8244) the GH13-family maltase BaAG2 with efficient transglycosylation activity on maltose. In the current study, transglycosylation of sucrose was studied in detail. The chemical entities of sucrose-derived oligosaccharides were determined using nuclear magnetic resonance. Several potentially prebiotic oligosaccharides with α-1,1, α-1,3, α-1,4, and α-1,6 linkages were disclosed among the products. Trisaccharides isomelezitose, erlose, and theanderose, and disaccharides maltulose and trehalulose were dominant transglycosylation products. To date no structure for yeast maltase has been deter-mined. Structures of the BaAG2 with acarbose and glucose in the active center were solved at 2.12 and 2.13 Å resolution, respectively. BaAG2 exhibited a catalytic domain with a (β/α)8-barrel fold and Asp216, Glu274, and Asp348 as the catalytic triad. The fairly wide active site cleft contained water channels mediating substrate hydrolysis. Next to the substrate-binding pocket an enlarged space for potential binding of transglycosylation acceptors was identified. The involvement of a Glu (Glu309) at subsite +2 and an Arg (Arg233) at subsite +3 in substrate binding was shown for the first time for α-glucosidases.This article belongs to the Special Issue: Fungal Enzymes 2021</p