Rational transformation of Lactobacillus reuteri 121 reuteransucrase into a dextransucrase

Glucansucrase or glucosyltransferase (GTF) enzymes of lactic acid bacteria display high sequence similarity but catalyze synthesis of different alpha-glucans (e.g., dextran, mutan, alternan, and reuteran) from sucrose. The variations in glucosidic linkage specificity observed in products of different glucansucrase enzymes appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. This notion was derived from mutagenesis of amino acids of GTFA (reuteransucrase) from Lactobacillus reuteri strain 121 putatively involved in acceptor substrate binding. A triple amino acid mutation (N1134S:N1135E:S1136V) in a region immediately next to the catalytic Asp1133 (putative transition state stabilizing residue) converted GTFA from a mainly alpha-(1-->4) ( approximately 45%, reuteran) to a mainly alpha-(1-->6) ( approximately 80%, dextran) synthesizing enzyme. The subsequent introduction of mutation P1026V:I1029V, involving two residues located in a region next to the catalytic Asp1024 (nucleophile), resulted in synthesis of an alpha-glucan containing only a very small percentage of alpha-(1-->4) glucosidic linkages ( approximately 5%) and a further increased percentage of alpha-(1-->6) glucosidic linkages ( approximately 85%). This changed glucosidic linkage specificity was also observed in the oligosaccharide products synthesized by the different mutant GTFA enzymes from (iso)maltose and sucrose. Amino acids crucial for glucosidic linkage type specificity of reuteransucrase have been identified in this report. The data show that a combination of mutations in different regions of GTF enzymes influences the nature of both the glucan and oligosaccharide products. The amino acids involved most likely contribute to sugar-binding acceptor subsites in glucansucrase enzymes.     

Molecular characterization of a novel glucosyltransferase from Lactobacillus reuteri strain 121 synthesizing a unique,highly branched glucan with alpha-(1-->4) and alpha-(1-->6) glucosidic bonds

Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the alpha-(1-->4) glucosidic type. The glucan also contains alpha-(1-->6)-linked glucosyl units and 4,6-disubstituted alpha-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M(r) of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.     

Highly hydrolytic reuteransucrase from probiotic Lactobacillus reuteri strain ATCC 55730

Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the alpha-(1-->4) glucosidic type ( approximately 70%). This reuteran also contains alpha-(1-->6)- linked glucosyl units and 4,6-disubstituted alpha-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing alpha-(1-->4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of alpha-(1-->4) linkages reported to date.     

Highly Hydrolytic Reuteransucrase from Probiotic Lactobacillus reuteri Strain ATCC 55730

Lactobacillus reuteri strain ATCC 55730 (LB BIO) was isolated as a pure culture from a Reuteri tablet purchased from the BioGaia company. This probiotic strain produces a soluble glucan (reuteran), in which the majority of the linkages are of the α-(1→4) glucosidic type (~70%). This reuteran also contains α-(1→6)- linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. The LB BIO glucansucrase gene (gtfO) was cloned and expressed in Escherichia coli, and the GTFO enzyme was purified. The recombinant GTFO enzyme and the LB BIO culture supernatants synthesized identical glucan polymers with respect to linkage type and size distribution. GTFO thus is a reuteransucrase, responsible for synthesis of this reuteran polymer in LB BIO. The preference of GTFO for synthesizing α-(1→4) linkages is also evident from the oligosaccharides produced from sucrose with different acceptor substrates, e.g., isopanose from isomaltose. GTFO has a relatively high hydrolysis/transferase activity ratio. Complete conversion of 100 mM sucrose by GTFO nevertheless yielded large amounts of reuteran, although more than 50% of sucrose was converted into glucose. This is only the second example of the isolation and characterization of a reuteransucrase and its reuteran product, both found in different L. reuteri strains. GTFO synthesizes a reuteran with the highest amount of α-(1→4) linkages reported to date.     

Molecular Characterization of a Novel Glucosyltransferase from Lactobacillus reuteri Strain 121 Synthesizing a Unique, Highly Branched Glucan with α-(1→4) and α-(1→6) Glucosidic Bonds

Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the α-(1→4) glucosidic type. The glucan also contains α-(1→6)-linked glucosyl units and 4,6-disubstituted α-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M_r of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.     

Characterization of a glucansucrase from Lactobacillus reuteri E81 and production of malto-oligosaccharides

Abstract

Glucansucrases, which can be produced by different Lactic Acid Bacteria (LAB), catalyze the synthesis of α-glucans with different structures and properties using sucrose as substrate. In this study, a novel glucansucrase (GTFA) from Lactobacillus reuteri E81 was identified and heterologously expressed. Alignments of GTFA with other glucansucrases revealed its novelty and a putative 3D model structure was obtained. The biochemical properties of the truncated enzyme without the N-terminal variable region, GTFA-ΔN, was characterized. The K_m and V_max were found to be 7.5?mM and 1.49?IU/mg, respectively, and it showed optimum activities at pH 7 and at 50?°C. The GTFA-ΔN produced in vitro an α-glucan with (α1 → 3) and (α1 → 6) glycosidic linkages using sucrose as the substrate. Importantly, GTFA-ΔN synthesized DP = 9 oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively, as detected by TLC, HPLC, LC-MS and NMR analysis.     

Structural analysis of the alpha-D-glucan (EPS35-5) produced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme

The neutral exopolysaccharide EPS35-5 (reuteran) produced from sucrose by the glucansucrase GTFA enzyme from Lactobacillus reuteri 35-5 was found to be a (1-->4,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis and 1D/2D 1H and 13C NMR spectroscopy of intact EPS35-5, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis and enzymatic hydrolysis, using pullulanase M1 (Klebsiella planticola), of EPS35-5, a composite model, that includes all identified structural elements, was formulated as follows: [Formula: see text].     

Differential inhibition of branching enzyme in a morphological mutant and in wild type Neurospora. Influence of carbon source in the growth medium.

1. A morphological mutant of Neurospora crassa, smco 9, (R2508) that exhibits colonial morphology when grown on sucrose or on maltose, showed a partial reversal of this morphology toward that of the wild type when it was grown on potato starch or on isomaltose. 2. A common feature of both potato starch and isomaltose is the presence of alpha-1, 6 glucosidic linkages. This suggested that these morphological effects might be due to differences in alpha-1,4 glucan: alpha-1,4 glucan 6 glycosyltransferase, (EC 2.4.1.18) commonly known as "the branching enzyme". 3. The branching enzyme was purified from wild type, Neurospora crassa, and from the semicolonial mutant, R2508, both grown on sucrose or on potato starch. It has a molecular weight of 140,000 as estimated by gel filtration on a Bio Gel A 1.5 m column. This enzyme plus phosphorylase a in an unprimed reaction catalyzes the synthesis of a branched polysaccharide in vitro. 4. No branching enzyme activity was apparent in extracts of the mutant R2508, grown on potato starch until a thermolabile inhibitor was removed by fractionation on a DEAE column. 5. This inhibitor has a molecular weight greater than 100,000 as estimated on a P-100 polyacrylamide gel column. The specificity of the inhibitor is not absolute in that it inhibits glycogen synthetase in addition to the branching enzyme in Neurospora.     

Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355

The gene encoding alternansucrase (ASR) from Leuconostoc mesenteroides NRRL B-1355, an original sucrose glucosyltransferase (GTF) specific to alternating alpha-1,3 and alpha-1,6 glucosidic bond synthesis, was cloned, sequenced and expressed into Escherichia coli. Recombinant enzyme catalyzed oligoalternan synthesis from sucrose and maltose acceptor. From sequence comparison, it appears that ASR possesses the same domains as those described for GTFs specific to either contiguous alpha-1,3 osidic bond or contiguous alpha-1,6 osidic bond synthesis. However, the variable region and the glucan binding domain are longer than in other GTFs (by 100 and 200 amino acids respectively). The N-catalytic domain which presents 49% identity with the other GTFs from L. mesenteroides possesses the three determinants potentially involved in the glucosyl enzyme formation.     

Three exopolysaccharides of the beta-(1-->6)-D-glucan type and a beta-(1-->3;1-->6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit

Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha, and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and (13)C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1-->3) linkages substituted at O-6 as shown in the putative structure below: [carbohydrate structure: see text]. The EPS of the other fungal isolates consisted of a linear chain of (1-->6)-linked glucopyranosyl residues of the following structure: [carbohydrate structure: see text]. FTIR spectra showed one band at 891 cm(-1), and (13)C NMR spectroscopy showed that all glucosidic linkages were of the beta-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. beta-(1-->6)-d-Glucans produced as exocellular polysaccharides by fungi are uncommon.     

Plant sucrose-H+ symporters mediate the transport of vitamin H

A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake-deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP), suggesting an energy-dependent biotin-H+ symport mechanism. The transport activity could contribute to the so-far uncharacterized plant sucrose-H+ symporter AtSUC5 which mediates the energy-dependent transport of biotin and sucrose, and restores growth of the biotin transport-deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin-H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.     

Molecular cloning and characterization of a novel glucansucrase from Leuconostoc mesenteroides subsp. mesenteroides LM34

Leuconostoc mesenteroides LM34 was isolated from kimchi, a traditional fermented Korean food. L. mesenteroides LM34 produced extracellular glucansucrase (DSRLM34), which is responsible for the synthesis of soluble glucan using sucrose. The DSRLM34 gene consists of a 4,503 bp open reading frame (ORF) and encodes an enzyme of 1,500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (98%) to that of glucansucrase of Lactobacillus lactis. The gene was over-expressed in Escherichia coli strain and the recombinant enzyme (rDSRLM34) was purified. Both DSRLM34 and rDSRLM34 synthesized glucan mainly containing α-1, 6 glucosidic linkage and branched α-1, 3 glucosidic linkages. The enzyme exhibited optimum activity at 30°C and pH 5.0. DSRLM34 has promising potential as a thickening agent in sucrose-supplemented milk.     

Podophyllum peltatum possesses a beta-glucosidase with high substrate specificity for the aryltetralin lignan podophyllotoxin

A beta-glucosidase with high specificity for podophyllotoxin-4-O-beta-D-glucopyranoside was purified from the leaves of Podophyllum peltatum. The 65-kDa polypeptide had optimum activity at pH 5.0 and was essentially inactive at pH 6.5 or above. Maximum catalytic activity of this glucosidase was obtained at 45 degrees C, but the enzyme was not heat stable. This beta-glucosidase displayed higher substrate specificity for podophyllotoxin-4-O-beta-D-glucopyranoside than for the other lignans tested, and for the (1-->3) linkage of laminaribiose than for other glucosidic linkages.     

The epidermis of Timarete filigera (Polychaeta, Cirratulidae): histochemical and ultrastructural analysis of the gland cells

The aim of this study was to verify whether different living conditions of Polychaeta are correlated with morphological and functional differences in the organization of the integument. For this purpose, we decided to study the epidermis of Timarete filigera, a non-tubicolous polychaete. With this objective in mind, we have identified the various cellular types responsible for mucous secretion in the epidermis of this species and defined the histochemical composition of the mucus produced by different types of gland cells. Three types of gland cells have been identified by histochemical and ultrastructural studies in the epidermis of this polychaete. The histochemistry was carried out using standard techniques and peroxidase-labelled lectins. In type 1 cells, the secretory granules contain neutral glycoproteins with glucosidic residues of GalNAc, Galbeta 1,3 GalNAc, glucosidic and/or mannosidic residues. In type 2 cells, the secretory granules contain acid glycoproteins mainly sulphated with glucosidic residues of GalNAc, Galbeta 1,3 GalNAc, glucosidic and/or mannosidic residues, and some terminal sialic acid. In type 3 cells, the residual granules have the same chemical composition as that of granules present in type 2 cells. The secretion of these glandular mucous cells consists of mainly sulphated acidic glycoproteins and GAG resistant to testis jaluronidase. In these cells, the residual granules have the same chemical composition as that of their secretion. The heterogeneity of mucus composition may be correlated with its different functions.     

Isolation of a gene from Leuconostoc citreum B/110-1-2 encoding a novel dextransucrase enzyme

The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for α-1,6 and α-1,3 glucosidic bond synthesis, with α-1,4 branching was cloned, sequenced, and expressed into Escherichia coli JM109. Recombinant enzyme catalyzed oligosaccharides synthesis from sucrose as donor and maltose acceptor. The dsrF gene encodes for a protein (DSR-F) of 1,528 amino acids, with a theoretical molecular mass of 170447.72 Da (~170 kDa). From amino acid sequence comparison, it appears that DSR-F possesses the same domains as those described for GTFs. However, the variable region is longer than in other GTFs (by 100 amino acids) and two APY repeats (a 79 residue long motif with a high number of conserved glycine and aromatic residues, characterized by the presence of the three consecutive residues Ala, Pro, and Tyr) were identified in the glucan binding domain. The DSR-F catalytic domain possesses the catalytic triad involved in the glucosyl enzyme formation. The amino acid sequence of this domain shares a 56% identity with catalytic domain of the alternansucrase ASR from L. citreum NRRL B-1355 and with the catalytic domain of a putative alternansucrase sequence found in the genome of L. citreum KM20. A truncated active variant DSR-F-∆SP-∆GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (~145 kDa), was obtained.     

Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.     

Efficient Screening Methods for Glucosyltransferase Genes in Lactobacillus Strains

Abstract

Limited information is available about homopolysaccharide synthesis in the genus Lactobacillus. Using efficient screening techniques, extracellular glucosyltransferase (GTF) enzyme activity, resulting in α-glucan synthesis from sucrose, was detected in variouslactobacilli. PCR with degenerate primers based on homologous boxes of known glucosyltransferase (gtf) genes of lactic acid bacteria strains allowed cloning of fragments of 10 putative gtf genes, similar to what has been observed for Leuconostoc and Streptococcus strains. Homologs of GTFA of Lb. reuteri 121 (synthesizing reuteran, a unique glucan with α-(1→4) and α-(1→6) glycosidic bonds) (Krajl et al., 2002) were found in three of the four other Lb. reuteri strains tested. The other Lactobacillus GTF fragments showed the highest similarity with GTF enzymes of Leuconostoc spp.     

Glucosyltransferase Mutants of Leuconostoc mesenteroides NRRL B-1355

Leuconostoc mesenteroides NRRL B-1355 produces dextrans and alternan from sucrose. Alternan is an unusual dextran-like polymer containing alternating α(1→6)/α(1→3) glucosidic bonds. Cultures were mutagenized with UV and ethyl methanesulfonate, and colony morphology mutants were selected on 10% sucrose plates. Colony morphology variants exhibited changes from parent cultures in the production of one or more glucosyltransferases (GTFs) and glucans. Mutants were characterized by measuring resistance of glucan products to dextranase digestion, by electrophoresis, and by high-pressure liquid chromatography of maltose acceptor products generated from sucrose-maltose mixtures. Some mutants produced almost pure fraction L dextran, and cultures exhibited a single principal GTF band on sodium dodecyl sulfate-acrylamide gels. Other mutants produced glucans enriched for alternan. Colony morphology characteristics (size, smoothness, and opacity) and liquid culture properties (clumpiness, color, and viscosity in 10% sucrose medium) were explained on the basis of GTF production. Three principal GTF bands were detected.     

Influencing the binding configuration of sucrose in the active sites of chicory fructan 1-exohydrolase and sugar beet fructan 6-exohydrolase

The hydrolytic plant enzymes of family 32 of glycoside hydrolases (GH32), including acid cell wall type invertases (EC 3.2.1.26), fructan 1-exohydrolases (1-FEH; EC 3.2.1.153) and fructan 6-exohydrolases (6-FEH; EC 3.2.1.154), are very similar at the molecular and structural levels, but are clearly functionally different. The work presented here aims at understanding the evolution of enzyme specificity and functional diversity in this family by means of site-directed mutagenesis. It is demonstrated for the first time that invertase activity can be introduced in an S101L mutant of chicory (Cichorium intybus) 1-FEH IIa by influencing the orientation of Trp 82. At high sucrose and enzyme concentrations, a shift is proposed from a stable inhibitor configuration to an unstable substrate configuration. In the same way, invertase activity was introduced in Beta vulgaris 6-FEH by introducing an acidic amino acid in the vicinity of the acid-base catalyst (F233D mutant), creating a beta-fructofuranosidase type of enzyme with dual activity against sucrose and levan. As single amino acid substitutions can influence the donor substrate specificity of FEHs, it is predicted that plant invertases and FEHs may have diversified by introduction of a very limited number of mutations in the common ancestor.     

Stimulatory effects of 2,4-dichlorophenoxyacetic acid and of 1-naphthylacetic acid on sucrose level,invertase activity and sucrose utilization in the latex ofHevea brasiliensis

Summary Treatment of the bark ofHevea brasiliensis with 2,4-dichlorophenoxyacetic acid (2,4-D) or l-naphthylacetic acid (NAA) greatly increases sucrose level, invertase activity and sucrose utilization in the latex; the efficacy of 2,4-D is considerably greater than that of NAA. The greater sucrose utilization is the consequence of increased invertase activity. The changes occur as soon as the first tapping following bark treatment. It is suggested that the rise in both sucrose level and utilization in the latex serum mediate the effect of auxins on latex production. This is most likely related to a faciliation of latex outflow resulting from an increase in the osmotic and turgor pressure in the laticiferous tissue, as well as to enhanced regeneration of latex.The latex invertase has been found to be of a weakly alkaline type, with a sharp pH optimum at 7.15–7.20 in citrate-phosphate buffer. Its activity falls of rapidly on the acid side, being almost zero at pH 6.4. Since the natural pH of latex generally varies between pH 6.5 and 7.0, it is suggested that pH is an important factor in the regulation of invertase activity in the latex, and that the limiting nature of invertase-mediated sucrose hydrolysis in latex serum is caused by unfavourable conditions for invertase activity rather than by a scarcity of this enzyme.Expert of the International Atomic Energy Agency.