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Tiina Alamäe - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans.
International Journal of Molecular Sciences, 2019Co-Authors: Triinu Visnapuu, Aivar Meldre, Kristina Põšnograjeva, Katrin Viigand, Karin Ernits, Tiina AlamäeAbstract:Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-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, α-methylglucoside) were not, confirming that BaAG2 is a Maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—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.
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Maltase protein of ogataea hansenula polymorpha is a counterpart to the resurrected ancestor protein ancmals of yeast Maltases and isoMaltases
Yeast, 2016Co-Authors: Katrin Viigand, Triinu Visnapuu, Karin Mardo, Anneli Aasamets, Tiina AlamäeAbstract:: 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 (Km ) and inhibition (Ki ) 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.
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clustering of mal genes in hansenula polymorpha cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and Maltase genes
Fems Yeast Research, 2005Co-Authors: Katrin Viigand, Kersti Tammus, Tiina AlamäeAbstract:Hansenula polymorpha uses Maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the Maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues. We sequenced the H. polymorpha maltose permease gene HPMAL2 of the cluster. The protein (582 amino acids) deduced from the HPMAL2 gene is predicted to have eleven transmembrane domains and shows 39–57% identity with yeast maltose permeases. The identity of the protein is highest with maltose permeases of Debaryomyces hansenii and Candida albicans. Expression of the HPMAL2 in a S. cerevisiae maltose permease-negative mutant CMY1050 proved functionality of the permease protein encoded by the gene. HPMAL1 and HPMAL2 genes are divergently positioned similarly to Maltase and maltose permease genes in many yeasts. A two-reporter assay of the expression from the HPMAL1–HPMAL2 intergenic region showed that expression of both genes is coordinately regulated, repressed by glucose, induced by maltose, and that basal expression is higher in the direction of the permease gene.
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regulation of the hansenula polymorpha Maltase gene promoter in h polymorpha and saccharomyces cerevisiae
Fems Yeast Research, 2003Co-Authors: Tiina Alamäe, Katrin Viigand, Pille Parn, Helen KarpAbstract:Hansenula polymorpha is an exception among methylotrophic yeasts because it can grow on the disaccharides maltose and sucrose. We disrupted the Maltase gene (HPMAL1) in H. polymorpha 201 using homologous recombination. Resulting disruptants HP201HPMAL1Δ failed to grow on maltose and sucrose, showing that Maltase is essential for the growth of H. polymorpha on both disaccharides. Expression of HPMAL1 in HP201HPMAL1Δ from the truncated variants of the promoter enabled us to define the 5′-upstream region as sufficient for the induction of Maltase by disaccharides and its repression by glucose. Expression of the Saccharomyces cerevisiae Maltase gene MAL62 was induced by maltose and sucrose, and repressed by glucose if expressed in HP201HPMAL1Δ from its own promoter. Similarly, the HPMAL1 promoter was recognized and correctly regulated by the carbon source in a S. cerevisiae Maltase-negative mutant 100-1B. Therefore we suggest that the transcriptional regulators of S. cerevisiae MAL genes (MAL activator and Mig1 repressor) can affect the expression of the H. polymorpha Maltase gene, and that homologues of these proteins may exist in H. polymorpha. Using the HPMAL1 gene as a reporter in a H. polymorpha Maltase disruption mutant it was shown that the strength of the HPMAL1 promoter if induced by sucrose is quite comparable to the strength of the H. polymorpha alcohol oxidase promoter under conditions of methanol induction, revealing the biotechnological potential of the HPMAL1 promoter.
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cloning of Maltase gene from a methylotrophic yeast hansenula polymorpha
Gene, 2001Co-Authors: Lele Liiv, Pille Parn, Tiina AlamäeAbstract:Abstract The Hansenula polymorpha Maltase structural gene (HPMAL1) was isolated from a genomic library by hybridization of the library clones with Maltase-specific gene probe. An open reading frame of 1695 nt encoding a 564 amino-acid protein with calculated molecular weight of 65.3 kD was characterized in the genomic DNA insert of the plasmid p51. The protein sequence deduced from the HPMAL1 exhibited 58 and 47% identity with Maltases from Candida albicans and Saccharomyces carlsbergesis encoded by CAMAL2 and MAL62, respectively, and 44% identity with oligo-α-1,6-glucosidase from Bacillus cereus. The recombinant Hansenula polymorpha Maltase produced in Escherichia coli hydrolyzed p-nitrophenyl-α- d -glucopyranoside (PNPG), sucrose, maltose and α-methylglucoside and did not act on melibiose, cellobiose, trehalose and o-nitrophenyl-β- d -galactopyranoside (ONPG). The affinity of the recombinant enzyme for its substrates increased in the order maltose
Dejan Bezbradica - One of the best experts on this subject based on the ideXlab platform.
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specificity of Maltase to maltose in three different directions of reaction hydrolytic vanillyl alcohol glucoside and vanillyl alcohol isomaltoside synthesis
Biotechnology Progress, 2012Co-Authors: Aleksandra Dimitrijevic, Dusan Velickovic, Nenad Milosavic, Dejan BezbradicaAbstract:Vanillyl alcohol glucoside is very attractive molecule due to its very powerful physiological activity. In this article, a detailed kinetic study of transglucosylation of vanillyl alcohol was performed. It was demonstrated that this reaction is very efficient (selectivity factor is 149) and occurred by a ping-pong mechanism with inhibition by glucose acceptor. At low concentration of vanillyl alcohol one additional transglucosylation product was detected. Its structure was determined to be α-isomaltoside of vanillyl alcohol, indicating that vanillyl alcohol glucoside is a product of the first transglucosylation reaction and a substrate for second, so the whole reaction mechanism was proposed. It was demonstrated that the rate of isomaltoside synthesis is two orders of magnitude smaller than glucoside synthesis, and that Maltase has interestingly high Km value to maltose when vanillyl alcohol glucoside is second transglucosylation substrate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012
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a highly efficient diastereoselective synthesis of α isosalicin by Maltase from saccharomyces cerevisiae
Process Biochemistry, 2011Co-Authors: Dusan Velickovic, Aleksandra Dimitrijevic, Filip Bihelovic, Dejan Bezbradica, Ratko M Jankov, Nenad MilosavicAbstract:Abstract In this report, α-isosalicin, a potent anticoagulant and skin whitening agent, was synthesized by a highly efficient chemoselective and diastereoselective reaction, catalyzed by Maltase from bakers’ yeast (Saccharomyces cerevisiae). The highest yield of this one-step transglucosylation reaction was achieved with 50 mM of salicyl alcohol as a glucose acceptor. The key reaction factors were optimized using response surface methodology (RSM) with an enzyme concentration of 10 U/mL. The optimum temperature of the reaction was determined as 36.5 °C, the optimal maltose concentration was 40% (w/v), the optimal pH was 6.5, and the optimal reaction time was 16 h. Under these conditions 75% of α-isosalicin was obtained, with a yield of 10 g/L, and no by product formation was observed.
Buford L Nichols - One of the best experts on this subject based on the ideXlab platform.
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metabolic impacts of Maltase deficiencies
Journal of Pediatric Gastroenterology and Nutrition, 2018Co-Authors: Buford L Nichols, Susan S Baker, Roberto QuezadacalvilloAbstract:: The mucosal Maltase enzymes are characterized by an activity that produces glucose from linear glucose polymers, assayed with the disaccharide maltose. The related enzyme isoMaltase produces glucose from branched glucose polymers, assayed with palatinose. Maltase and isoMaltase activities are part of the 4 disaccharidases assayed from clinical duodenal biopsy homogenates. The reported Maltase activities are more difficult to interpret than lactase or sucrase activities because both the sucrase-isoMaltase and Maltase-glucoamylase proteins have overlapping Maltase activities. The early work of Dahlqvist identified 4 Maltase activities from human small intestinal mucosa. On one peptide, sucrase (Maltase Ib) and isoMaltase (Maltase Ia) activities shared Maltase activities but identified the enzymes as sucrase-isoMaltase. On the other peptide, no distinguishing characteristics of the 2 Maltase activities (Maltases II and III) were detected and the activities identified as Maltase-glucoamylase. The nutritional/clinical importance of small intestinal Maltase and isoMaltase activities are due to their crucial role in the digestion of food starches to absorbable free glucose. This review focuses on the interpretation of biopsy Maltase activities in the context of reported lactase, sucrase, Maltase, and palatinase biopsy assay activity patterns. We present a classification of mucosal Maltase deficiencies and novel primary Maltase deficiency (Ib, II, III) and provide a clarification of the role of Maltase activity assayed from clinically obtained duodenal biopsies, as a path toward future clinical and molecular genomic investigations.
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contribution of the individual small intestinal α glucosidases to digestion of unusual α linked glycemic disaccharides
Journal of Agricultural and Food Chemistry, 2016Co-Authors: David R Rose, Buford L Nichols, Roberto Quezadacalvillo, Bruce R HamakerAbstract:The mammalian mucosal α-glucosidase complexes, Maltase–glucoamylase (MGAM) and sucrase–isoMaltase (SI), have two catalytic subunits (N- and C-termini). Concurrent with the desire to modulate glycemic response, there has been a focus on di-/oligosaccharides with unusual α-linkages that are digested to glucose slowly by these enzymes. Here, we look at disaccharides with various possible α-linkages and their hydrolysis. Hydrolytic properties of the maltose and sucrose isomers were determined using rat intestinal and individual recombinant α-glucosidases. The individual α-glucosidases had moderate to low hydrolytic activities on all α-linked disaccharides, except trehalose. Maltase (N-terminal MGAM) showed a higher ability to digest α-1,2 and α-1,3 disaccharides, as well as α-1,4, making it the most versatile in α-hydrolytic activity. These findings apply to the development of new glycemic oligosaccharides based on unusual α-linkages for extended glycemic response. It also emphasizes that mammalian mucosal α-...
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dietary phenolic compounds selectively inhibit the individual subunits of Maltase glucoamylase and sucrase isoMaltase with the potential of modulating glucose release
Journal of Agricultural and Food Chemistry, 2015Co-Authors: Meric Simsek, Buford L Nichols, Roberto Quezadacalvillo, Mario G Ferruzzi, Bruce R HamakerAbstract:In this study, it was hypothesized that dietary phenolic compounds selectively inhibit the individual C- and N-terminal (Ct, Nt) subunits of the two small intestinal α-glucosidases, Maltase-glucoamylase (MGAM) and sucrase-isoMaltase (SI), for a modulated glycemic carbohydrate digestion. The inhibition by chlorogenic acid, caffeic acid, gallic acid, (+)-catechin, and (−)-epigallocatechin gallate (EGCG) on individual recombinant human Nt-MGAM and Nt-SI and on mouse Ct-MGAM and Ct-SI was assayed using maltose as the substrate. Inhibition constants, inhibition mechanisms, and IC50 values for each combination of phenolic compound and enzymatic subunit were determined. EGCG and chlorogenic acid were found to be more potent inhibitors for selectively inhibiting the two subunits with highest activity, Ct-MGAM and Ct-SI. All compounds displayed noncompetitive type inhibition. Inhibition of fast-digesting Ct-MGAM and Ct-SI by EGCG and chlorogenic acid could lead to a slow, but complete, digestion of starch for impr...
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luminal starch substrate brake on Maltase glucoamylase activity is located within the glucoamylase subunit
Journal of Nutrition, 2008Co-Authors: Roberto Quezadacalvillo, Claudia C Robayotorres, Zihua Ao, Bruce R Hamaker, Andrea Quaroni, Gary D Brayer, Erwin E Sterchi, David R Rose, Buford L NicholsAbstract:The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isoMaltase complex (SI) and human Maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "Maltase" activity of the internally duplicated enzyme.
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the Maltase glucoamylase gene common ancestry to sucrase isoMaltase with complementary starch digestion activities
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Buford L Nichols, Stephen E Avery, Dagmar Hahn, Dallas M Swallow, Erwin E SterchiAbstract:Brush-border Maltase-glucoamylase (MGA) activity serves as the final step of small intestinal digestion of linear regions of dietary starch to glucose. Brush-border sucrase-isoMaltase (SI) activity is complementary, through digestion of branched starch linkages. Here we report the cloning and sequencing of human MGA gene and demonstrate its close evolutionary relationship to SI. The gene is ≈82,000 bp long and located at chromosome 7q34. Forty-eight exons were identified. The 5′ gene product, when expressed as the N-terminal protein sequence, hydrolyzes maltose and starch, but not sucrose, and is thus distinct from SI. The catalytic residue was identified by mutation of an aspartic acid and was found to be identical with that described for SI. The exon structures of MGA and SI were identical. This homology of genomic structure is even more impressive than the previously reported 59% amino acid sequence identity. The shared exon structures and peptide domains, including proton donors, suggest that MGA and SI evolved by duplication of an ancestral gene, which itself had already undergone tandem gene duplication. The complementary human enzyme activities allow digestion of the starches of plant origin that make up two-thirds of most diets.
Nenad Milosavic - One of the best experts on this subject based on the ideXlab platform.
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specificity of Maltase to maltose in three different directions of reaction hydrolytic vanillyl alcohol glucoside and vanillyl alcohol isomaltoside synthesis
Biotechnology Progress, 2012Co-Authors: Aleksandra Dimitrijevic, Dusan Velickovic, Nenad Milosavic, Dejan BezbradicaAbstract:Vanillyl alcohol glucoside is very attractive molecule due to its very powerful physiological activity. In this article, a detailed kinetic study of transglucosylation of vanillyl alcohol was performed. It was demonstrated that this reaction is very efficient (selectivity factor is 149) and occurred by a ping-pong mechanism with inhibition by glucose acceptor. At low concentration of vanillyl alcohol one additional transglucosylation product was detected. Its structure was determined to be α-isomaltoside of vanillyl alcohol, indicating that vanillyl alcohol glucoside is a product of the first transglucosylation reaction and a substrate for second, so the whole reaction mechanism was proposed. It was demonstrated that the rate of isomaltoside synthesis is two orders of magnitude smaller than glucoside synthesis, and that Maltase has interestingly high Km value to maltose when vanillyl alcohol glucoside is second transglucosylation substrate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012
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a highly efficient diastereoselective synthesis of α isosalicin by Maltase from saccharomyces cerevisiae
Process Biochemistry, 2011Co-Authors: Dusan Velickovic, Aleksandra Dimitrijevic, Filip Bihelovic, Dejan Bezbradica, Ratko M Jankov, Nenad MilosavicAbstract:Abstract In this report, α-isosalicin, a potent anticoagulant and skin whitening agent, was synthesized by a highly efficient chemoselective and diastereoselective reaction, catalyzed by Maltase from bakers’ yeast (Saccharomyces cerevisiae). The highest yield of this one-step transglucosylation reaction was achieved with 50 mM of salicyl alcohol as a glucose acceptor. The key reaction factors were optimized using response surface methodology (RSM) with an enzyme concentration of 10 U/mL. The optimum temperature of the reaction was determined as 36.5 °C, the optimal maltose concentration was 40% (w/v), the optimal pH was 6.5, and the optimal reaction time was 16 h. Under these conditions 75% of α-isosalicin was obtained, with a yield of 10 g/L, and no by product formation was observed.
Katrin Viigand - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans.
International Journal of Molecular Sciences, 2019Co-Authors: Triinu Visnapuu, Aivar Meldre, Kristina Põšnograjeva, Katrin Viigand, Karin Ernits, Tiina AlamäeAbstract:Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-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, α-methylglucoside) were not, confirming that BaAG2 is a Maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—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.
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Maltase protein of ogataea hansenula polymorpha is a counterpart to the resurrected ancestor protein ancmals of yeast Maltases and isoMaltases
Yeast, 2016Co-Authors: Katrin Viigand, Triinu Visnapuu, Karin Mardo, Anneli Aasamets, Tiina AlamäeAbstract:: 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 (Km ) and inhibition (Ki ) 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.
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clustering of mal genes in hansenula polymorpha cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and Maltase genes
Fems Yeast Research, 2005Co-Authors: Katrin Viigand, Kersti Tammus, Tiina AlamäeAbstract:Hansenula polymorpha uses Maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the Maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues. We sequenced the H. polymorpha maltose permease gene HPMAL2 of the cluster. The protein (582 amino acids) deduced from the HPMAL2 gene is predicted to have eleven transmembrane domains and shows 39–57% identity with yeast maltose permeases. The identity of the protein is highest with maltose permeases of Debaryomyces hansenii and Candida albicans. Expression of the HPMAL2 in a S. cerevisiae maltose permease-negative mutant CMY1050 proved functionality of the permease protein encoded by the gene. HPMAL1 and HPMAL2 genes are divergently positioned similarly to Maltase and maltose permease genes in many yeasts. A two-reporter assay of the expression from the HPMAL1–HPMAL2 intergenic region showed that expression of both genes is coordinately regulated, repressed by glucose, induced by maltose, and that basal expression is higher in the direction of the permease gene.
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regulation of the hansenula polymorpha Maltase gene promoter in h polymorpha and saccharomyces cerevisiae
Fems Yeast Research, 2003Co-Authors: Tiina Alamäe, Katrin Viigand, Pille Parn, Helen KarpAbstract:Hansenula polymorpha is an exception among methylotrophic yeasts because it can grow on the disaccharides maltose and sucrose. We disrupted the Maltase gene (HPMAL1) in H. polymorpha 201 using homologous recombination. Resulting disruptants HP201HPMAL1Δ failed to grow on maltose and sucrose, showing that Maltase is essential for the growth of H. polymorpha on both disaccharides. Expression of HPMAL1 in HP201HPMAL1Δ from the truncated variants of the promoter enabled us to define the 5′-upstream region as sufficient for the induction of Maltase by disaccharides and its repression by glucose. Expression of the Saccharomyces cerevisiae Maltase gene MAL62 was induced by maltose and sucrose, and repressed by glucose if expressed in HP201HPMAL1Δ from its own promoter. Similarly, the HPMAL1 promoter was recognized and correctly regulated by the carbon source in a S. cerevisiae Maltase-negative mutant 100-1B. Therefore we suggest that the transcriptional regulators of S. cerevisiae MAL genes (MAL activator and Mig1 repressor) can affect the expression of the H. polymorpha Maltase gene, and that homologues of these proteins may exist in H. polymorpha. Using the HPMAL1 gene as a reporter in a H. polymorpha Maltase disruption mutant it was shown that the strength of the HPMAL1 promoter if induced by sucrose is quite comparable to the strength of the H. polymorpha alcohol oxidase promoter under conditions of methanol induction, revealing the biotechnological potential of the HPMAL1 promoter.
