Hansenula polymorpha MAL-lookus (original) (raw)
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FEMS Yeast Research
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 HP201HPMAL1v 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 HP201HPMAL1v from the truncated variants of the promoter enabled us to define the 5P-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 HP201HPMAL1v 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.
Regulation of the maltase gene promoter in and
FEMS Yeast Research, 2003
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 HP201HPMAL1v 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 HP201HPMAL1v from the truncated variants of the promoter enabled us to define the 5P-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 HP201HPMAL1v 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.
FEMS Yeast Research, 2013
A high-throughput approach was used to assess the effect of mono-and disaccharides on MOX, FMD, MPP1 and MAL1 promoters in Hansenula polymorpha. Site-specifically designed strains deficient for (i) hexokinase, (ii) hexokinase and glucokinase, (iii) maltose permease and (iv) maltase were used as hosts for reporter plasmids in which β-glucuronidase (Gus) expression was controlled by these promoters. The reporter strains were grown on agar plates containing varied carbon sources and Gus activity was measured in permeabilized cells on microtitre plates. We report that monosaccharides (glucose, fructose) repress studied promoters only if phosphorylated in the cell.
Yeast, 2000
The methylotrophic yeast H. polymorpha is a popular system for the expression of recombinant proteins using the strong and regulatable methanol oxidase (MOX) promoter. Here we show that the constitutive PMA1 promoter can programme the expression of two heterologous proteins, glucose oxidase and human serum albumin. A constitutive promoter provides a useful additional facility to the H. polymorpha expression system because it allows a simpli®ed fermentation regime, avoids the use of methanol, which is both toxic and an explosive hazard, and allows more¯exibility for ectopic gene expression during the course of academic studies. A fragment previously isolated in a promoter screen, using glucose oxidase (GOD) as a reporter gene, was shown to consist of the promoter region and the ®rst 659 bp of the H. polymorpha PMA1 gene, encoding the plasma membrane H + -ATPase. When the PMA1 promoter was optimally aligned with the GOD coding region, it produced 185 mg/l glucose oxidase in high cell density fed batch fermentations, whereas in previous experiments using the MOX promoter, a yield of 500 mg/l was recovered. The PMA1 promoter was also used to express recombinant human serum albumin (rHA) in H. polymorpha. In high cell density fermentations the PMA1 promoter produced 460 mg/l rHA, whereas 280 mg/l rHA was obtained using the MOX promoter. Taken together, these experiments show that the HpPMA1 programmes the constitutive expression of recombinant proteins and provides a yield comparable to that from the MOX promoter.
Journal of Bacteriology
Saccharomyces yeast strains able to ferment maltose carty at least one member of a family of MAL loci: MAL], MAL2, MAL3, MAL4, and MAL6. The MAL6 locus has been cloned and shown to be a cluster of at least three transcribed regions, all of which are required for maltose fermentation. Transcription at two of these genes, MAL61 and MAL62, is both induced by maltose and repressed by glucose. The third gene, MAL63, appears to encode a regulatory product controlling mnaltose fermentation. In this report, we demonstrate that the MAL62 gene is the structural gene coding for the enzyme maltase. Strain 332-5A is a maltose fermenter of the genotype MAL6 mad)0. Integrative disruption of the MAL62 gene of the MAL6 locus produces a strain which is still capable of fermenting maltose, but which synthesizes a more heat-labile form of maltase than the undisrupted strain. Synthesis of this more heat-labile maltase was shown to be linked to the mall°locus present in the strain. Integrative disruption of both the MAL62 gene and the MAL62-homologous sequence present at the mall°locus produces a nonfermenter which is unable to synthesize maltase. These results identify MAL62 as the maltase structural gene.
FEMS Yeast Research, 2005
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.
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.
Genetic control of maltase synthesis in yeast
Molecular and General Genetics MGG, 1979
A new series of maltase negative mutants have been isolated from yeast strains carrying the MAL4 gene. These mutants are allelic to the MAL4 gene and fail to ferment maltose, sucrose, and alphamethylglucoside. Most revertants isolated from these mutants restore the ability to ferment above sugars, and also produce the same levels of maltase as the parental strains. One of the revertants (NA-520-R1), however, ferments maltose slowly, and produces 24 fold less enzyme than the parental strain. Genetic studies revealed that revertant (NA-520-R1), is not a true back mutation but is carrying an extra-genic suppressor, which suppresses the real4 allele in mutant (NA-520). Since several lines of published evidence indicate that the MAL4 gene is a regulatory gene, it is suggested that the MAL4 gene codes for a regulatory protein, which acts as positive regulatory element in maltase synthesis.
FEMS Yeast Research, 2007
The HpMAL2 gene of the MAL gene cluster of Hansenula polymorpha codes for a permease similar to yeast maltose and a-glucoside transporters. Genomic disruption of HpMAL2 resulted in an inability of cells to grow on maltose, sucrose, trehalose, maltotriose and turanose, as well as a lack of p-nitrophenyl-a-Dglucopyranoside (PNPG) transport. PNPG uptake was competitively inhibited by all these substrates, with K i values between 0.23 and 1.47 mM. Transport by HpMal2p was sensitive to pH and a protonophore carbonyl cyanide-m-chlorophenylhydrazone (CCCP), revealing its energization by the proton gradient over the cell membrane. Although HpMAL2 was responsible for trehalose uptake, its expression was not induced during trehalose growth. A maltase disruption mutant did not grow on maltotriose and turanose, whereas it showed normal growth on trehalose, demonstrating the dispensability of maltase for intracellular hydrolysis of trehalose. In a Genolevures clone pBB0AA011B12, the promoter region and the N-terminal fragment of the putative transactivator of MAL genes is located adjacent to HpMAL2. A reporter gene assay showed that expression from that promoter was induced by maltose and sucrose, repressed by glucose, and derepressed during glycerol and trehalose growth. Therefore, we presume that the gene encodes a functional regulator.
Molecular and General Genetics MGG, 1972
1. The p-nitrophenyl-~-D-glucopyranoside (PNPG) hydrolyzing capacity synthesized in protoplasts of Saccharomyces carlsbergensis under conditions of induction and (de)repression has been characterized. With respect to their substrate-speeificities it is possible to distinguish between a maltase and an ~-methyl-glucosidase, both enzymes being able to hydrolyze PNPG. 2. The induction of both ~-glucosidases has been examined: maltose induces maltase and to a less extent ~-methylglucosidase in strains containing the MAZ 6 gene, whereas a-methylglucoside induces ~-methylglucosidase in strains containing MGLa, MGL b and MGL e. 3. Catabolite repression for each of these enzymes was found to be somewhat different: maltase synthesis is more sensitive to changes in the degree of catabolite repression than a-methylglucosidase synthesis.