Further study of theHansenula polymorpha MALlocus: characterization of the α-glucoside permease encoded by theHpMAL2gene

Abstract

The HpMAL2 gene of the MAL gene cluster of Hansenula polymorpha codes for a permease similar to yeast maltose and alpha-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-alpha-D-glucopyranoside (PNPG) transport. PNPG uptake was competitively inhibited by all these substrates, with Ki 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.

Selection of Saccharomyces pastorianus variants with improved fermentation performance under very high gravity wort conditions

“…The a-glucoside transport rates and ADH activities of control strain and variants under VHG condition Sugars transport is the first step in the utilization of a-glucosides (maltose and maltotriose) and the limiting step for fermentation by brewer's yeast cells Viigand and Alamäe 2007). The ADH catalytic reaction in S. cerevisiae links fermentative carbon metabolism under anaerobic condition (de Smidt et al 2008).…”

“…At a desired time point, the reaction was terminated by addition of the equal volumes of 2 M NaHCO 3 (pH 10.0), and the cells were removed by centrifugation, and then the amount of p-nitrophenol present in the supernatant fluid was determined at 400 nm (Viigand and Alamäe 2007). The a-glucoside transport rate was expressed as nmol transported substrate min -1 mg DCW -1 .…”

Characterization and expression analysis of a maltose-utilizing (MAL) cluster in Aspergillus oryzae

“…In addition to Saccharomyces yeasts, there are some reports that describe a cluster of three genes encoding a maltose permease, an a-glucosidase (maltase), and a transcription factor in Hansenula polymorpha (Viigand et al, 2005;Viigand and Alamäe, 2007), or a clustering of maltase and maltose permease genes in Torulaspora delbrueckii (Alves-Araújo et al, 2004) and Kluyveromyces lactis (Fairhead and Dujon, 2006). Thus far, however, there are no reports concerning such a cluster in filamentous fungi, and hence this is the first report that characterizes the functional maltose-utilization cluster in filamentous fungi.…”

Repression vs. activation ofMOX,FMD,MPP1andMAL1promoters by sugars inHansenula polymorpha: the outcome depends on cell's ability to phosphorylate sugar

“…Hansenula polymorpha strain 201 ( HXK1 GLK1 ura3‐1 leu2‐2 met4‐220 ; Lahtchev et al ., ) isogenic to CBS 4732 was used as a wild type. In this strain, maltase‐disruption mutant mal1 and maltose permease disruption mutant mal2 were constructed by us earlier (Alamäe et al ., ; Viigand & Alamäe, ). The plasmids used in this work and their construction are described in Table .…”

“…One hexose transporter gene, HXT1 , which encodes a permease for glucose and fructose, has also been cloned from H. polymorpha (Stasyk et al ., ). We have characterized transport of glucose (Karp & Alamäe, ), maltose and sucrose (Viigand & Alamäe, ) in H. polymorpha , and cloned the genes for glucokinase (Laht et al ., ), hexokinase (Karp et al ., ), maltase (Liiv et al ., ) and maltose (α‐glucoside) permease (Viigand & Alamäe, ). Data in the literature suggest that sugar repression in H. polymorpha cannot be described on the basis of the baker's yeast model.…”