Hansenula Ofunaensis Research Articles

Purification, characterization, and gene cloning of glycerol dehydrogenase from Hansenula ofunaensis, and its expression for production of optically active diol

Optically active alcohol is an important building block as a versatile chiral synthon for the asymmetric synthesis of pharmaceuticals and agrochemicals. We purified and characterized glycerol dehydrogenase from Hansenula ofunaensis and prepared optically active 1,2-octanediol using a recombinant Escherichia coli harboring the glycerol dehydrogenase gene. The deduced amino acid sequence was investigated for identities with those of other alcohol dehydrogenases in the NCBI databank. The identification of the unknown product of a resting-cell reaction was performed by GC-MS. In the deduced amino acid sequence composed of 376 residues, the NAD(H) binding pattern and cysteine residues that correspond to the cysteine ligands at the zinc atom were conserved as they are in alcohol dehydrogenases from other origins. Glycerol dehydrogenase from Hansenula polymorpha DL-1 (Pichia angusta, DDBJ/EMBL/GenBank accession no. BAD32688) had the highest identity to our enzyme, showing 73% identity. Our glycerol dehydrogenase catalyzed the NAD(+)-dependent oxidation of long-chain secondary alcohols such as 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol. Activities toward 2,4-pentanediol and 2,5-hexanediol were hardly detected. From these results, it was confirmed that our enzyme requires two hydroxyl groups on adjacent carbon atoms for oxidation. 2,3-Pentanedione, 2,3-hexanedione, and 3,4-hexanedione were significantly reduced. The transformants oxidized only (R)-1,2-octanediol in 50 mM racemate (R:S=52:48), and produced (S)-1,2-octanediol (24 mM, <99.9% e.e.) after 24 h of incubation. The reaction product was suggested to be 1-hydroxy-2-octanone by GC-MS, which showed secondary hydroxyl groups oxidized. Glycerol dehydrogenase from H. ofunaensis could be useful for the production of long-chain optically active secondary alcohols.

Gene Cloning of Dihydroxyacetone Reductase from a Methylotrophic Yeast, Hansenula ofunaensis, and its Expression in Escherichia coli HB101 for Production of Optically Active 2‐Pentanol

AbstractOptically active alcohols are important building blocks as versatile chiral synthons for asymmetric syntheses of pharmaceuticals and agrochemicals. The aim of this paper is to efficiently prepare chiral 2‐pentanol by means of microorganisms. The gene of dihydroxyacetone reductase (EC 1.1.1.6) from a methylotrophic yeast, Hansenula ofunaensis, was cloned and chiral 2‐pentanol was produced by the recombinant Escherichia coli harboring the gene. The gene encoding the enzyme was cloned from an H. ofunaensis genomic library. In the deduced amino acid sequence of 364 residues, the NAD(H) binding motif and the cysteine residues that correspond to the cysteine ligands in the zinc atom were conserved, as they are in alcohol dehydrogenases from other origins. Dihydroxyacetone reductase was similar to alcohol dehydrogenases of prokaryotes. For the production of chiral compounds, an E. coli HB101 strain was transformed. The H. ofunaensis gene product, dihydroxyacetone reductase, catalyzed the NAD+‐dependent oxidation of 2‐pentanol to 2‐pentanone as well as the corresponding reverse reactions, showing specificity towards the secondary alcohol in (R)‐configuration. From 100 mM 2‐pentanone, (R)‐2‐pentanol (98 mM, &gt; 99.9 % enantiometric excess, e.e.) was obtained in a 30‐min reaction with resting cells of the E. coli HB101 strain harboring the expression plasmid, pSG‐HOD1, which possesses the genes of both dihydroxyacetone reductase and glucose dehydrogenase as an NADH reproducing system. The stereospecificity changed during the reduction, depending on the pH. E. coli HB101 was also transformed by the expression plasmid, pSE‐HOD4, in which the gene of glucose dehydrogenase was removed from pSG‐HOD1, and designated as E. coli HB101 (pSE‐HOD4). E. coli HB101 (pSE‐HOD4) oxidized only (R)‐2‐pentanol in 100 mM of the racemate (R:S = 52:48), and the reaction medium was enriched with (S)‐2‐pentanol (48 mM, 98 % e.e.) after 30 min of incubation. The reaction was sufficiently promoted without the other additives. E. coli transformants expressing the gene of this enzyme could be particularly advantageous to the production of optically active 2‐pentanol.

Polyol production by culture of methanol-utilizing yeast

Four methanol-utilizing yeasts, Candida boidinii, Hansenula polymorpha, Hansenula ofunaensis, and Pichia pinus, produced polyols from corresponding sugars in a methanol medium. H. polymorpha produced larger amounts of xylitol than the other yeasts. Productivity was the highest at pH 8 when 5 g (dry)/ l cultured cells were incubated with 2.5 g/ l urea as the nitrogen source in a medium containing 1% (v/v) methanol and 1 g/ l MgSO 4·7H 2O. Under these conditions, 57 g/ l xylitol was obtained from 110 g/ l d-xylose after 3 d of cultivation. The largest amount of xylitol (58 g/ l; yield, 0.62 g/g) was produced from 125 g/ l, d-xylose and 5% (w/v) glycerol instead of methanol after 4 d of cultivation.

Purification and characterization of an enzyme that has dihydroxyacetone-reducing activity from methanol-grown Hansenula ofunaensis

An intracellular enzyme having reduction activity towards dihydroxyacetone (DHA), and that was induced by DHA, was purified and characterized from a methanol-grown yeast, Hansenula ofunaensis. After harvesting cells grown in a 1% methanol medium until the early stationary phase, the enzyme was purified through ammonium sulfate fractination and a series of ion-exchange, hydrophobic, and gel-filtration column chromatographies. SDS-PAGE and HPLC showed the enzyme to be a homo dimer composed of two identical subunits, each with a molecular mass of 38 kDa. The optimum pHs for DHA reduction and glycerol oxidation were 6.0 and 7.0, respectively. The optimum temperature for enzyme activity was 55°C. The enzyme reduced several other compounds, including acetaldehyde, acetol, 2-butanone and 3-methyl-2-butanone, more effectively than it did DHA, while its oxidation activity was higher towards ethanol, 2-propanol, 1,2-propanediol, 2,3-butanediol and 1,3-butanediol than towards glycerol. The K m values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The K m values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The purified enzyme had high K m values for glycerol and DHA and low K m values for 2-butanol and butanone, although physiologically it had a role in DHA metabolism. There were similarities between the purified enzyme and sec-alcohol dehydrogenases reported previously in their behavior towards inhibitors and metal ions, as well as in their K m values for 2-butanol and 2-butanone, but differences in their subunit molecular masses and activities for ethanol. At pH 9.8, the oxidative activity of the purified enzyme for l-2-butanol was about eleven times higher than that for d-2-butanol.

Dihydroxyacetone reductase responsible for less-aerobic growth of a methylotrophic yeast, Hansenula ofunaensis.

Correlation between the activity of dihydroxyacetone reductase (DHAR) and the intracellular content of glycerol in a methylotrophic yeast, Hansenula ofunaensis, was observed when it was grown on methanol. The DHAR activity and the glycerol synthesis were low in the presence of a high concentration of NaCl or KC1. DHAR activity increased with growth under less-aerobic conditions in the methanol medium. The less-aerobic conditions after aerobic cultivation increased the glycerol synthesis. Methanol was the most effective carbon source for DHAR synthesis and glycerol did not repress the synthesis. From these results, it was concluded that DHAR in methanol-grown cells of H. ofunaensis might be responsible for the growth under less-aerobic conditions, but not for that under salt stress conditions or for glycerol synthesis itself.

Glycerol dehydrogenase and dihydroxyacetone reductase of a methylotrophic yeast, Hansenula ofunaensis.

NAD+-Linked glycerol dehydrogenase (GDH) activity in Hansenula ofunaensis grown on glycerol was separated into three enzymes by a procedure involving DEAE-cellulose and DEAE-Sephadex A-50 column chromatographies. The oxidative activities toward glycerol of GDH I-1 and GDH I-2 were only detected when the concentration of glycerol was high, while GDH II showed significant activity with a low concentration of glycerol. The Km values for glycerol of GDH I-1, GDH I-2 and GDH II were 0.56M, 2.9M and 0.16M, respectively. The Km values for dihydroxyacetone of GDH I-1, GDH I-2 and GDH II were 0.40M, 0.36M and 1.7 mM, respectively. The oxidative activities toward 1, 2-propanediol of GDH I-1 and GDH II were 0.37- and 1.5-fold those toward glycerol, respectively. GDH I-2 showed significant oxidative activity toward ethanol, while GDH I-1 and GDH II showed no such activity. GDH I-2 was identical to dihydroxyacetone reductase induced in methanol-grown cells.

Diversity in glycerol metabolism of methylotrophic yeasts

Methylotrophic yeasts showed 3 different patterns in regard to the initial step of glycerol dissimilation. Hansenula polymorpha DL-1 had activities of NAD+-linked glycerol dehydrogenase (GDH) and glycerol kinase (GK), which were induced by glycerol and methanol and by glycerol, respectively. Hansenula ofunaensis had the GDH activity, which was induced by glycerol but not by methanol. Candida boidinii No. 2201 had the GK activity, which was induced by glycerol. It was concluded that the phosphorylative pathway in C. boidinii No. 2201, the oxidative pathway in H. ofunaensis, and both the pathways in H. polymorpha DL-1 may be essential for glycerol dissimilation.

Dihydroxyacetone reductase of a methylotrophic yeast, Hansenula ofunaensis.

Dihydroxyacetone reductase of a methylotrophic yeast, Hansenula ofunaensis.

Glycerol metabolism in methylotrophic yeasts.

Enzyme activities involved in the initial step of glycerol metabolism were determined in cells of methylotrophic yeasts grown on glycerol, methanol or glucose. In Candida boidinii (Kloeckera sp.) No. 2201, the activities of glycerol kinase and dihydroxyacetone kinase were detected in cells grown on glycerol and methanol, respectively. The activity of NAD+-linked glycerol dehydrogenase of Hansenula polymorpha dl-1 was induced by glycerol and methanol, while that of Hansenula ofunaensis was induced by glycerol. The enzymes of both strains were subject to catabolite repression by glucose.The yeasts tested were divided into three groups as to the glycerol dissimilation patterns. Strains of the genera Candida, Saccharomyces, Pichia and Torulopsis had the phosphorylative pathway, in which glycerol is first phosphorylated. H. ofunaensis had the oxidative pathway, in which glycerol is first oxidized. H. polymorpha dl-1 had both the phosphorylative and oxidative pathways.

NEW SPECIES OF METHANOL-ASSIMILATING YEASTS

In the course of investigations on yeast cell production and on the metabolisms of methanol, two new species, Torulopsis nagoyaensis and Hansenula ofunaensis, were found in the yeasts isolated from various natural sources in Japan. Taxonomical studies of these new species are described.