FEMS Microbiology Letters 40 (1987) 151-153 151 Published by Elsevier

FEM 02655

Diversity in glycerol metabolism of methylotrophic yeasts

Yoshiki Tani and Keiko Yamada

Research Center for Cell and Ttssue Culture. b~tcultv of Agriculture. Kvoto Unt~,ersitv. Kvoto. Japan

Key words: Methylotrophic

Received 23 September 1986 Accepted 6 October 19~6

yeast; Glycerol dehydrogenase" Dihydroxyacetone kinase: Glycerol kinase

1. SUMMARY

Methylotrophic yeasts showed 3 different pat- terns in regard to the initial step of glycerol dis- similation. Hansenula polymorpha DL-1 had activ- ities 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. poly- morpha DL-I may be essential for glycerol dissim- ilation.

2. INTRODUCTION

Several pathways for the dissimilation of glycerol in microorganism are known; the phos- phorylative pathway, involving glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase

Correspondence to: Yoshiki Tani, Research Center for Cell and Tissue Culture. Faculty of Agriculture, Kyoto University. Sakyo-ku, Kyoto, Japan.

(G3PDH) [1]; the oxidative pathway involving NAD + or NADP '-linked glycerol dehydrogenase (GDH) and dihydroxyacetone kinase (DHAK) [1]; or a pathway involving NADP'-linked GDH and D-glyceraldehyde kinase or glycerate kinase [2]. The phosphorylative pathway is generally found in eukaryotes, with the exception of Neurospora crassa, in which both the phosphorylative and oxidative pathways may operate [3-6], and Schizosaccharomyces pombe and Candida valida H122. in which only the oxidative pathway may operate [7-9]. The xylulose monophosphate path- way, which is responsible for assimilation of methanol in yeasts, involves dihydroxyacetone (DHA) as the intermediate [10]. DHA is the oxidized product of glycerol in the oxidative path- way for the glycerol dissimilation. The presence of GDH and absence of GK were indicated in some methylotrophic yeasts [9]. However, no detailed investigation of the glycerol dissimilation of meth- ylotrophic yeast has been reported. In this paper, the diversity of glycerol metabolism in methylo- trophic yeasts will be demonstrated.

3. MATERIALS AND METHODS

3.1. Microorganisms and cultit, ation H. polymorpha DL-1, H, ofunaensis and C.

0378-1097/87/$03.50 ~::' 1987 Federation of European Microbiological Societies

152

hoidinii (KIoeckera sp.) No. 2201 were from our laboratory collection [11 ].

3.2. Cultivation Culture medium consisted of 1.0 g carbon

source. 0.4 g NH4CI, 0.1 g K2HPO 4, 0.1 g KH2PO 4, 0.05 g MgSO 4- 7H20 and 0.1 g yeast extract in 100 ml of distilled water, pH 6.5. Each yeast was inoculated into the seed culture (5 ml medium in a 16.5-cm test tube) and incubated at 28°C under reciprocal shaking for 1 2 days until the culture reached the late logarithmic phase. The seed culture was then transferred into 100 ml of the medium in a 500-ml shaking flask.

3.3. Enzyme preparation and assay After cultivation, cells were harvested at several

growth periods by centrifugation at 5000 × g . Yeast cells from 100 ml culture were suspended in 15 ml of 0.1 M potassium phosphate buffer, pH 7.0, containing 0.5 mM D' IT and disrupted with a Kubota lnsonator Model 200M (9 kHZ) for 30 rain below 15°C at 1.0-1.5 A. Cell-free extract was prepared by centrifugation at 8000 x g for 15 min at 5°C.

GDH, G K and D H A K activities were de- termined by measuring the reduction of N A D * or N A D P and oxidation of NADH, respectively. and the unit was defined as described previously

[121. The reaction mixture for G D H assay contained

300 >mol of NH4CI-NH4OH buffer, pH 9.0, 300

p, mol glycerol, 0.5 p, mol N A D ' or NADP" and 100 ~1 of cell-free extract in a total volume of 3 ml. The reaction mixture without glycerol was used as a reference. The reaction mixture for GK assay contained 600 p, mol glycine-NaOH buffer, pH 9.8, 12.3 mmol of hydrazine hydrate, 12 p, mol of MgCI_, • 6H20, 5 p, mol of ATP. Na 2, 1.4/,tmol of NAD ~, 60 p, mol of glycerol, 18 U of G3PDH (Oriental Yeast Co.) and 100 p.1 of cell-free extract in a total volume of 3.38 ml. As a reference, the reaction mixture without G3PDH was used. The reaction mixture for the DHAK assay' contained 300 ~umol of imidazole-HCl buffer, pH 7.0, 15 p, mol of MgSO4-7H20, 15 p, mol of A T P - N a ~ , 1.5 p, mol of DHA, 0.5 p, mol of NADH, 18 U of G3PDH and 100 >1 of cell-free extract in a total volume of 2.83 ml. The reaction mixture without DHA was used as a reference. Concentration of protein was determined by measuring the ab- sorbance at 280 nm and with a Bio-Rad protein assay kit.

4. RESULTS AND DISCUSSION

The activities of GDH, G K and DHAK in extracts from H. polymorpha DI,-1, H. ofunaensis and C. hoidinii No. 2201 grown on glucose, glycerol and methanol were determined at different growth phases. The maximum values for each culture are indicated in Table I.

High NAD '-linked G D H activity was found in

1able 1

Enz,,me activities of methylotrophic yeast.,, grov,n on different carbon sources

Yea~,t Growth substrate

Specific activitie.,, ( U / r a g protein × I(XR})

( IDH I)HAK (iK

('. h o u h m i No. 2201 Glycerol Methanol (;lucose

I t . o li~naen~t~ ( ilvcerol Methanol

(Jluc~'~se

II. p o l v m o r p h a DL-1 (Jlvccrol Methanol Glucose

1.6 2o 3Ix) l.g 130 34

1.8 4.5 46

76 180 6.7 3.8 120 7.4 4.4 24 6.1

55 73 120

57 48O 5~ 23 17 22

C. b o l d i n i i N o . 2 2 0 1 G L ~ D H A

t G 3 P ~ D H A P

M e t h a n o l

H. o f u n a e n s i s G L ~ D H A ~ M e t h a n o l

l G 3 P D H A P

H. po/ymof_pha DL-1 G L ~ D H A ~ M e t h a n o l

G 3 P ~ D H A P

Fig 1. (il,,cerol ((iI.) di,s,similation in methylotrophic yca,sts.

extracts from H. polymorpha DL-1 and H. ofunaensis grown on glycerol, but not in extracts from C. boidinii No. 2201. D H A K activity was found in cell-free extracts from all strains grown on methanol. D H A K is used to regenerate the C~ acceptor, xylulose 5-phosphate, by making DHA- phosphate (DHAP) in methylotrophic yeasts. D H A K activities in extracts from H. ofunaensis and II. polymorpha DL-1 grown on glycerol, were found to be significantly higher than those on glucose. NADP ' - l i nked G D H activities could hardly be observed in extracts from all strains at any growth phase. This result showed that NAD' - l i nked G D H and D H A K might play an essential role in glycerol metabolism of tt. poly- morpha DL-1 and tl. ofunaensis. The significance of G D H in the methanol metabolism in yeasts was not clear: Candida methylica could not utilize glycerol, but did have G D H activity [9]. The specific activity of the enzyme in extracts from H. polymorpha I)L-1 grown on methanol was found to be at the same level as on glycerol, and to be induced by the transfer from the glucose medium to the methanol medium. Thus, G D H in H. poly- morpha DL-I is probably involved in methanol metabolism, in contrast to G D H in H. ofunaensis

153

and C. boidinii No. 2201. Induction of an enzyme activity which catalyzed the reduction of DHA was detected in extracts from all strains grown on methanol.

C. boidinii No. 2201 showed high GK activity, as demonstrated in Candida utilis, which assimi- lates glycerol via the phosphorylative pathway [13]. 1t. polymorpha DL-I also showed high activity at the mid-logarithmic phase in the glycerol medium. tl. ofunaensis did not show significant G K activity when grown on glycerol at any growth phase.

From these results, it was considered that these 3 methylotrophic yeasts might have different path- ways for the initial step of glycerol assimilation, as shown in Fig. 1: C. hoidinii No. 2201 has the phosphorylative pathway, H. ofunaensis has the oxidative pathway, and H. polymorpha DL-I has both the phosphorylativc and oxidative pathways.

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