Appl Microbiol Biotechnol (1989) 30:170-175 Applied Microbiology

Biotecknology © Springer-Verlag 1989

Influence of ethanol on the activities of 3-hydroxy-3-methylglutaryl-coenzyme A-reductase and squalene-hopene-cyclase in Zymomonas mobilis

Andrea Schmidt 1, Stephanie Bringer-Meyer 1, Karl Poralla 2, and Hermann Sahm ~

1 Institut for Biotechnologie der Kernforschungsanlage Jtilich GmbH, Postfach 1913, D-5170 Jiilich, Federal Republic of Germany 2 Institut ftir Biologie, Universitat T~ibingen, Auf der Morgenstelle 28, D-7400 Ttibingen, Federal Republic of Germany

Summary. The influence of different primary ali- phatic alcohols on the activities of two key en- zymes in hopanoid biosynthesis of Zymomonas mobilis was investigated. By use of 14C- and 3H- labelled substrates the enzymes 3-hydroxy-3-me- thylglutaryl-CoA-reductase and squalene-hopene- cyclase were detected with activities of 1.6 pmol × (min × mg protein)- 1 and 2.3 pmol × - (minx mg protein) - 1, respectively. Cells grown in the presence of 6% (v/v) ethanol did not show higher activities of these enzymes than cells grown in the presence of 1% (v/v) ethanol. Fur- thermore, 3-hydroxy-3-methylglutaryl-CoA-reduc- tase was not activated by ethanol. However, ethanol activated the squalene-hopene-cyclase when added to the enzyme test system. Besides ethanol, propanol also had a positive effect on the squalene-hopene-cyclase: the enzyme's activity increased 1.7-fold in the presence of either alco- hol at a concentration of 6% (v/v). This corre- sponded with a similar increase of hopanoid con- tent of whole cells when grown in the presence of 6% (v/v) added ethanol or propanol. These results indicated that the squalene-hopene-cyclase has a regulatory function in the alcohol dependent ho- panoid biosynthesis of Z. mobilis.

Introduction

Among bacteria, Z. mobilis has an exceptionally high ethanol tolerance and is capable of produc- ing ethanol in concentrations up to 13% (w/v) (Rogers et al. 1982). Ethanol is known to affect

Offprint requests to: H. Sahm

Abbreviation: HMG-CoA-reductase, 3-hydroxy-3-methylgluta- ryl-coenzyme A-reductase

the integrity of biological membranes by intercal- ation into the lipid bilayer which results in an in- crease of membrane fluidity (Ingram and Buttke 1984; Ingram 1986). Recently it was shown that the cell membrane of Z. mobilis contains hopa- noids and that the contributions of these penta- cyclic triterpenoids to the total lipid fraction de- pends strongly on ethanol concentration (Bringer et al. 1985; Schmidt et al. 1986). Hopanoids are considered to be the structural and functional equivalents of sterols in prokaryotes (Ourisson et al. 1984; Poralla and Kannenberg 1987); sterols are known to be partly responsible for the high alcohol tolerance of yeast (Thomas et al. 1978; Ohta and Hayashida 1983). Since hopanoids have a potential to counteract the increase of mem- brane fluidity and permeability caused by ethanol (Rowe 1987; Dombek and Ingram 1984) it was surmised that the ethanol dependent hopanoid formation has a protective function at high etha- nol concentrations (Bringer et al. 1985; Schmidt et al. 1986; Bringer-Meyer and Sahm 1988).

Since the triterpene squalene is an interme- diate common to sterol as well as to hopanoid biosyntheses, it can be assumed that the enzy- matic pathways up to squalene are similar in ste- rol and hopanoid producing organisms. The 3-hy- droxy-3-methylglutaryl-coenzyme A reductase, E.C. 1.1.1.34 (HMG-CoA-reductase) (Fig. 1) is a key enzyme in the regulation of cholesterol biosynthe- sis and has intensively been investigated in mam- mals (Schroepfer 1981). Microbial HMG-CoA-re- ductases have so far only been examined in yeast (Ferguson et al. 1958), Pseudomonas (Bensch and Rodwell 1970), Halobacterium halobium (Cabrera et al. 1986), and Streptomyces arenae (Maurer 1984). Yeast and bacterial HMG-CoA-reductases have similar molecula r weights which are in turn greater than that of the mammalian enzyme (Boll

A. Schmidt et al. : Influence of ethanol in Zymomonas mobilis 171

1. 3-Hydroxy-3-methylglutaryl-coenzyme A-reductase

( HMG-CoA-reductase )

o U ~HeOH C--S--CoA I ?H 2

HO--C--CH] HO--C--CH 3 [

COO CO0-

3-Hydroxy-3-methyl- glutaryI-CoA Mevalonate

Until now little information exists on the regu- lation of hopanoid biosynthesis in Z. mobilis. This paper describes the influence of ethanol and some other primary alcohols on the activities of HMG- CoA-reductase and squalene-hopene-cyclase in this organism.

Materials and methods

Bacterial strain and culture conditions

2. Squalene-hopene-cyclase

Squalene

/ \

g~ %-..

Hopan-22-ol Hop-22(29)-ene

Fig. 1. Two enzymatic key reactions in hopanoid biosynthe- sis

1983). The enzyme from Pseudomonas is induced by growth on mevalonate and uses NADH in con- trast to all other known HMG-CoA-reductases which require NADPH (Bensch and Rodwell 1970).

The intermediate squalene is the branching point of the biosynthetic pathways for sterols and hopanoids (Taylor 1984). While sterols are syn- thesized by cyclization of epoxysqualene formed from squalene by O2-dependent oxidation, in the biosynthesis of hopanoids squalene is cyclized without prior epoxidation (Seckler and Poralla 1986). The enzyme responsible for the direct cycli- zation of squalene to pentacyclic hopanoids, a squalene-hopene-cyclase (Fig. 1), has recently been investigated (Anding et al. 1976; Bouvier 1978; Rohmer et al. 1980a, b; Neumann and Si- mon 1986; Seckler and Poralla 1986). In Bacillus acidocaldarius the cyclase is a membrane-bound enzyme and converts squalene to two pentacyclic triterpenoids, hopene (hop-22(29)-ene) and di- plopterol (hopan-22-ol) (Fig. 1) in a molar ratio of hopene:hopanol, 5-6:1 (Seckler and Poralla 1986; Neumann and Simon 1986).

Z. mobilis subsp, mobilis (ATCC 29191) was grown anaerobi- cally as previously described (Bringer et al. 1985). Cultivation in presence of different alcohols was performed according to Schmidt et al. (1986).

Analytical methods

Cell densities were measured at 560 nm with a Beckman pho- tometer. Hopanoids were determined as described by Poralla et al. (1984) and Schmidt et al. (1986).

Enzyme assays

3-Hydroxy-3-methylglutaryl-CoA-reductase

The assay was performed according to the method of Shapiro et al. (1974), modified by Jenke et al. (1981) and Maurer (1984). Cells were harvested in the exponential phase of growth, washed twice with cold buffer containing 60 mM KHEPO4, 29 mM Na2HPO4, pH 7.0 and resuspended in cold buffer containing 100 mM KH2PO4, 100 mM KC1, 25% glycer- ol, 10 mM EDTA, 10 mM dithioerythritol, pH 7.0. At this stage the cells were concentrated to an OD56onrn of approx. 200 (approx. 40 mg dry weight/ml). The cells were disrupted by ultrasonification at 0 ° C for 2 × 30 s with 1 rain interval. Resid- ual cells were removed by low-speed centrifugation (30 min at 4°C and 48000 9) and 60-170 ~tl of the cell-free extract were used for the enzyme assay. The cell-free extract was preincu- bated for 20 min at 30°C in the assay mixture to restore full enzymatic activity (Kleinsek and Porter 1979).

The assay mixture contained in a total volume of 250 ktl: 60-170 p.1 crude extract 20 ixl 0.5 M glucose-6-phosphate 10 l.tl 0.1 M NADP 10 ~tl 0.1 M NAD 10 t.tl glucose-6-phosphate-dehydrogenase (2 mg/ml) 10 Ixl BSA (10 mg/ml)

110-0 lxl 0.25 M KHzPO4-buffer. The enzyme reaction was started by addition of 20 F1 of

substrate solution containing 1 mM DL-3-hydroxy-3-methyl- glutaryl-CoA and 77 ktM DL-3-hydroxy-3-methyl[3-14C]gluta- ryl-CoA (specific activity: 51.3 mCi/mmol). After incubation for 15 min at 30°C 75 l.tl of 1.0 M mevalonolactone and 25 I-tl of 0.1 M HMG-acid were added as carriers for the thin layer chromatography and 100 I-tl of 6 M HESO4 as stop reagent. The mixture was incubated for 20 min at 50°C to complete lacton- ization and subsequently centrifuged to remove the protein. 200 txl of the supernatant was applied on an activated silica gel plate (silica gel 60, 0.5 mm, Merck AG, Darmstadt, F.R.G.). The separation of products and substrate by thin-layer chro- matography was achieved by development in benzene-acetone

172 A. Schmidt et al. : Influence of ethanol in Zymomonas mobilis

(1:1). Mevalonic acid and mevalonolactone were detected by staining with bromocresolgreen/bromophenolblue/potas- siumpermanganate (Berndt and Gaumert 1971). Mevalonic acid became visible as violet, mevalonolactone as blue and HMG-acid as pink spots. The areas containing mevalonic acid and mevalonolactone were removed and the radioactivity was counted in dioxan scintillation solution.

Squalene-hopene-cyclase

Cells were washed and resuspended in 10 mM Tris-HC1 buf- fer, pH 8.0. The conditions of cell harvest and disruption were as described for the HMG-CoA-reductase. Membrane prepa- ration, solubilization of the enzyme, and the assay were per- formed according to Seckler and Poralla (1986). The assay mixture contained in a total volume of 1.0 ml: 100 mM sodium citrate buffer pH 6.0, 50~M [3H] squalene (1 x 105 dpm), 0.1 mg Trixon X-100 and 1-5 mg protein. The mixture was in- cubated for 60 min at 30°C. The reaction was stopped by ex- tracting the chilled vial with 2ml hexane/2-propanol 3:2 (v/v). After concentrating the organic phase to about 100 ~tl, hopene, diplopterol and bacteriohopenetetrol were added as carriers. The separation was performed on thin-layer plates (silica gel 60, 0.25 mm, Merck, Darmstadt, FRG) with chloro- form. The chromatography was stopped when the solvent front reached ½ of the distance to the upper edge. Afterwards the dried plate was further developed in n-hexane. Squalene, hopene and diplopterol were detected with berberinchloride (10~tg/ml in ethanol:water 95:5 v/v) under UV light at 340 nm.

Protein determination

Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.

Results and discussion

The HMG-CoA-reductase was detected in crude extracts of Z. mobilis with a specific activity of 1.6 pmol x (minx mg protein)- 1. This low enzyme activity could only be measured with labelled HMG-CoA and detection of the labelled product mevalonic acid (Fig. 1). The photometric assay coupled with the NADPH-dependent reduction of HMG-CoA to mevalonic acid (Siedel 1983) was disturbed by a strong NAD(P)H oxidase ac- tivity of the crude extracts (Bringer et al. 1984). Anaerobic assay performance could not suffi- ciently increase the sensitivity of the photometric assay for measurement of the HMG-CoA-reduc- tase activity. Furthermore, the determination of HMG-CoA-reductase activity in crude extracts of Z. mobilis was rendered difficult by other HMG- CoA converting enzymes. This effect has also been described by Young et al. (1982). Moreover, lactonization of mevalonic acid to mevalonolac- tone, described by Jenke et al. (1981) was incom- plete. Staining of the thin-layer-chromatogramms

with bromocresolgreen/bromophenolblue/potas- siumpermanganate (Berndt and Gaumert 1971) as well as GC/MS-measurements showed that meva- lonolactone as well as mevalonic acid were reac- tion products of HMG-CoA and therefore both had to be taken into account for calculation of the HMG-CoA-reductase activity.

In comparison to other bacterial HMG-CoA- reductases the specific activity of the Z. mobilis enzyme -- 1.6 pmol x (minx mg protein)- 1 _ was in the same range as the activ!ty of the HMG- CoA-reductase of S. arenae TU 469 (Maurer 1984). Dependent on the age of the culture the HMG-CoA-reductase activity in S. arenae fluc- tuated between 1 and 19 pmol x (minx mg pro- tein) -1 (Maurer 1984). The Halobacterium halo- bium reductase showed a much higher specific ac- tivity of 4 nmol × (min x mg protein)- 1 (Cabrera et al. 1986).

Activation of the Z. mobilis HMG-CoA-reduc- tase by ethanol was tested by addition of ethanol to the assay to a final concentration of 6% (v/v). However, the specific enzyme activity of 1.6 pmol × (min × mg protein)- 1 remained unaltered. Alcohol-dependent induction of HMG-CoA-re- ductase in Z. mobilis was examined by cultivation of cells at different ethanol concentrations and measurement of the corresponding enzyme activi- ty. Cells grown in batch- or continuous cultures at medium to high ethanol concentrations (47 to 74 g/1 ethanol) showed an even lower HMG-CoA- reductase activity than cells grown with 20 g/1 glucose (producing ethanol concentrations ~< 1% v/v) without externally added ethanol (Fig. 2). These results suggest that the HMG-CoA-reduc- tase of Z. mobil& is not induced by ethanol. How- ever, an induction of HMG-CoA-reductase by ethanol was observed in yeast already at a low ethanol concentration. Addition of 1% (v/v) etha- nol to the culture medium led to an increase in HMG-CoA-reductase activity of 85% compared to the ethanol-free control (Boll et al. 1975). The HMG-CoA-reductase in chinese hamster fibro- blastes likewise was induced by ethanol (Sinensky and Kleiner 1981). The mechanism by which etha- nol induces the synthesis of this enzyme is not known.

Squalene-hopene-cyclase, the first specific en- zyme of hopanoid biosynthesis also was tested. By use of radioactive labelled squalene this en- zyme was detected in crude-extracts of Z. mobilis with a specific activity of 2.27 pmol x (min ×mg protein)-1. As product of the enzymatic reaction hopene was measured. A high background ra- dioactivity presumably overlayed the radioactivity

A. Schmidt et al. : Influence of ethanol in Zymomonas mobilis 173

6 -

c

© E 4- c~

' ~ 3 -

o

W 1 -

0 0

a ) - - / "

. .

I i 2 3

Protein ( mg )

Fig. 2. Influence of ethanol concentration in the culture me- dium on the HMG-CoA-reductase activity in cell-flee extracts of Z. mobil&. Batch cultures were grown with 20 g/1 glucose a) and 47 g/1 externally added ethanol b). The continuous cul- ture c) was grown with 37 g/1 glucose and 69 g/1 added etha- nol (total ethanol concentration: 74 g/l) at a dilution rate of 0.22 h - 1

6,0 O3 E &

"~ 5,0

5 E

4 , 0 -

• --> 3,0 0

[:: 2 ,0

U C

1,0

O. 09

o

20 40 6 0

Ethanol in the assay (g/I)

Fig. 3. Activation of squalene-hopene-cyclase in cell-flee ex- tracts of Z. mobil& by addition of ethanol to the assay. Cells were grown with 20 g / l glucose and harvested in the mid-ex- ponential growth phase

of diplopterol, which is produced from squalene by the squalene-hopene-cyclase from B. acidocal- darius concurrently with hopene (Seckler and Po- ralla 1986; Neumann and Simon 1986). This en- zyme forms hopene and hopanol in a molar ratio of 5-6:1 (Seckler and Poralla 1986; Neumann and Simon 1986).

Compared with the squalene-hopene-cyclase activity of 0.62 pmol x (rain x mg protein)- 1 in Acetobacter rancens (Bouvier 1978) the cyclase ac- tivity in Z. mobilis was 4-fold higher. However, the activity of the Z. mobilis-enzyme was 100-fold lower than that of the squalene-hopene-cyclase in cell-flee extracts of B. acidocaldarius (Neumann and Simon 1986; Seckler and Poralla 1986). By ul- tracentrifugation of the cell-free extract and solu- bilization of the membrane fraction with Triton X-100 the squalene-hopene-cyclase could be en-

riched 6.5-fold. This indicates that the squalene- hopene-cyclase of Z. mobilis is a membrane-asso- ciated protein, as has already been shown for the squalene-hopene-cyclase of B. acidocaldarius (Seckler and Poralla 1986; Neumann and Simon 1986; G/irtner 1987) and Acetobacter rancens (Bouvier 1978).

The activity of squalene-hopene-cyclase was the same in crude extracts of Z. mobilis cells which were grown in the presence of 2.5% (v/v) or 8.5% (v/v) ethanol. A significant increase of en- zyme activity was caused by addition of ethanol to the assay (Fig. 3). An ethanol concentration of 60 g/1 (7.6% v/v) led to enzyme activities of up to 5.8 pmol x (minx mg protein) -1. As shown in Ta- ble 1 not only addition of ethanol but also of pro- panol to the assay increased the enzyme's activity. With methanol no alteration could be observed.

Table 1. Squalene-hopene-cyclase (SHC)-activity in cell-free extracts of Z. mobilis and hopanoid content of total cells in the presence of 6% (v/v) of different added alcohols

Additons to Spec. SHC- THBH a (% the culture activity of total medium/assay (pmol/rnin × mg) lipids)

Enhancement factor

of SHC- of THBH "- activity content

Control 4.24 19.9 1.00 1.00 Methanol 4.18 20.2 0.98 1.02 Ethanol 7.32 32.9 1.72 1.65 Propanol 7.19 32.5 1.69 1.63

a THBH; Tetrahydroxybacteriohopane

174 A. Schmidt et al. : Influence of ethanol in Zyrnomonas mobilis

The increase in squalene-hopene-cyclase activity, obtained by addition of ethanol or propanol to the assay correlated with the increase in hopanoid content of whole cells, cultivated in the presence of ethanol or propanol (Table 1). This correlation suggests a main regulatory function of squalene- hopene-cyclase in the alcohol dependent hopa- noid biosynthesis of Z. mobilis.

In contrast to the activation effect of ethanol on the squalene-hopene-cyclase of Z. mobilis the corresponding enzyme of B. acidocaldarius is in- hibited in the presence of 2% ethanol by 50% (G/irtner 1987). However, ethanol activation of other membrane-bound enzymes by ethanol has already been reported. For example, addition of ethanol or butanol to membranes prepared from L6 muscle cells resulted in a dose-dependent in- crease in adenylate-cyclase activity (Rabin et al. 1986). Concentrations of 0.25 M butanol (2.4% v/v) and 1.6 M ethanol (9.3% v/v) caused an 1.8- 1.9-fold increase of the maximum enzyme activity compared to the control. Whereas these alcohol concentrations are unphysiological for the adeny- late-cyclase, they usually accompany growth of Z. mobilis at high sugar concentrations.

For the activation of membrane-bound en- zymes by short chain alcohols several mecha- nisms have been suggested which are based on the lipid solubility of alcohols and their potential to decrease the order of the membrane lipid bilayer. The activity of membrane-bound enzymes could be influenced by changes in bulk membrane or- der, i.e. membrane fluidity (Gordon et al. 1980; Luthin and Tabakoff 1984), but also by interac- tions of the alcohols with hydrophobic regions of the enzymes themselves or with the direct lipid environment of these enzymes (Tabakoff et al. 1987; Rabin et al. 1986). Thus, the mechanism of activation of the squalene-hopene-cyclase can be studied by using membranes with artificially changed bilayer fluidity.

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Received June 28, 1988/Accepted August 26, 1988