A Labeling Study To Elucidate the Biosynthesis of 4-(4-Hydroxyphenyl)-Butan-2-one (Raspberry Ketone) by Nidula niveo-tomentosa

Submerged cells of the basidiomycete Nidula niveo-tomentosa,a microbial producer of 4-(4-hydroxyphenyl)-butan-2-one, weresupplemented with ¹³C-labeled L-phenylalanines and with [1-¹³C]glucose.Labeled transformation products were detected by a novel methodof analyzing stable isotope-labeled metabolites, gas chromatography(GC) coupled to an atomic emission detector, and by GC-massspectrometry. A benzoate moiety was side chain elongated accordingto the poly-ß-keto scheme. The presence of an acetylcoenzyme A-carboxylase inhibitor shifted the spectrum of productsto benzyl compounds. Hence, the fungal pathway differs fromthe one established for plant tissues.

INTRODUCTION

Top

Introduction

Free or glycosidically bound 4-(4-hydroxyphenyl)-butan-2-one(p-HPB) has been found in numerous plant genera, such as Artemisia,Capparis, Dendrobium, Hippophae, Larix, Limonium, Pinus, Prunus,Rheum, Rubus, Saxifraga, Taxus, Vaccinium, Vanilla, and Vitis,and in the glands of the melon fly, Dacus cucurbitae, and ofthe North American beaver, Castor canadensis (4, 8, 9, 13, 17,20, 24). Surface cultures of the basidiomycete Nidula niveo-tomentosawere reported as another source as early as 1980 (2).

The methyl ketone showed antibacterial activity (19) and activityas an attractant for pollinating bees (10) and fruit flies (21).In mammalian metabolism, a skin-lightening activity was causedby a selective growth inhibition on human melanotic cells (14),and a lipid degradation activity was explained by a promotionof norephedrine-induced fatty acid release from rat adipocytes(15). The strongest interest in the compound, however, sinceits structural identification as the aglycon of rheosmin, isin its character impact function in raspberry flavor (3). Asits concentration in food plants is far too low for economicalisolation, efforts were made to open a biotechnological route,based on submerged culture of N. niveo-tomentosa (4). The naturalform of this sought-after aroma compound may cost up to $10,000/kg.

Supplementation of a fungal production medium with either L-phenylalanineor L-tyrosine increased the yields of phenylbutanoid compoundssignificantly to levels beyond 100 mg liter^-1. However, immediatedevelopment of the bioprocess to a productivity level sufficientfor industrial-scale production failed (5). It was thereforedecided to elucidate the fungal pathway in more detail. Thisshould provide sound biochemical knowledge to aid in selectingmetabolic precursors for further optimization. The phenylpropanoylprecursor in plant biochemical studies was p-coumaryl coenzymeA (CoA) (1, 6, 7). Decarboxylative condensation with labeledmalonyl-CoA was demonstrated for cell extracts of raspberryfruit and tissue cultures. Intracellular levels of intermediatesand further metabolism of p-HPB were monitored in raspberrysuspension cultures (18). The key enzyme benzalacetone synthasewas enriched from ripe raspberries (7), and the same plant-specificenzyme and a cDNA encoding it were isolated from rhubarb (1).

By using the more food-compliant and convenient precursor L-phenylalanineas the phenylpropanoyl moiety, it was attempted to confirm theplant pathway to p-HPB in the high-yielding fungal metabolismof N. niveo-tomentosa (12). The pattern of ring-labeled L-[²H₅]phenylalaninewas recovered from p-HPB, from the corresponding 4-(4-hydroxyphenyl)-butan-2-ol(betuligenol and rhododrendrol), from the ${alpha}$ ,ß-enone,and from 3-hydroxy-5-(4-hydroxyphenyl)-pentanoic acid, demonstratingthe role of L-phenylalanine as a facultative precursor and alsoindicating a side chain-elongating capability of the fungus.When the cells were exposed to L-[1-¹³C]phenylalanine in concentrationsup to 10 mmol liter^-1, no isotope effects occurred (12). Ifthe fungus had used the plant pathway, the label of L-[1-¹³C]phenylalaninewould have had to be recovered from fungal p-HPB and the relatedvolatile metabolites; this, however, was not observed (12).The objective of the present study was to resolve this apparentcontradiction and to attract attention to a novel method ofanalyzing stable isotope-labeled metabolites, gas chromatography-atomicemission detection (GC-AED).

MATERIALS AND METHODS

Top

Materials and Methods

Microorganisms.
The examined strains of N. niveo-tomentosa were from the Centraalbureauvoor Schimmelcultures (CBS strain 380.80), Baarn, The Netherlands,and the American Type Culture Collection (ATCC 38357), Manassas,Va. All quantitative data refer to the CBS strain. The ATCCstrain gave comparable results; both strains are ex soil underpteridium.

Chemicals.
All chemicals for culture media were purchased from Merck (Darmstadt,Germany). Raspberry ketone and acetonitrile (high-performanceliquid chromatography [HPLC] grade) were supplied by Aldrich(Taufkirchen, Germany). Labeled phenylalanines were from Promochem,Wesel, Germany; labeled glucose was from Chemotrade, Düsseldorf,Germany. (±)-Diclofop-methyl {propanoic acid, 2-[4-(2,4-dichlorophenoxy)phenoxy]-,methyl ester} was from Riedel-de Haen, Seelze, Germany. Thesolvents used for extraction were of analytical grade and distilledbefore use.

Cultivation.
Strains were inoculated (homogenized mycelium) into 100 ml ofculture medium (20 ml for the experiments carried out with D-[¹³C]glucose)and grown aerobically at 24°C in 300-ml Erlenmeyer flaskson a rotary shaker (Multitron; Infors, Bottmingen/Basel, Switzerland).The standard culture period was 14 to 16 days, after which theformation of volatile metabolites leveled off. Each day aliquotsof the culture broth were centrifuged, glucose consumption anddry weight were measured according to standard protocols, andthe supernatants were extracted immediately for metabolite analysis.The culture medium contained 30 g of glucose monohydrate liter^-1,6 g of soy peptone liter^-1, 1.5 g of yeast extract liter^-1,2.5 g of KH₂PO₄ liter^-1, 0.5 g of MgSO₄ liter^-1, 73.5 mg ofCaCl₂ · 2H₂O liter^-1, and 1.0 ml of trace element solution(Fe, Zn, Cu, and Mn ions) liter^-1 (soy peptone medium; pH 6.0).

Metabolite extraction.
The centrifuged culture broths were extracted with pentane-diethylether (azeotropic mixture). The organic phases were dried overanhydrous Na₂SO₄; concentrated in a Vigreux column; and analyzeddirectly or after methylation, silylation, or acetylation bycapillary gas-liquid chromatography with different systems,as indicated in the text.

GC analysis.
The solvent extracts were analyzed by capillary gas-liquid chromatographywith a FISONS 8000 gas chromatograph. Chromatographic conditionswere as follows: splitless injection; injection port, 230°C;flame ionization detector, 270°C; carrier gas, hydrogen;flow rate, 3.8 ml min^-1. Chromatograms were evaluated with HPChemStation software (Agilent Technologies, Palo Alto, Calif.).

An HP 6890 gas chromatograph with a Gerstel KAS 4 injectionsystem, coupled with an HP G2350 atomic emission detector (GC-AED),was used for isotope analysis with the following conditions:programmed temperature vaporization, 40 to 240°C (10°C/s),atomic emission detector, 280°C; carrier gas, helium; inletpressure, 20 lb/in². The GC-AED technique provides rapid andselective, in part also simultaneous, measurement of most elementsof the periodic table and of some stable isotopes including²H, ¹³C, and ¹⁵N, even in the presence of a large excess ofthe lighter isotope (23). The eluting compounds are atomizedin a microwave-induced plasma tube. Light emitted by the excitedatoms passes a holographic grating and is then recorded by amovable 512-diode array. The minimum detectable level is 1 pgof ²H/s at 655,990 nm and 10 pg of ¹³C/s at 176,934 nm. Signalsderived from concurrently occurring lighter or heavier isotopeshave to be subtracted electronically or manually. This is particularlytrue for ¹³C measurement, as the natural abundance (1.108% oftotal C) is much higher than that for deuterium (0.0145% oftotal H). Because of the selective detection of isotopes, chromatographicoverlap of isotopomers is irrelevant, a significant advantageover GC-MS. Quantification is carried out as usual by electronicintegration of peak areas (HP ChemStation software).

A FISONS 8000 gas chromatograph coupled with an FISONS MD 800mass spectrometer (GC-mass spectrometry [MS]) was used to analyzelabeling patterns, and conditions were as follows: splitlessinjection; cool on column (40°C); ion source, 200°C,70 eV; carrier gas, helium; inlet pressure, 50 kPa. Chromatographicconditions were the same for all three systems: Optima 5-MScapillary column (30 m by 0.25 mm [inside diameter] by 0.25µm; Macherey & Nagel, Düren, Germany); injectionvolume, 1 µl; temperature program, 40°C for 3 min,to 280°C at a rate of 5°C/min, 10 min. The retentionindices of the sample components were compared to those of referencesubstances. All quantitative data are means of two analysesof independently grown shake-flask cultures. The coefficientof variation was <7% for all experiments. The inhibitor studywith diclofop-methyl was repeated four times.

HPLC analysis.
The L-phenylalanine concentration was analyzed by HPLC. Thesystem consisted of a Jasco PU-980 Intelligent HPLC gradientpump, a Jasco LG-980-02 gradient former, and a Jasco MD-910photodiode array detector (Tokyo, Japan). Clear culture mediawere injected on a Nucleosil 120-5 C₁₈ (250- by 4-mm) column(Macherey & Nagel), and conditions were as follows: injectionvolume, 20 µl; mobile phase flow rate, 0.8 ml min^-1. Thegradient was initiated with 5% acetonitrile and 95% water andincreased linearly to 50% acetonitrile over a period of 16 min.The system was kept constant at 50% acetonitrile for 4 min.The detection was performed at a ${lambda}$ value of 216 nm.

RESULTS AND DISCUSSION

Top

Results and Discussion

Labeling experiments with L-phenylalanines.
Suspended pellets of N. niveo-tomentosa tolerate exogenous L-phenylalaninein concentrations up to 10 mmol liter^-1 without microscopicchanges or effects on growth. A previous time course study ofthe incorporation of ring-perdeuterated phenylalanine over 16days did not show a pronounced isotope effect or scramblingof the added isotope label (12). The addition of 3 mmol of sterile-filteredL-[2-¹³C]phenylalanine liter^-1 resulted in an empty AED chromatogramfor the ¹³C trace (data not shown), just like the addition ofthe 1-¹³C-isotopomer in the preceding study (12). This indicatedcorrect subtraction of the natural ¹³C background but also nonincorporationof 1- and 2-¹³C of the phenylalanine side chain. Accordingly,no isotope labeling of phenylbutanoid compounds was found byGC-MS. ¹³C-labeled target compounds occurred only upon feedingof L-[3-¹³C]phenylalanine. Figure 1 compares ¹²C and ¹³C tracesof the GC-AED: the chromatographic ¹³C trace almost perfectlymirrored the ¹²C trace, although only 1 out of the 10 carbonsof the phenylbutanoid products could contain a ¹³C atom derivedfrom the precursor molecule and although nonlabeled phenylalaninewas present in the soy peptone used as the nitrogen source.Evaluation of the ¹²C/¹³C labeling ratios of the benzylic carbonatom of both p-HPB and betuligenol by GC-AED is possible, ifequal response factors of the two carbon isotopes are assumedin integrating the peak areas of the ¹²C and the ¹³C trace.A proportion of 45% of the phenylbutanoids was found to be labeled.MS data resulted in a calculated labeling proportion of about44%. This meant that each 23rd (10 carbon atoms: 44 x 100) atomof a phenylbutanoid compound actually was a ¹³C atom. Higherelectronic amplification was therefore necessary for equal signalheight, which caused the higher signal-to-noise ratio seen inthe lower trace of Fig. 1.

View larger version (15K):
[in this window]
[in a new window]
FIG. 1. GC-AED chromatogram of an extract from N. niveo-tomentosa upon addition of 3 mmol of L-[3-¹³C]phenylalanine. Compound designations: 1, p-HPB; 2, 4-(4-hydroxyphenyl)-butan-2-ol; 3, 4-(4-hydroxyphenyl)-3-buten-2-one; 4 and 5, structure isomers of hydroxymethoxyphenyl-butan-2-ol. ¹²C was detected at 177.491 nm, and ¹³C was detected at 176.934 nm

p-[4-¹³C]HPB showed an intense molecular ion at m/z 165 whichupon ${alpha}$ -cleavage of the CC bond in benzylic position yielded aring-extended hydroxytropylium ion at m/z 108 (nonlabeled counterpart,m/z 107); other important fragment ions were HOPh[¹³CH₂]CH_2CO⁺at m/z 150 and HOPh[¹³CH₂]CH₂⁺ at m/z 122. The similar massspectrum of betuligenol is distinguished by a fragment ion resultingfrom elimination of water at m/z 149 (nonlabeled counterpart,m/z 148) and by the propenylic fragment ion HOPh[¹³CH_2]CHCH⁺at m/z 134 resulting from the loss of the terminal methyl group(and water).

The presence of labeled 4-(4-hydroxyphenyl)-3-buten-2-one isconsistent with the general route of polyketide synthesis: reductionof the ß-keto function followed by elimination ofwater would yield just this intermediate. Hydrogenation of the ${alpha}$ ,ß double bond concludes the sequence, deliveringa saturated hydrocarbon chain.

Formation of the side chain.
The growth of a hydrocarbon chain is brought about by Claisen-typecondensation reactions of activated acetate or acetate plusmalonate units (11). A partial replacement of glucose in thenutrient medium by equimolar amounts of acetate, malonate, dimethylmalonate, pyruvate, L-alanine, oxalacetate, 2-oxoglutarate,hexadecanoic acid, or plant oils resulted in similar or decreasedconcentrations of p-HPB and betuligenol during the growth cycle.As the endogenous acetyl pool appears to be replenished by glucoseas the main C source of the nutrient medium, D-[1-¹³C]glucosewas supplied together with 3 mmol of nonlabeled L-phenylalanineliter^-1. This should yield 2-¹³C-labeled acetyl-CoA and, uponcarboxylation, 2-¹³C-labeled malonyl-CoA. If the benzyl starterunit was elongated by twofold condensation of malonyl-CoA units,C₁- and C₃-labeled phenylbutanoid compounds had to be received.Indeed, a GC-AED chromatogram very similar to that in Fig. 1was obtained after 12 days of cultivation in the presence ofD-[1-¹³C]glucose. From the intensity of the fragment ions aproportion of 24.4% of labeled p-HPB was derived. The mass spectrumof p-HPB showed additional isotope fragments at m/z 165 and,with clearly lesser intensity, m/z 166 (nonlabeled molecularion M^·⁺ at m/z 164) resulting from monolabeled (1- or3- position) and from double-labeled (1- and 3- position) metabolites,respectively. With the label in positions 1 and 3 of the butylside chain, ${alpha}$ cleavage of the CC bond in benzylic position yieldedagain the (here nonlabeled) hydroxytropylium ion at m/z 107and the phenyl cation at m/z 77. An ${alpha}$ cleavage next to the ketogroup led to HOPhCH₂[¹³CH_2]CO⁺ at m/z 150, HOPhCH₂[¹³CH_2]⁺ atm/z 122, and [¹³CH_3]CO⁺ at m/z 44 (nonlabeled m/z 43). The massspectrum of betuligenol analogously showed intense isotope fragmentsat m/z 167 and 168 and further labeled fragment ions at m/z150, 149 (loss of water), 134, 122, and 46 ([¹³CH_3]CH⁺OH). Themass spectral data further indicate that scrambling of the glucoselabel did not occur: a labeled carbon in the 2- position wouldhave been detected in the mass spectrum of the ketone by m/z45 (¹³CO-¹³CH₃) and in betuligenol by m/z 47 (¹³CHOH-¹³CH₃),but both ions were completely absent. As the fourth carbon ofthe chain is obviously derived from phenylalanine, the glucoselabel cannot occur there. The elongation of a benzyl moietyby addition of a three-carbon unit, an alternative route thatcould be envisaged, for example, through a reduction of theterminal carboxylate of added malonate, is also excluded bythe observed labeling patterns. These patterns were obtainedthroughout the entire cultivation period.

Other labeled metabolites.
According to GC-AED and GC-MS analyses the ¹³C label was alsocontained in the ${alpha}$ ,ß-enone (peak 3 in Fig. 1), presumablythe immediate progenitor of betuligenol (see above discussion),and in two metabolites with M^•⁺ 196 and isotope fragmentsat m/z 197 and 198. Evaluation of the mass spectra pointed tothe presence of 4-(4-hydroxy-3-methoxyphenyl)-butan-2-ol (zingerol)and another methoxy derivative of betuligenol (compounds 4 and5 in Fig. 1). The concentrations were too low for GC-preparativeenrichment and nuclear magnetic resonance spectrometry. Microchemicalcharacterization gave the following results: methylation didnot alter the structure of both compounds, silylation and acetylationindicated the presence of two hydroxy groups, and the acetylatedderivatives yielded fragment ions typical of one aromatic (M^•⁺42) and aliphatic hydroxy (M^•⁺ 60) groups (Fig. 2). Hydroxylationof aromatic rings and subsequent methylation of the phenolicfunction are common in basidiomycete biochemistry (22). Thesecompounds may contribute to the pool of monomers used by thefungus for the assembly of higher-molecular-weight metabolites(2).

View larger version (12K):
[in this window]
[in a new window]
FIG. 2. ¹³C-labeled metabolites upon addition of L-[3-¹³C]phenylalanine (for compound designations see the Fig. 1 legend).

Inhibition of acetyl-CoA-carboxylase.
Several arylphenoxypropanoates, such as diclofop-methyl, inhibitthe carboxylation of acetyl-CoA and can thus be expected todecrease the formation of phenylbutanoids by N. niveo-tomentosa.In the presence of 3 mmol of L-phenylalanine liter^-1 and 0.5mmol of diclofop-methyl liter^-1, growth was not retarded; onlyafter more than 2 weeks of cultivation was premature entranceinto stationary phase, compared to that of a control culture,observed. A concentration of 0.1 mmol of inhibitor liter^-1 onlyslightly altered the spectrum of products, but at a concentrationof 1 mmol liter^-1 the inhibitor blocked the formation of thephenylbutanoid compounds in favor of the formation of mainlybenzyl compounds (Table 1). This inhibition could not be overcomeby the addition of up to 20 mmol of dimethyl malonate. Evenin the absence of an inhibitor, millimolar supplements of sodiummalonate or malonyl dimethylester did not increase the concentrationof phenylbutanoid compounds. This failure to cross-access ametabolic pathway is a common observation when multienzyme complexesare involved.

View this table:
[in this window]
[in a new window]
TABLE 1. Compounds identified in gas chromatograms of extracts from nutrient media of N. niveo-tomentosa upon supplementation with diclofop-methyl, an acetyl-CoA-carboxylase inhibitor

Further indications for the suggested pathway.
Benzoates or 4-HO-benzoates, respectively, could serve as anexogenous benzyl starter for further chain elongation by thefungus. Supplementation in millimolar amounts did, however,not increase the formation of phenylbutanoid compounds but retardedgrowth. In view of the worldwide use of these compounds as antifungalfood additives, no other result was expected. Likewise, no precursoractivity was found for 3-HO-3-phenylpropanoic acid and its 3-ketoanalogue, when supplied as ethyl esters in millimolar amounts.These hypothetical intermediates will occur as activated, enzyme-boundmetabolites, saturating the following enzyme on site. Hence,exogenous substrate will be ignored because of the multienzymecomplex nature of the catalysis involved. The lack of measurableconcentrations of free intermediates other than those shownin Fig. 1 is in line with this view.

N-Benzoyl glycine (hippuric acid) was able to replace L-phenylalanineas a facultative precursor of p-HPB (Table 2). Growth curvesindicated little acute toxicity (data not shown). While feedingwith 10 mmol liter^-1 reduced the yields of phenylbutanoid compounds,indicating an initial adverse effect, 1 mmol liter^-1 resultedin product formation almost as high as that by 3 mmol of L-phenylalanineliter^-1 (molar conversion rate of about 52%). The ratio of phenylbutanoidalcohol to ketone was around 91/9 throughout these experiments.It is supposed that the superior precursor efficiency of N-benzoylglycine originated from the metabolic use of both the benzyland the glycine moiety of the amide. N-Benzoyl glycine hydrolasesoccur widely in microorganisms (16). However, high hydrolaseactivity would be suicidal; 1 mmol of free benzoic acid alreadywas exerting toxic effects as judged by growth inhibition. Thus,the observed efficiency of N-benzoyl glycine might rather beattributed to its function as a chemical cache of a benzyl moietyunder feedback regulation by the fungus. Alternatively, themonocharged amide may pass membrane barriers more easily thanphenylalanine might.

View this table:
[in this window]
[in a new window]
TABLE 2. N-Benzoyl glycine and L-phenylalanine as precursors of phenylbutanoid compounds in N. niveo-tomentosa^a

The studies feeding labeled and nonlabeled precursor substratessuggest the fungal provision of a benzyl starter which is thenside chain elongated by a twofold condensation to form compoundspossessing a phenylbutanoid skeleton, with D-glucose actingas an efficient malonyl donor (Fig. 3). As was presumed previously(12), this pathway is obviously different from the one wellestablished previously for raspberry (6, 7, 18) and rhubarb(1), where phenylbutanoids were formed via one-step decarboxylativecondensation of 4-coumaryl-CoA with malonyl-CoA. While benzalacetonesynthase from rhubarb was cloned and sequenced, the respectivefungal enzyme was not reported yet. The reductive decarboxylationof a phenylpentanoic moiety with accumulation of phenylbutanoidsis a singular step in microbial biochemistry, and no microorganismbesides N. niveo-tomentosa is known to be capable of this reaction.The formation of phenylbutanoid compounds may be explained bya premature release of intermediates from a polyketide synthasecomplex incapable of regularly handling longer-chain intermediates.This deficiency, however, becomes easily observable only inthe presence of a large supply of exogenous benzyl precursorleading to pathway overflow: in the absence of added precursor,the total concentration of phenylbutanoids in a submerged culturein standard nutrient medium without complex ingredients is onlyin the range of 2 to 3 mg/liter (12 days of cultivation).

View larger version (20K):
[in this window]
[in a new window]
FIG. 3. Formation of p-PHB and 4-(4-hydroxyphenyl)-butan-2-ol by condensation of a benzoyl-CoA moiety and two malonyl-CoA units. Positions of ¹³C labels are indicated (for compound designations see the Fig. 1 legend).

Preliminary attempts to reproduce the bioconversion by usingcell extracts prepared according to a standard protocol (celldisintegration with a glass ball mill, 0°C; phosphate bufferpH 7.5), or according to the method in reference 6, have sofar been unsuccessful but will be the subject of future experiments.The use of other specifically labeled precursors, such as L-3-D-phenylalanine,could provide additional evidence for the suggested metabolicpathway. The role of N-benzoyl glycine and of N-benzoyl glycinehydrolases will be investigated by monitoring the time courseof liberation of benzoic acid in the nutrient medium. Anotherobjective will be to locate experimental conditions for shiftingthe formation of 4-(4-hydroxyphenyl)-butan-2-ol more towardthe actual biotechnological target, raspberry ketone.

ACKNOWLEDGMENTS

This work was supported by the former Tastemaker BV, Barneveld,The Netherlands, and by the Fonds der Chemischen Industrie,Frankfurt, Germany. The project was part of the joint initiativeproject "Biologisch aktive Naturstoffe—Chemische Diversität"at the University of Hannover.

FOOTNOTES

* Corresponding author. Mailing address: Zentrum Angewandte Chemie, Universität Hannover, Institut für Lebensmittelchemie, Wunstorfer Straße 14, D-30453 Hannover, Germany. Phone: 49511 762 4581. Fax: 49511 762 4547. E-mail: rg.berger{at}lci.uni-hannover.de.

REFERENCES

Top