Tryptophol Formation by Zygosaccharomyces Priorianus

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APPLIED MICROBIOLOGY, JUly 1973, p. 98-105 Vol. 26, No. 1 Copyright 0 1973 American Society for Microbiology Printed in U.S.A. Tryptophol Formation by Zygosaccharomyces priorianus

J. P. ROSAZZA, R. JUHL, AND P. DAVIS Pharmacognosy Research Laboratories, The University of Iowa, College of Pharmacy, Iowa City, Iowa 52242 Received for publication 26 February 1973

Zygosaccharomyces priorianus converted L-tryptophan to tryptophol and to small quantities of indole-3-acetic acid. Neither tryptophol nor indole-3-acetic acid was metabolized when added separately to growing cultures. The possible intermediacy of indole-3-pyruvic acid, indole-3-acetaldehyde, and tryptamine in the degradation of L-tryptophan was tested by feeding these compounds to Z. priorianus and Saccharomyces cerevisiae. Indole-3-pyruvic acid and indole-3- acetaldehyde were converted to tryptophol and indole-3-acetic acid, with the latter accumulating only in small amounts. Tryptamine was converted to its N-acetyl derivative by these organisms. A qualitative study was made on the metabolism of L-phenylalanine, L-tyrosine, and L-5-hydroxytryptophan by these organisms. Like L-tryptophan, these amino acids were metabolized to their respective alcohol and acid derivatives. Of a large number of organisms tested, the yeasts possessed the highest capacity for degrading L-tryptophan to tryptophol.

The metabolic disposition of L-tryptophan In our laboratories, preliminary studies on the (Trp) by microorganisms has been widely stud- metabolism of Trp by shaken cultures of Zygo- ied. Besides being incorporated nearly intact saccharomyces priorianus resulted in the iden- into secondary metabolites such as violacein (5, tification of Trp-ol and indole-3-acetic acid 31), the ergot alkaloids (24), neoechinuline (3), (IAA) as major metabolites. According to initial and other compounds (28, 54), numerous degra- qualitative data, Trp-ol was formed to a much dation products of this amino acid are also greater extent than was IAA by this organism. formed by microorganisms. Common metabolic We became interested in the potential use of reactions which lead to the accumulation of Trp yeasts for the synthesis of Trp-ol and some degradation products include tryptophanase (6, Trp-ol derivatives which have been found to 19, 32, 42), deamination (2, 4, 14, 15, 26, 43), possess interesting pharmacological activity and oxidative fission of the pyrrole ring of the (13, 51). indole nucleus (23, 36, 47, 48, 52, 55). A few However, results of other studies on the bacterial systems metabolize Trp by means of metabolism of Trp by yeasts indicated that decarboxylation as the primary step (30, 34). Trp-ol and IAA were indeed formed, but that Yeasts accomplish the degradation of amino the reaction could be complicated by the forma- acids principally via the Ehrlich mechanism tion of other Trp metabolites such as indole-3- (18, 37, 44, 53). In this general metabolic lactic (ILA) (15), indole-3-carboxylic acid, and pathway, amino acids undergo transamination indole-3-pyruvic acid (IPA) (14). These studies to their respective keto acids. These are decar- also indicated that the ratios of various Trp boxylated, and the resulting aldehydes are re- metabolites obtained was dependent on fermen- duced to their alcohol equivalents (44). Ehrlich tation conditions. was the first to demonstrate the formation of This work was therefore undertaken to deter- tryptophol (Trp-ol) (11), tyrosol, and phenethyl mine the stoichiometry of the degradation of alcohol (10) from their respective amino acid Trp by Z. priorianus. An attempt was also made precursors. These alcohol derivatives have been to determine whether the accumulation of found in the higher-boiling fractions of fusel oils Trp-ol and IAA was peculiar to particular and are generally thought to be formed by way organisms or to groups of organisms. The abili- of the Ehrlich mechanism (37, 50, 53). ties of Z. priorianus and Saccharomyces 98 VOL. 26, 1973 TRYPTOPHOL FORMATION BY Z. PRIORIANUS 99 cerevisiae to convert known Trp metabolites contained in 125-ml cotton-plugged Erlenmeyer and certain other amino acids to their respec- flasks. The 48-h growth started from slants was used tive alcohol derivatives was also examined. as inoculum for the fermentation proper. The inoculum volume was 10% of the volume of fermentation medium in all cases. Substrates were added to these MATERIALS AND METHODS secondary cultures after 24 h of growth. Trp and other Melting points are corrected and were obtained in amino acids were added as their HCl salts, and all open-ended capillary tubes. Infrared spectra were other substrates were added in dimethylformamide obtained on a Beckman 10 spectrophotometer by us- solutions (0.1 ml/25 ml of medium). ing KBr disks. Nuclear magnetic resonance spectra Procedure for the separation of Trp-ol, IAA, and were obtained with a Varian T-60 instrument in CDCl1 Trp. Samples of fermentations (5 ml) were adjusted with tetramethylsilane as an internal standard. to pH 2 with 5 N HCl and extracted three times with A number of chemicals were purchased. The iden- 5-ml portions of ethyl acetate. The extracts were tity of these compounds was confirmed either by combined, and 10-ml samples were partitioned with 5 melting point or by the preparation of suitable deriva- ml of 0.1% NaHCOs. The resulting ethyl acetate tives. Each compound was also examined by thin- solution (extract A) was assayed for Trp-ol. The layer chromatography (TLC) to verify purity. Chemi- NaHCOs layer was adjusted to pH 2 and extracted cals used in this study were: Trp and phenethyl alco- with two 5-ml portions of ether, the ether extracts hol (Eastman); IAA and L-tyrosine (Tyr), (Fisher); were combined, and samples were assayed for IAA L-5-hydroxytryptophan (5-OH-Trp), Trp-ol, 5-hydroxy- (extract B). The aqueous portion which remained indole-3-acetic acid (5-OH-IAA), IPA, ILA, tryp- after the initial ethyl acetate extractions was used for tamine (Trp-amine), indole-3-acetaldehyde (I-ace- analysis of Trp (extract C). tald), L-phenylalanine, phenylacetic acid, and 4-hy- This overall extraction procedure was slightly mod- droxyphenylacetic acid (Sigma Chemical Co.) and 5- ified for the quantitative determination of Trp. Media hydroxytroptophol (5-OH-Trp-ol) (Regis). samples were diluted to known volumes with 0.01 N Tyrosol (4-hydroxyphenethyl alcohol) was prepared HCl and extracted three times with ethyl acetate (as from 4-hydroxyphenylacetic acid according to previ- above) to remove all Trp-ol and IAA. Suitable sam- ously described methods (20, 41). The compound was ples of the remaining aqueous portions were then crystallized from chloroform (mp 88-90 C; reported assayed for Trp. mp 91.5 C [41]). Each extract was examined by TLC to estimate its Synthetic N-acetyltryptamine was obtained by purity. Extracts A, B, and C were found to contain refluxing 500 mg of Trp-amine-hydrochloride with a only Trp-ol, IAA, and Trp, respectively. No other mixture of 1 ml of glacial acetic acid, 1 ml of acetic indole compounds were detected in any of these anhydride, and 10 mg of zinc dust for 30 min. The hot extracts in the experiments to be described. reaction mixture was poured into 25 ml of cold water, Trp-ol assay. Trp-ol was determined by a modifi- and the crude solid product was filtered. Crystalliza- cation of the procedure of Harbo and Aashiem (17). tion was achieved with a benzene-hexane mixture Suitable samples of extract A were evaporated to (mp 76.5-77 C, reported 77 C [461: nuclear magnetic dryness in a test tube under a stream of air. To the resonance (CDCL,) 6 1.95 (s, 3, COCH3), 2.9 (q, 2,- resulting residue was added 2.75 ml of methanol and -CH,2-NH-), 3.6 (q,2, Ar-CH2), 7.32 (m,5, Aro- 1.5 ml of a solution containing 2 g of p-dimeth- matic); infrared (KBr disk) q 2.97, 3.26, 6.25, 6.41, ylaminobenzaldehyde (PDAB) in 84 ml of 95% 9.17, 9.41, 12.55, 13.48). ethanol and 16 ml of concentrated HCl. The test tubes The benzyl ester of Trp was prepared by known were placed in a water bath maintained at 95 C for 2 procedures (mp 71-72 C, reported 71 C [33]). min. The samples were removed from the water bath, Cultures and general growth procedures. Cul- 0.25 ml of 0.1% NaNO2 was added, the solutions were tures used extensively in this study were Zygosac- allowed to cool for 30 min at room temperature, and charomyces priorianus SP-WISC 22, which was ob- absorbances were measured at 530 nm. The Trp-ol tained from the culture collection of C. J. Sih at the standard curve was linear in the concentration range University of Wisconsin, School of Pharmacy; and of 0 to 50 Atg. Recoveries of added samples of Trp-ol Saccharomyces cerevisiae NRRL Y-2034. Organisms through the entire extraction procedure averaged 100 were maintained on Sabouraud-maltose agar slants ± 4%. and stored in a refrigerator at 4 C prior to use. All IAA assay. IAA was determined by the spectroflu- organisms mentioned in this work are maintained in orometric procedure of Stoessel and Venis (49). Rela- the culture collection at the College of Pharmacy of tive fluorescent intensities were measured in an the University of Iowa. Aminco-Bowman spectrophotofluorometer at an exci- Cultures were grown in a soybean meal-glucose tation wavelength of 460 nm and an emission wave- medium of the following composition: soybean meal, 5 length of 490 nm. A slit width of 3 was used through- g; glucose, 20 g; yeast extract, 5 g; NaCl, 5 g; K2HPO4, out. Concentrations were related to a standard curve 5 g; distilled water, 1 liter; pH 7.0, adjusted with 5 N which was linear in the range of 0 to 30 gg of IAA. HCl. Media were autoclaved at 121 C for 15 min. Recoveries of added IAA samples through the extrac- Fermentations were conducted on rotary shakers tion procedure averaged 100 + 10%. (model G-25, New Brunswick Scientific Co.) (250 Trp assay. A modification of the procedure of rpm, at 27 C) usually in 25-ml amounts of medium Udenfriend and Peterson was used (56). Suitable 100 ROSAZZA, JUHL, AND DAVIS APPL. MICROBIOL. volumes of extract C were diluted to 0.5 ml with 5 N each of medium. The benzyl ester of Trp (1.22 HCl, PDAB reagent (3.0 ml of a 0.5% solution in g) was distributed evenly among 24 flasks, and concentrated HCl) was added, samples were allowed one was kept as a control. Culture samples were to stand for 10 min, and 0.25 ml of 0.1% NaNO2 and taken at 24, 48, and 72 h after substrate 3.0 ml of anhydrous ethanol were added to them. addition and were examined by TLC (solvent After 10 absorbances were measured at 620 nm. min, for the presence of the unknown The standard curve was linear between Trp concen- system A) trations of 10 to 100 ug, and recoveries of added Trp indole. At 72 h, most of the substrate had been through the extraction process averaged 100 4 5%. consumed, and the unknown product was the TLC. TLC was performed on 0.25-mm thick silica major indolic metabolite. The control contained gel GC254 (Merck) plates. Modifications of published a trace of this metabolite. TLC solvent systems (35) were used to develop The cultures were filtered through a Celite chromatograms. Indolic compounds were visualized pad, and the filtered solids were washed with by spraying developed plates with 0.1% PDAB in 500 ml of distilled water. The combined filtrates concentrated HCl, drying with a heat gun, and (3 liters) were adjusted to pH 8.25 with 1 N spraying with 0.1% aqueous NaNO2. Most indoles three times with 800 ml gave blue to purple spots on chromatograms. Non- NaHCO3 and extracted indolic compounds were visualized with a p-anisalde- of ethyl acetate. The extract was dried over hyde-H2SO4-MeOH (1: 1: 48, vol/vol/vol) spray rea- Na2SO, and evaporated to a viscous brown gent. Different compounds displayed a variety of residue (0.934 g) which was adsorbed onto colors when sprayed plates were heated. Table 1 alumina (Fisher A-540, 100 g, column dimen- shows the solvent systems, and R. values of com- 2ions 2.5 by 26 cm). Elution of the column was pounds involved in this work. accomplished with benzene-95% ethanol (25: 1, A routine procedure was used in the TLC analysis vol/vol), and 10-ml fractions were collected at a of fermentations. Samples were adjusted to pH 2 to 90 with 5 N HCl and extracted with 0.25 volume of ethyl flow rate of 1 ml/min, fractions 36 yielding acetate, and approximately 50 pliters of the extracts 0.257 g of the indole product. The metabolite a was spotted on TLC plates. Extracts were always was crystallized from mixture of equal parts of co-chromatographed with standards to facilitate iden- benzene and hexane: mp 58 to 59 C, reported for tification of unknowns. In most cases, two or three Trp-ol, 59 C (45); infrared (KBr disk) qs 2.92, different solvent systems were used to verify the 3.0 to 3.35 (broad band), 6.13, 6.74, 6.89, 7.04, identify of metabolites. 7.43, 7.49; nuclear magnetic resonance (CDCl1) 6 1.17 (s,1,OH), 3.0 (t,2,-CH2OH), 3.9 RESULTS (t, 2, Ar-CH2), 6.9-7.78 (m,5, aromatic), 8.13 Production and isolation of the major me- (s,broad, NH). The peak at 1.17 disappeared tabolite of Trp esters. Z. priorianus was grown upon equilibration with D20. Elemental com- in 500-ml Erlenmeyer flasks containing 100 ml position was: calculated for Trp-ol, C 74.51, H TABLE 1. Thin-layer chromatography solvent systems and R, values of substrates and metabolites of Z. priorianus and other microorganisms R. values in Solvent systemsa Compound A B C D E F Tryptophol 0.50 0.40 0.90 0.71 Indole-3-acetic acid 0.30 0.54 0.20 0.75 Indole-3-Propionic acid 0.68 0.85 0.55 Indole-3-acetaldehyde 0.62 0.72 0.90 0.89 Tryptamine 0.15 0.05 Indole-3-pyruvic acid 0.54 Many 0.81 spots Indole-3-Lactic acid 0.08 0.12 0.19 0.23 N-Acetyltryptamine 0.40 0.20 1.00 0.60 5-Hydroxytryptophol 0.35 0.51 0.85 0.45 5-Hydroxyindole-3-acetic acid 0.10 0.95 0.35 0.20 Phenylethyl alcohol 0.73 0.96 0.92 0.90 Phenylacetic acid 0.62 0.57 0.46 0.54 Tyrosol 0.30 0.96 0.91 0.50 4-Hydroxyphenylacetic acid 0.40 0.38 0.69 0.35 a Solvent systems used (vol/vol/vol ratios): A, benzene-ethanol (4:1); B, chloroform-glacial acetic acid, (95:5); C, ethyl acetate-isopropanol-25% NH4OH (9:5:4); D, chloroform-glacial acetic acid-95% ethanol (90:5:5); E, ethyl acetate-acetone (10:1); F, benzene-methanol (5:1). VOL.26, 1973 TRYPTOPHOL FORMATION BY Z. PRIORIANUS 101 6.88, N 8.69; found, C 74.99, H 6.78, N 8.68. The priorianus cells. Based on the amount of Trp metabolite was spectrally and chromatograph- originally added to the cultures, the 46-h analy- ically (solvent systems A, B, and C) identical to ses gave a 96% recovery, expressed as total a purchased sample of Trp-ol. indoles. Of the Trp consumed in the fermenta- Quantitative analysis of the degradation of tion at this time, 93% of it was converted to Trp to Trp-ol and IAA. According to TLC Trp-ol. In a separate, identical experiment, it analysis (solvent systems A, B, and C), IAA was was determined that the number of viable cells identified as a minor metabolite in the previous of Z. priorianus remained constant during the experiment, and it was the only other indole course of the fermentation and that the orga- compound detected. We learned that Z. nism was in the stationary growth phase. priorianus could convert free Trp to Trp-ol and Metabolism of Trp-ol, IAA, I-acetald, IPA, IAA in proportions similar to those obtained and Trp-amine by Z. priorianus and S. with the benzyl and ethyl esters. Thus, Trp was cerevisiae. A qualitative study was made to used as substrate in all subsequent work. This determine the capacity of Z. priorianus and S. quantitative study was conducted to determine cerevisiae to metabolize Trp-ol, IAA, I-acetald, the stoichiometry of the conversion of Trp to IPA, and Trp-amine to recognizable indole Trp-ol and IAA during the course of a fermenta- products. This was done to show whether IAA or tion. Trp-ol were interconvertible and if I-acetald, Z. priorianus was grown according to the IPA and/or Trp-amine could be utilized, thus usual procedure except that the fermentation supporting the general pathway for Trp degra- proper was conducted in 1-liter Erlenmeyer dation by Z. priorianus and other yeasts. Fer- flasks containing 200 ml of the Soybean-glucose TABLE 2. medium. Trp was added as substrate to obtain a Quantitative analysis of the degradation of Trp to Trp-ol and LAA by Zygosaccharomyces concentration of 4.90 gmol/ml (1 mg/ml) in each priorianus of three cultures. A fourth culture containing no Trp was kept as a control. Samples (5 ml) were Yields (Mumol/ml) withdrawn from the fermentations at time in- Hours after tervals between 0.5 and 77.5 h after substrate Addition of Trypto- Trypto- Total tryptophan IAAa phol phan indolesb additions. These were stored at -10 C until recovered required for analysis. Each sample was assayed for Trp, Trp-ol, and IAA by the methods 0.5 0.006 0.061 3.86 3.93 described. (Separate analyses showed that 20 ,ug 9.0 0.016 0.856 3.39 4.26 of Trp per ml was present as a normal compo- 22.0 0.025 1.99 2.51 4.53 32.5 nent of the Soybean-glucose medium, presuma- 0.035 3.38 1.34 4.75 46.0 0.069 3.62 1.01 4.69 bly being introduced via the yeast extract. 56.5 0.117 3.55 0.60 4.33 Traces of Trp-ol found in control flasks in 69.5 0.172 3.38 0.32 3.87 earlier experiments were undoubtedly produced 77.5 0.191 3.27 0.28 3.74 from this "endogenous" source of Trp. It should be noted that the amounts of Trp, Trp-ol, and a Values for IAA and Trp represent the averages of IAA found in the control culture in this experi- triplicate samples, while those for Trp-ol are dupli- ment were too low to be detected by the cates. described in Materials and "A 4.90-Mumol amount of Trp per ml was added to analyses Methods.) the cultures at zero time. The results of this experiment are shown in Table 2. TABLE 3. The metabolism of Trp-ol, IAA, IPA, Trp decreased steadily throughout the fer- I-acetald and Trp-amine by Z. priorianus and S. mentation from 3.86 to 0.28 ,gmol/ml at the end. cerevisiae Trp-ol was rapidly formed during the first half Substratea Trp-ol IAA Other of the fermentation, reaching a maximum of 3.62 gmol/ml at 46 h. Trp-ol levels decreased Trp-ol _ _ slightly until the end of the fermentation. IAA _ _ _ Relatively small amounts of IAA were pro- IPA + ++++ duced, and the ratio of Trp-ol to IAA at 32.5 h I-Acetald ++++ + was nearly 100. Trp-amine ++++ Assays at 0.5 h accounted for only 3.93 jsmol a Fermentations were conducted in duplicate with of total indoles per ml, mostly as Trp. Trp was suitable controls. Substrates were added to cultures in added to cultures in levels of 4.90 Mmol/ml. This concentrations of 0.5 mg/ml of medium. Samples apparent low recovery may be attributed to the taken at 48 h were examined by TLC (solvent systems uptake of Trp from the medium by Z. A, B, C, and D). 102 ROSAZZA, JUHL, AND DAVIS APPL. MICROBIOL. mentations were conducted in duplicate with as well. While the conversion of Trp to Trp-ol suitable controls, and substrates were added in was not restricted entirely to the yeasts, they concentrations of 0.5 mg/ml of medium. Sam- demonstrated a greater capacity for performing ples taken at 48 h were examined by TLC this metabolic transformation than other orga- (solvent systems A, B, and C, and D for nisms examined. tryptamine-containing cultures) (Table 3). Metabolism of other amino acids by Z. Z. priorianus and S. cerevisiae converted priorianus and S. cerevisiae. We examined Trp-amine into an indolic compound different the abilities of Z. priorianus and S. cerevisiae to than Trp-ol or IAA. To obtain a quantity of the metabolize L-phenylalanine, Tyr, and L-5-OH- metabolite sufficient for identification, Z. Trp to their respective alcohol and acid deriva- priorianus was grown in 24, 500-ml Erlenmeyer tives. Fermentations were conducted in dupli- flasks containing 100 ml of medium each. Trp- cate with controls, and the amino acid sub- amine-hydrochloride (930 mg) was distributed strates were added as their HCl salts in levels of equally among the flasks. Samples were taken 0.5 mg/ml of culture medium. Samples of the at various time intervals after substrate addi- fermentations were withdrawn at 72 h and tion, and the 48-h TLC analysis (solvent sys- examined by TLC (solvent systems E and F for tems A and D) showed that all of the substrate the phenylalanine fermentation, and solvent was utilized. systems C and F evaluate the 5-OH-Trp and The cultures were pooled, adjusted to pH 2 Tyr containing fermentations). 5-OH-Trp-ol, with 5 N HCl, and extracted with 3 liters of 5-OH-IAA, phenethyl alcohol, phenylacetic ethyl acetate. After drying, the extract was acid, tyrosol, and p-hydroxyphenylacetic acid evaporated under vacuum to a tarry residue of were used as standards for comparison on chro- 2.1 g. The tar was adsorbed onto silica gel (100 matograms. By estimation, these amino acids g, column dimensions 2.5 by 55 cm), and was were converted into the expected products in eluted with benzene-95% ethanol (15:1, vol! approximately the same proportions as Trp-ol vol). Fractions of 10 ml were collected at a flow and IAA by these yeasts. rate of 2.5 mlmin, fractions 48 to 84 yielding a total of 214 mg of the indole metabolite. The compound was crystallized from a benzene-hex- TABLE 4. Cultures which convert Trp to Trp-ol and ane mixture: mp 75 C, reported for N-acetyl- IAAa tryptamine 77 C (46); infrared (KBr disk) g 2.97, 3.10, 3.26, 6.25, 6.41, 9.17, 12.55, 13.48; Organism Taxonomicorder Trp-ol IAA nuclear magnetic resonance (CDCl) 61.95 (s, 3,- COCH) 2.9 (q,2,-CH2NH-), 3.59 (q,2,Ar- Zygosaccharo- Endomycetales + + + + + 7.32 (m,5,aromatic). The isolated prod- myces CH2), priorianus (22) uct was spectrally and chromatographically Saccharomyces Endomycetales + + + + + (solvent systems A and D) identical to synthetic cerevisiae N-acetyltryptamine (Materials and Methods). (NRRL Y-2034) Formation of Trp-ol and IAA by other Saccharomyces Endomycetales + + + + + sp. (6187) microorganisms. Although the formation of Schizosaccharo- Endomycetales + ++ + Trp-ol by yeasts and a few other microorga- myces octo- nisms is known, no general investigation on the sporus (NRRL taxonomic distribution of this metabolic con- Y-854) S. versatilis Endomycetales + + + + + version has been reported. To determine if other (NRRL Y-1026) microorganisms were capable of forming Trp-ol Nadsonia species Endomycetales + + + from Trp, 144 organisms representing five major (6188) + were in the Ascobolus Pezizales + classes and eight orders grown stercorarius presence of 1 mg of Trp per ml. Estimation of (237a) the relative amounts of Trp-ol and IAA pro- Candida Moniliales + + + duced was made by TLC (solvent systems A, B, lipolytica and C) (Table 4). (6189) Sporobolomyces Moniliales + + Only 9 of the 144 cultures produced detecta- species (1584) ble quantities of Trp-ol. Two of 79 Monililiales, and 1 of 3 Pezizales produced Trp-ol in this a Fermentations were conducted in duplicate according to of the 13 the usual procedure, and TLC assays were performed on study. Six organisms Actinomycetales, samples taken 48 h after addition of substrate by using sol- of Sphaeriales, 7 of Eurotiales, and 8 Agaricales vent systems A, B, and C. Microorganisms are designated by failed to produce Trp-ol. All cultures which genus and species names where possible, culture collection formed Trp-ol also produced quantities of IAA numbers which are in parentheses, and taxonomic order. VOL. 26, 1973 TRYPTOPHOL FORMATION BY Z. PRIORIANUS 103

DISCUSSION COON This work began in connection with studies NH2_2 * H on the abilities of microorganisms to hydroxyl- H _ H ate N- and carboxyl-blocked Trp derivatives. I rypto. pha-A n Indole-3-pyruvic acid When incubated with certain esters of Trp, Z. priorianus produced relatively large amounts of a metabolite which reacted blue with PDAB- NaNO2 spray reagents and gray with FeCl, on TLC plates. The identification of the metabo- OOHI lite was originally pursued because the mi- I OH HO crobial conversion of Trp esters to this com- H H pound was unusually complete and uncompli- Indole-3-lactic acid cated. Z. priorianus converted both the benzyl- Indole-3-acetaldehyde and ethyl-esters of Trp to this unknown indole product, which was isolated and identified as Trp-ol. The quantitative study showed that, when H20H 5 O OOH Trp was used as substrate, much larger amounts of Trp-ol were produced in comparison H to IAA. The formation of Trp-ol by microorga- H nisms has been reported (2, 7, 11, 14, 15, 22, 25, Tryptopho Indole-3-acetic acid 27, 29, 38, 40), and its formation in greater FIG. 1. General pathway for the degradation of Trp amounts than IAA has also been observed before by yeasts. (14, 15, 25, 39, 40). Aeration rates are known to influence the ratios of IAA to Trp-ol (39, 40). In similar to those obtained when Trp was used as cell-free preparations of plant tissue, pH af- substrate. IPA-containing cultures produced fected the ratios of IAA to Trp-ol produced from much larger amounts of IAA than Trp-ol and, I-acetald (39). This finding indicated that two by estimation, the ratio of IAA to Trp-ol was enzymes were capable of utilizing I-acetald- about 4. However, controls containing only one which produces Trp-ol and a second which culture medium and IPA were found to yield produces IAA. Two crude enzyme preparations large amounts of IAA simply by agitating the were obtained which were capable of selectively solutions on the rotary shaker. No Trp-ol was forming either Trp-ol or IAA. The existence of formed in the control flask. two such separate enzymes in microorganisms The great instability of IPA in aqueous media has not yet been demonstrated. has been reported before (14, 15, 26, 29), and it ILA has also been reported as a major Trp- has been shown that IPA spontaneously decom- degradation product of yeasts (15, 16), where poses to IAA under acidic conditions (26), with only minor amounts of Trp-ol and IAA were as many was seven different spots on chromato- obtained. These results suggest that, in yeasts, grams under alkaline conditions (15, 29). In the major pathway for Trp degradation involves controlled experiments, it has been shown that transamination as the initial step and subse- IPA does convert to Trp-ol in yeasts (15). quent formation of I-acetald and other products Trp-amine was not metabolized to Trp-ol and as shown in Fig. 1. Dialyzed cell-free extracts IAA but was converted into N-acetyltryptamine from yeast have been shown to catalyze the by Z-priorianus and S. cerevisiae. Trp-amine is transfer of the amino group from several amino converted into its N-acetyl derivative by Bacil- acids, including Trp, to alpha-ketoglutarate (4). lus cereus (34). Other microorganisms have Pyridoxal has been shown to facilitate the reac- been reported to N-acetylate D-tryptophan (16) tion. Others have demonstrated that proposed and other amino acids (57). Ehrlich suggested intermediates such as IPA (14) and I-acetald that amines may represent the intermediates in (38, 40) serve well as substrates for yeasts in the formation of Trp-ol, tyrosol, and other the formation of ILA, Trp-ol, and IAA. alcohols formed from amino acids (12). Others The possible intermediacy of EPA, I-acetald, have demonstrated that Trp-amine is converted and Trp-amine in the degradation of Trp was to Trp-ol and IAA by several microorganisms (8, tested by feeding these compounds to Z. 9). It is apparent that Trp-amine does not serve priorianus and S. cerevisiae. I-Acetald served as a substrate in the formation of Trp-ol and well as a substrate for these yeasts and was IAA with Z. priorianus and S. cerevisiae. metabolized to Trp-ol and IAA in proportions N-Acetyltryptamine arises as an artifact dur- 104 ROSAZZA, JUHL, AND DAVIS APPL. MICROBIOL. ing ethyl acetate extraction of Trp-amine from synthesized by Diplodia natalensis. Plant. Physiol. biological samples, especially at alkaline pH 37:439-445. 3. Barbetta, M., G. Casnati, A. Pochini and A. Selva. 1969. (21). Our metabolite was isolated by ethyl Neoecninuline: A new indole metabolite from Aspergil- acetate extraction at pH 2. A control study lus amstelodami. Tetrahedron Lett. 1969:4457-4460. showed that N-acetyltryptamine could not have 4. Bigger-Gehring, L. 1955. Transamination reactions in been formed by our extraction procedure. Saccharomyces fragilis. J. Gen. Microbiol. 13:45-53. 5. DeMoss, R. D. 1967. Violacein, p. 77-81, In D. Gottlieb Of a large number of organisms tested, the and P. D. Shaw (ed.), Antibiotics, vol. 2. Springer-Ver- yeasts possessed the highest capacity for de- lag, Berlin. grading Trp to Trp-ol. Only 9 of 144 microor- 6. DeMoss, R. D., and K. Moser. 1969. Tryptophanase in ganisms were found to produce Trp-ol when diverse bacterial species. J. Bacteriol. 98:167-171. 7. Drews, B., H. Specht, and E. Schwartz. 1966. Ober die incubated with Trp. Rajagopal incubated 11 bei der alkoholischen Garung der Hefe entstehenden microorganisms and several plant tissues with aromatischen Alkohole. Monatschr. Brau. 19:76-87. I-acetald under aerobic conditions and showed 8. Dvornikova, T. P., G. K. Skryabin, and N. N. Suvarov. of 1968. Metabolism of tryptamine by Aspergillus niger. that all these living systems formed Trp-ol Mikrobiologiya 37:228-232, Chem. Abstr. 69:17012c. and that all of the microorganisms produced 9. Dvornikova, T. P., G. K. Skryabin, and N. N. Suvarov. IAA as well (38). We used several microorgan- 1970. Enzymic transformation of tryptamine by fungi. isms from within the same genera and orders as Mikrobiologiya 39:42-46, Chem. Abstr. 72:118652d. 10. Ehrlich, F. 1906. Uber die Bedingungen der Fuesldlbil- those in Rajagopal's study and obtained Trp-ol dung und uber ihren Zusammenhang mit dem Eiweis- only from the yeasts. We found no evidence sanfbau der Hefe. Chem. Ber. 40:1027-1047. indicating that other extractable indole metab- 11. Ehrlich, F. 1912. Uber Tryptophol (fl-Indolylathylakohol) olites were formed by microorganisms which did ein neues Garprodukt der Hefe aus Aminosauren. It is Chem. Ber. 45:883-889. not produce Trp-ol. likely that these 12. Ehrlich, F., and P. Postschimuka. 1912. lberfuhrung von cultures either did not metabolize the Trp aminen in Alkohole durch Hefe- und Schimmelpilze. substrate or that they degraded it via other Chem. Ber. 45:1006-1012. metabolic routes such as the kynurenine path- 13. Feldstein, A., F. H. Chang, and J. M. Kurcharski. 1970. Tryptophol, 5-hydroxytryptophol and 5-methoxytryp- way. tophol induced sleep in mice. Life Sci. 9:323-329. Z. priorianus and S. cerevisiae metabolized 14. Glombitza, K. W. 1965. Dunnschichtchromatogra- L-phenylalanine, Tyr, and L-5-OH-Trp to their phischer Nachweis der Indolyl-3-Brenztraubensaure respective alcohol and acid derivatives. In aus Biologischen Material. J. 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