APPLIED MICROBIOLOGY, Oct. 1969, p. 622-627 Vol. 18, No.

4
Copyright © 1969 American Society for Microbiology Printed in U.S.A.

Biosynthesis of Ochratoxin A'

J. W. SEARCY, N. D. DAVIS, AND U. L. DIENER
Botany and Plant Pathology Department, Auburn University Agricultural
Experiment Station, Auburn, Alabama 36830
Received for publication 15 August 1969

Biosynthesis of ochratoxin A by Aspergillus ochraceus Wilh. was investigated by

radiolabeling experiments in which phenylalanine-1-'4C and sodium acetate-2-14C
were supplied to the fungus in sucrose-yeast extract medium. Results showed that
phenylalanine was incorporated unaltered into the phenylalanine moiety of ochra-
toxin A, whereas the isocoumarin moiety of ochratoxin A was mostly derived via
acetate condensation.

Aspergillus ochraceus Wilh. has been isolated containing 4% sucrose and 2% yeast extract, in
from a variety of agricultural commodities (4-7, which the fungus produced 29 mg of ochratoxin
19, 24, 27). Symptoms of chronic or acute toxicity A per 100 ml of medium in stationary cultures.
have been observed in test animals when A. They also observed that the proportions of ochra-
ochraceus was grown in pure culture on corn, toxins A and B could be altered by appropriate
wheat, rye, sorghum, rice, buckwheat, soybeans, variation of the sucrose and yeast extract concen-
and peanuts and the molded substrates fed to tration, thus simplifying the procedure for subse-
the test animals (5-7, 17, 27). Shotwell et al. (20) quent purification of ochratoxin A.
found that ochratoxin A was a natural con- From a theoretical consideration of the struc-
taminant of a corn sample. Van Walbeek et al. ture of ochratoxin A, it appears probable that
(28) reported ochratoxin A production by a the phenylalanine portion of the molecule is
Penicillium species and by A. ochraceus. synthesized via the shikimic acid pathway. On
The toxic agent was first isolated and named the other hand, the isocoumarin portion could
ochratoxin by van der Merwe et al. (27). A. arise either via the shikimate pathway as in cou-
ochraceus strain K-804, originally isolated from marin synthesis of higher plants, or from acetate
sorghum grain, was grown in bulk on sterilized units via an isoprenoid type of condensation as in
corn meal, the toxic principle was quantitatively oosponal (18), oospolactone (18), and aflatoxin
extracted with methanol-chloroform (1:1), and a (1). This research was conducted to determine
chemical structure was suggested (27). This struc- the pattern of incorporation of the theoretical
ture (Fig. 1, I) was later confirmed by chemical 4IC precursors, phenylalanine and acetate, into
synthesis of ochratoxins A and B (23). Ochra- ochratoxin A, thereby revealing the pathway of
toxin A was shown to be 7-carboxy-5-chloro- biosynthesis of the metabolite.
8 - hydroxy - 3,4 - dihydro - 3 - methylisocoumarin,
linked through the 7-carboxy-group to L-f- MATERIALS AND METHODS
phenylalanine by an amide bond (26). A. ochraceus NRRL 3174, obtained from C. W.
Microbiological aspects of A. ochraceus growth Hesseltine, Northern Regional Research Laboratory,
and ochratoxin production were investigated by U.S. Department of Agriculture, Peoria, Ill., was
Ferreira (10), who formulated a synthetic medium used throughout this investigation. Cultures were
suitable for the production of 100 mg of ochra- maintained at 28 C on Czapek solution agar with 20%
toxin per liter in shaken flasks and 10-liter sucrose supplemented with 0.7% Difco yeast extract
fermentors. He obtained 50 mg of ochratoxin per (8).
liter of medium in a 100-liter pilot plant fermenta- Radiolabeled ochratoxin A was prepared by grow-
tion. The preferred carbon and nitrogen sources ing A. ochraceus in stationary cultures on a 4% su-
were sucrose (3%) and glutamic acid (1%), re- crose and 2% yeast extract (SYE) medium containing
spectively. Ferreira (11) further investigated the phenylalanine-1-14C or sodium acetate-2-'4C at 25C
effect of amino acids on ochratoxin production. (8). Ochratoxin A was extracted from the culture
medium, purified by preparative thin-layer chroma-
Davis et al. (8) developed a semisynthetic medium tography (TLC), and hydrolyzed to separate the
I Portions of this study were submitted to Auburn University phenylalanine and isocoumarin moieties. The iso-
by J. W. Searcy in partial fulfillment of the requirements of the coumarin moiety was selectively degraded to deter-
Ph.D. degree. mine the position of the 14C label derived from the
622
VOL. 18, 1969 BIOSYNTHESIS OF OCHRATOXIN A 623
sodium acetate-2-14C. The numbering system used for
these compounds is shown in Fig. 2.
Quantitative determinations of ochratoxin. Culture
filtrates were extracted four times with 100 ml of
I ~ ~
QSONCH
43F~~~Q ~ ~ ~ Q~HI
~ CHEZ-NH2

chloroform in a 500-ml separatory funnel. Extracts Cl

were combined, dried by passage through a sodium
sulfate column, and evaporated to dryness on a water
bath. The residue was then dissolved in 5 ml of
chloroform and streaked on Silica Gel TLC sheets
(Eastman 6061) for separation of ochratoxin A. The KOH-Fusion
CH3-COOH
521
525C
9 02
(9)0
chromatograms were developed in unlined chroma- +
tography tanks containing a 2-cm layer of toluene- H0l"GOH CHi- -CH3
ethyl acetate-90% formic acid (5:4:1, v/v). They were n~~~~~~s ~ ~ ~ ~ ~ 19KI
|
then air-dried and examined with a shortwave (254 Cl (,0)CHI3
nm) ultraviolet lamp; ochratoxin A was located by
comparison with an external standard of authentic H3 P04
ochratoxin A. The ochratoxin A standard solution
was prepared in this laboratory and was calibrated co0(IIl)
against a standard supplied by L. J. Vorster, National
Nutrition Research Institute, South African Council 3 3
for Scientific and Industrial Research, Pretoria. HH Q 3

Ochratoxin A was separated from the TLC sheets

and purified as described below. Confirmation of the
X)0H3WK)H3 CI
22

MIR
identity of the extracted compound was ascertained
by TLC with authentic ochratoxin A as a control in 9OC03(3,5) I
three different solvent systems, by exposure of + 8(0M~
So
fluorescent zones to ammonia fumes, and by the BrPGNCi(2,4,6)
preparation and co-chromatography of methyl esters
of extracts and authentic ochratoxin A (9; J. W. FIG. 2. Stepwise degradation of the isocoumarin
Searcy, Ph.D. Thesis, Auburn Univ., Auburn, Ala., moiety of ochratoxin A radiolabeled with sodium ace-
1969). tate-2-14C.
Experiment 1. Radiolabeled ochratoxin A was
prepared by placing 100 ;c of phenylalanine-J-14C
(Calbiochem, Los Angeles, Calif.) in 100 ml of sterile scending manner. The eluates were combined, evapo-
SYE medium. After inoculation with A. ochraceus rated to dryness, and redissolved in a known volume
conidia, the culture was incubated at room tempera- of chloroform for measurement of radioactivity in a
ture for 8 days. The culture filtrate was extracted four liquid scintillation spectrometer (Beckman model
times with 200 ml of chloroform. Extracts were evapo- 1650). Duplicate 100-Muliter samples were p'aced in
rated to 5 ml and streaked on TLC sheets for develop- liquid scintillation (LS) vials and evaporated to dry-
ment and separation in the toluene-ethyl acetate-90% ness before adding the LS cocktail to reduce quenching
formic acid solvent system. Ochratoxin A was located to a minimum. Radioactivity measurements were
by comparison with a standard spotted beside the corrected to counts per minute (counts/min) by con-
extract. The areas containing ochratoxin A were cut structing a quenching curve with acetone and sodium
from the sheets and eluted by the same solvent system acetate-I, 2-14C, and by counting a standard 14C
running at right angles to the solvent front in a de- sample and a reference background sample with each
set of counts made. The chloroform solution of 14C-
ochratoxin A was then transferred to a 50-ml boiling
HO-NH-$tt
flask and evaporated to dryness for acid hydrolysis.
C > X + 0CH2-0H-NH2 Radiolabeled ochratoxin A was hydrolyzed by the
method of van der Merwe et al. (26). Ochratoxin A
was suspended in 25 ml of 6 N HCl and heated under
(Ochratoxin A reflux for 30 hr. Chloroform extraction (4 X 100 ml)
of the cooled homogenous mixture and evaporation
of the solvent from the sodium sulfate-dried organic
COOH~~~~ phase gave 5-chloro-3 ,4-dihydro-8-hydroxy-3-methyl-
isocoumarin-7-carboxylic acid (II) as described by van
der Merwe et al. (26). The identity of this compound
was confirmed by ultraviolet and infrared spectra and
radioautography. The aqueous phase, containing the

CH,-CHO
gCH2-C-NH2 Ninhydrin
f)C~~~V~~HA< K)
02 + NH3- Nrnhydrin
Conv~ead phenylalanine moiety (III), was adjusted to pH 5.85
with NaOH, and a portion was spotted directly on
TLC sheets adjacent to a standard solution of phenyl-
FIG. 1. Degradation scheme of ochratoxin A radio- alanine. Sheets were developed in n-butyl alcohol-
labeled with phenylalanine-1-'4C. acetic acid-water (4:1:1, v/v), dried, sprayed with
624 SEARCY, DAVIL .S, AND DIENER APPL. MICROBIOL.

ninhydrin spray reagent, and heated at 110 C for 10 boxy-5-chloro-6-methyl salicylic acid (IV) by refluxing
min to confirm the presence of phenylalanine. for 30 min in 25 ml of 85% H3PO4 by the method of
A portion of the remaining aqueous phase contain- Nitta et al. (18). The top of the condenser was at-
ing the 14C-phenylalanine moiety (4.15 X 105 counts/ tached to a barium hydroxide trapping solution. A
min) from ochratoxin A was treated with ninhydrin stream of C02-free nitrogen was flushed through the
solution reagent in a closed 250-ml, side-arm flask, system to force evolved CO2 into the barium hydroxide.
with the side chamber containing 5 mJ of 1 N NaOH. Carbon dioxide was evolved and reabsorbed in fresh
This allowed the determination of the proportion of barium hydroxide by acidification before it was iso-
radioactivity remaining in carbon 1 of the hydrolyzed lated by filtration and the radioactivity was measured.
phenylalanine moiety. The aqueous solution (20 ml, The phosphoric acid solution was diluted to 100 ml
2.99 X 105 counts/min) was heated in a water bath and extracted with 4 X 200 ml of ethyl acetate which
with the stopper removed from the flask. Ninhydrin was then evaporated to near dryness in a water bath
solution (40 ml) was added, the stopper was quickly at reduced pressure. The remaining solution was
put in place, and the system was allowed to heat for transferred to a 50-ml flask and evaporated to dryness
20 min. The flask was removed from the water bath for nitration.
and allowed to stand for 30 min, while the remaining Methylpicric acid for the final degradation reaction
14CO2 was absorbed in NaOH. This procedure was was prepared by dechlorinating 2-chloro-5-hydroxy-
repeated until no increase in radioactivity in CO2 was toluene (V). Dechlorination was facilitated by nitra-
noted. tion of the ring positions ortho to the hydroxyl group.
Experiment 2. Radiolabeled ochratoxin A was A 1-ml amount of fuming nitric acid was added to
prepared by culturing A. ochraceus on SYE medium compound V in a 100-ml, round-bottom boiling flask
containing 250 Mc of sodium acetate-2-'4C (Calbio- and then was evaporated to dryness on a water bath
chem, Los Angeles, Calif.). Ochratoxin A was ex- (12). In the same flask, now containing 2,4-dinitro-3-
tracted and hydrolyzed as in experiment 1. After hydroxy-6-chloro toluene (VI), we placed 2.8 mg of
hydrolysis, the '4C-isocoumarin moiety (II) was red phosphorus and 20 ml of hydriodic acid (22). The
identified as in experiment 1 and selectively degraded mixture was refluxed for 14 hr, cooled, and filtered
to determine the radioactivity of individual carbons through a sintered-glass funnel to remove the phos-
in the isocoumarin nucleus (see flow chart in Fig. 2.) phorus. The flask and the solid were washed with two
The radiolabeled isocoumarin moiety (II) was sub- 20-ml portions of chloroform. The clear filtrate was
jected to KOH fusion to remove carbons 9 and 10 as transferred to a flash-evaporator flask and concen-
acetate (18). A chloroform solution of isocoumarin trated to near dryness by heating in a water bath at
was evaporated to dryness in a three-hole boiling flask. reduced pressure. The residue was washed into a
A 4-g amount of KOH and 1 ml of demineralized beaker with a small portion of hot chloroform. The
water were then added. The flask was attached to a chloroform solution containing 2,4-dinitro-3-hy-
reflux condenser; a thermometer was placed in one droxytoluene (VII) was next evaporated to dryness.
port through a ground-glass stopper, and the other The third nitro group was introduced para to the hy-
port was plugged with a cork stopper. The mixture droxy group by twice repeating the procedure pre-
was heated carefully as the temperature increased and viously described for nitration of compound V. This
was maintained at 208 to 212 C for 10 min. As the procedure yielded methyl picric acid (VIII), which
mixture cooled, it was dissolved in 10 ml of water consisted of carbons 2, 3, 4, 5, 6, 7, and 8 of the iso-
which was added slowly through the condenser. After coumarin moiety of ochratoxin A.
cooling to room temperature, the cork was replaced The final reaction of the stepwise degradation of the
by a buret, and H3PO4 was added to neutralize the isocoumarin moiety of '4C-labeled ochratoxin A was
KOH. This was done slowly to maintain the tempera- accomplished by using barium hypobromite to con-
ture below 60 C. A 10-fold excess of H3PO4 was then vert methyl pici ic acid to barium carbonate and
added, and the mixture was steam-distilled to remove nitrobromomethane (bromopicrin; 12). Barium hypo-
the acetate. The distillate was adjusted to pH 8 with bromite was prepared by mixing 100 mg of barium
barium hydroxide and evaporated to dryness on a hydroxide octahydrate and 0.38 ml of bromine (99.5%
boiling-water bath. Dried barium acetate was de- pure) in 60 ml of water. A 30-ml amount of barium
graded by pyrolysis to CO2 (carbon 9) and acetone hypobromite at 0 C was added to the methyl picric
(Fig. 2). The acetone was next converted to iodoform acid, brought to room temperature, and allowed to
(carbon 10) by the procedure of Calvin et al. (3). stand for 30 min. Carbons 3 and 5 of the original
Pyrolysis was conducted at 525 C for 10 min in a isocoumarin were removed as barium carbonate,
nitrogen atmosphere flowing though the chamber of a which was then converted to carbon dioxide. The
pyrolyzer (model PY-2, Barnes Engineering Co., liquid containing barium carbonate was decanted,
Stamford, Conn.). Evolved acetone was trapped in 10 and the residual bromopicrin was washed with 3 small
ml of water. The aqueous acetone was made strongly volumes of water. Both liquid and washings were
basic by addition of 5 ml of saturated NaOH. Po- placed in a side-arm flask the side chamber of which
tassium tri-iodide was added in portions until the pre- contained 5 ml of 1 N NaOH. The carbonate was then
cipitation of iodoform was completed. The radio- recycled by acidification into NaOH, and the radio-
activities on the separated iodoform and barium activity was measured by liquid scintillation. Bromo-
carbonate fractions, respectively, were counted in the picrin (carbons 2, 4, and 6) was dissolved in ethyl
liquid scintillation system. ether for measurement of radioactivity by liquid scin-
Carbons 1 and 11 were removed as CO2 from 3-car- tillation. The presence of bromopicrin was confirmed
VOL. 18X 1969 BIOSYNTHESIS OF OCHRATOXIN A 625
by comparing ultraviolet and infrared spectra of the (0.2% incorporation into ochratoxin A). Hy-
radioactive material with authentic bromopicrin pre- drolysis of the '4C-ochratoxin A with 6 N HCl
pared by hypobromite degradation of picric acid (12). and separation of the phenylalanine and iso-
Safety precautions. Because of the explosive nature coumarin moieties revealed that 91% (4.15 X
of derivatives of picric acid, the reactions involving
these compounds were conducted behind safety glass 105 counts/min) of the incorporated phenyl-
of a closed chemical fume hood. Reaction vessels were alanine was in the pyenylalanine moiety of ochra-
placed in an ice bath during reactions that liberated toxin A (Table 1). Only 0.95% (4.3 X 103 counts/
heat and then were allowed to come slowly to room min) of the incorporated radioactivity was pres-
temperature. ent in the isocoumarin moiety of ochratoxin A.
The remaining 8% of the radioactivity was lost,
RESULTS presumably through incomplete extraction of the
Radioactivity from phenylalanine-1-_4C was hydrolysis products.
readily incorporated into ochratoxin A by A. Radioactivity from sodium acetate-2-_4C was
ochraceus (Table 1). After 8 days of incubation on incorporated into ochratoxin A by A. ochraceus
SYE medium containing 100 juc (2.22 X 108 (Table 2). After 8 days of incubation on SYE
counts/min) of phenylalanine-1-'4C, chloroform medium containing 250 ,uc (5.55 X 108 counts/
extraction, and TLC purification, radiolabeled min) of sodium acetate-2-14C, chloroform extrac-
ochratoxin A with 4.55 X 105 dpm was obtained tion, and TLC purification, radiolabeled 14C-

TABLE 1. Distribution of radioactivity in ochratoxin A produced by A. ochraceus from phenylalanine-1-_4Ca

Material degraded Degradation products Total radioactivity Radioactivity as per cent of
material degraded
Ochratoxin A (I)b 4.55 X 105 i 2% 100.0
Isocoumarin (II) 0.04 X 105 i1 5% 0.9
Phenylalanine (III) 4.15 X 105 =1= 2% 91.0
Phenylalanine 4.15 X 105 ± 2% 100.0
CO2 (carboxyl group) 2.56 X 105 i 2% 61.2
a Incubation was for 8 days in SYE medium containing 100 ,c of phenylalanine-1-_4C.
I Numbers refer to structures in Fig. 1.

TABLE 2. Distribution of radioactivity in ochratoxin A produced by A. ochraceus from sodium acetate-2-l4Ca

Material degraded Degradation products Total radioactivity Radioactivity as per cent

of material degraded

counts/min
Ochratoxin A ()b 769.0 X 105 - 2% 100.0
Isocoumarin (II) 478.0 X 105 - 2%o 62.0
Phenylalanine (III) 127.0 X 105 i 2% 16.5
Isocoumarin (II) 478.0 X 105 - 2% 100.0
Acetate (C9, 10)c 0.82 X 105 4- 2% 0.17
Acetate (C9, 10) 0.82 X 101 ± 5 + 100.0
BaCO3 (C9) 0.33 X 105 i 5% 40.2
CHI3 (CIO) 0.30 X 105i 5% 36.6
3-Carboxy-5-chloro-6-methyl salicylic 450.0 X 105 i 2% 100.0
acid (IV) ,2 CO2 (Cl, 11) 13.5 X 105 it 5% 3.0
Methyl picric acid (VIII) 145.5 X 105 2% 100.0
BaCO3 (C3, 5) 1.0 X 105 i 5 % 0.69
Br3CNO2 (C2, 4, 6) 119.0 X 105 33%76 81.8
a Incubation was for 8 days in SYE medium with 250,Uc of sodium acetate-2-'4C.
h Numbers refer to structures in Fig. 2.
c Carbon numbers refer to numbering scheme of ochratoxin A in Fig. 2.
626 SEARCY, DAVIS, AND DIENER APPL. MICROBIOL.

ochratoxin A with 7.69 X 105 counts/min was ochratoxin A. Ninety-one per cent of this activity
obtained (1.39% incorporation into ochratoxin was contained in the phenylalanine moiety, with
A). Acid hydrolysis of '4C-ochratoxin A and 61.2 % of the '4C-phenylalanine radioactivity
separation of the isocoumarin and phenylalanine being in the carboxyl group. The isocoumarin
moieties showed that 62% (4.78 X 105 counts/ nucleus was not comparably labeled from phenyl-
min) of the radioactivity incorporated from alanine-1-'4C. The fungus did degrade phenyl-
acetate appeared in the isocoumarin moiety, with alanine to some extent and appeared to incor-
only 16.5% (1.27 X 105 counts/min) being pres- porate some radioactivity randomly into both the
ent in the phenylalanine moiety of ochratoxin A. phenylalanine ring and the isocoumarin ring.
After acid hydrolysis of the acetate-labeled However, the isocoumarin moiety contained only
ochratoxin A, the isocoumarin moiety was iso- 0.9% of the radioactivity present in the ochra-
lated and degraded stepwise to determine the toxin. A. ochraceus can degrade phenylalanine to
radioactivity of various individual carbons in the some extent, since uniformly labeled 14C-phenyl-
isocoumarin nucleus. The degradation scheme is alanine, supplied to the fungus, was catabolized
illustrated in Fig. 2, and the results are shown in by 38% to 14CO2 (unpublished data).
Table 2. The barium acetate that was recovered Results of experiment 2 show that A. ochraceus
after KOH fusion of isocoumarin yielded only incorporated sodium acetate-2-_4C into ochra-
0.17% (820 counts/min) of the radioactivity in toxin A. The fungus utilized 1.39% of the 250 ,ic
the parent compound. The radioactivity was of radiolabeled acetate supplied in the medium in
found to be distributed equally in the acetate the synthesis of ochratoxin A, with 62% being
between carbons 9 and 10 of isocoumarin (Table incorporated into the isocoumarin moiety and
2). 16.5% being incorporated into phenylalanine.
Decarboxylation of 3-carboxy-5-chloro-6- The 62 % incorporation into isocoumarin strongly
methyl salicylic acid (IV), by refluxing for 30 min supports the acetate pathway hypothesis. The
in 85% H3PO4, produced 14CO2 (carbons 1 and incorporation of acetate into phenylalanine may
11) with 3% (1.35 X 104 counts/min) of the have resulted from the metabolism of acetate
radioactivity originally present in IV. through the citric acid cycle or the glyoxylate
The 2-chloro-5-hydroxytoluene (V) that re- cycle to malate or oxalacetate with subsequent
sulted from decarboxylation of IV was next decarboxylation to phosphoenolpyruvate. These
dechlorinated and trinitrated to produce methyl intermediates could then have been incorporated
picric acid (VIII). The picric acid was then sub- into phenylalanine via the shikimic acid pathway.
jected to barium hypobromite degradation to Such pathways are known to occur in micro-
yield barium carbonate (carbons 3 and 5) and organisms (13, 21, 29).
bromopicrin (carbons 2, 4, and 6). The barium The stepwise degradation of the acetate-labeled
carbonate was found to contain only 0.69% isocoumarin moiety revealed that most of the
(1.03 X 103 counts/min) of the methyl picrate incorporated radioactivity was contained in ring
activity, whereas the bromopicrin contained carbons 2, 4, and 6, with little or no radioactivity
81.8% (1.19 X 105 counts/min). in carbons 1, 3, 5, 9, 10, or 11. This pattern of
DISCUSSION
labeling is consistent with the hypothesis that the
major portion of the isocoumarin moiety of
A strictly theoretical consideration of the ochratoxin A is synthesized via acetate condensa-
structure of the ochratoxin A molecule suggests tion. The absence of any significant amount of
that both an aromatic and an aliphatic pathway radioactivity in carbon 10 suggests that this
could be involved in its biosynthesis. The pres- methyl group is not derived from acetate. This
ence of phenylalanine in the molecule strongly absence of radioactivity raises the possibility
implies that the shikimic acid pathway is re- that the carbons of the lactone ring of the iso-
sponsible for the biosynthesis of that portion of coumarin arose from a phenylpropanoid pre-
ochratoxin A. The isocoumarin moiety could cursor in a manner similar to that of several
possibly be synthesized either via shikimate, as in carbon atoms in the isocoumarin portion of
coumarin synthesis in higher plants (16), or from hydrangenol from the plant Hydrangea macro-
acetate units via acetate condensation, as in the phylla (14, 15).
formation of oosponol and oospolactone by The small but significant amount of radioac-
Oospora astringenes (18) and of several other tivity found in isocoumarin carbons 1 and 11 sug-
similar fungal metabolites (1, 16). gests that the lactone carbonyl group results from
Results of experiment 1 show that A. ochraceus a head-to-head condensation of acetate, with sub-
readily incorporated phenylalanine-1_14C into sequent decarboxylation and oxidation of the
ochratoxin A. Of the 100 ,uc of radioactivity sup- remaining methyl group to a carboxy group with
plied in the medium, 0.2% was recovered in 14C- subsequent closure of the ring. This oxidation of
VOL. 18, 1969 BIOSYNTHESIS OF OCHRATOXIN A 627
a methyl group would then be similar to the 13. Herbert, D. 1955. Oxalacetic carboxylase of Micrococcus
situation reported by Gatenbeck (12) for the bio- lysodelktlcus, p. 753-757. In S. P. Colowick and N. O.
Kaplan (ed.), Methods in enzymology, vol. 1. Academic
synthesis of 3-hydroxy-phthalic acid. If this is Press Inc., New York.
true, the carboxyl group attached to carbon 4 14. Ibrahim, R. K., and G. H. N. Towers. 1960. Studies of hy-
probably arose from the one-carbon pool of the drangenol in Hydrangea macrophylla Ser. I. Isolation,
organism, as demonstrated by Birch et al. for a identification, and biosynthesis from C1Llabeled com-
pounds. Can. J. Biochem. PhysioL 38:627-634.
corresponding carboxyl group in citrinin from 15. Ibrahim, R. K., and G. H. N. Towers. 1962. Studies of hy-
A. candidus (2). drangenol in Hydrangea macrophylla Ser. II. Biosynthesis
ACKNOWLEDGMENTS of hydrangenol from C04labeled compounds. Can. J.
Biochem. Physiol. 40:449-453.
We thank F. J. Stevens, Auburn University, for advice con- 16. Neish, A. C. 1964. Major pathways of biosynthesis of phenols,
cerning organic chemical reactions utilized in this research. p. 294-359. In J. B. Harborne (ed.), Biochemistry of phe-
This investigation was supported by Public Health Service nolic compounds. Academic Press Inc., New York.
research grant UI-00147 from the National Center for Urban 17. Nesheim, S. 1967. Note on ochratoxin. J. Ass. Offic. Anal.
and Industrial Health. Chem. 50:370-371.
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