The biosynthesis of ochratoxin A and other structurally related polyketides by Aspergillus ochraceus.
A thesis submitted in fulfilment of the requirements for the degree of Ph.D. of the University of London.
by Jonathan Peter Harris 1996
Department of Biochemistry
Imperial College of Science, Technology and Medicine
London, SW7 2AY Abstract.
This study has revealed new information on the pathway and the dynamics of the biosynthesis of the important mycotoxin ochratoxin A by a particular isolate of
Aspergillus ochraceus in the context of the production of other structurally related polyketides including ochratoxins B, a, and 13, mellein, diaporthin and orthosporin. Orthosporin and diaporthin had never been previously found as A. ochraceus metabolites and the metabolic oiigin of diaporthin was found to be from both acetate and methionine. New physico-chemical data were obtained for diaporthin, ochratoxins a and 13, 0-methyl, methylochratoxins A and a, 0-methylochratoxin A and mellein.
[ 14C] Labelled primary metabolite precursors and the methylation inhibitor ethionine were used in feeding studies to shaken liquid cultures of A. ochraceus to reveal a distinct early phase of ochratoxin A biosynthesis principally involving acetate. Further isotopic labelling experiments using [ 14C] mellein, [1O- 14C] ochratoxins a and 13 and
[1O-14C, phenylalanyl-3H] ochratoxins A and B, all prepared biosynthetically, showed that i) ochratoxin 13 was incorporated into both ochratoxins A and B, ii) ochratoxin a was incorporated only into ochratoxin A, iii) mellein may not be an advanced intermediate in either ochratoxins A or B biosynthesis (which questions the accuracy of proposed biosynthetic schemes) and iv) there was detectable inter-conversion between ochratoxins A and B, albeit non-specifically and at a low rate. Optimised shaken shredded wheat cultures of A. ochraceus yielded astonishing amounts of ochratoxins A and B (up to 12 and 3 mg/g substrate, respectively). This finding led to the development of this unusual fermentation system to facilitate the use of this technique in the above biosynthetic studies. A pilot study on the mycobiota of fine green coffee beans from the major producing regions revealed a low incidence of A. ochraceus and, of those isolated, none was ocbratoxinogenic. However, an apparently novel metabolite (C31H27N0), produced by all A. ochraceus strains isolated from
coffee beans was partially charactensed.
II To Barbara and Khan, my special little family.
111 Table of contents.
Title page 1
Abstract. 11
Dedication. 111
Table of contents. iv
List of figures. ix
List of tables. xvi Acknowledgements. xix
1: Introduction. 1 1.1: The ochratoiins. 1
1.2: Polyketides. 4
1.3: Polyketide biosynthesis. 4
1.4: The biosynthesis of the ochratoxins. 12
1.5: The melleins. 17
1.6: The biosynthesis of the melleins. 20 1.7: Diaporthin and orthosporin. 28 1.8: The biosynthesis of diaporthin and orthosponn. 29
2: Dynamics of the biosynthesis of Aspergillus ochraceus D2306
polyketide metabolites in liquid and solid substrate fermentations. 32
2.1: Introduction. 32 2.2: Materials and methods. 34 2.2.1: Dynamics of the biosynthesis of Aspergillus ochraceus
D2306 polyketide metabolites in liquid substrate fermentations. 34 2.2.2: Intra- and extracellular polyketide concentrations versus time
for Aspergillus ochraceus D2306 on either potato dextrose broth or yeast extract-sucrose. 36
2.2.3: Aspergillus ochraceus D2306 solid substrate fermentations. 39
iv 2.2.4: Dynamics of the biosynthesis of Aspergillus ochraceus D2306
polyketide metabolites in solid substrate fermentations. 41 2.2.5: Effectiveness of shaken shredded wheat culture for other
ochratoxinogenic fungi. 42 2.3: Results and discussion. 45 2.3.1: Dynamics of the biosynthesis of Aspergillus ochraceus D2306
polyketide metabolites in liquid substrate fermentations. 45 2.3.2: Intra- and extracellular polyketide concentrations versus time
for Aspergillus ochraceus D23 06 on either potato dextrose broth or
yeast extract-sucrose. 47 2.3.3: Aspergillus ochraceus D2306 solid substrate fermentations. 51 2.3.4: Dynamics of the biosynthesis of Aspergillus ochraceus D2306
polyketide metabolites in solid substrate fermentations. 55 2.3.5: Effectiveness of shaken shredded wheat culture for other
ochratoxinogenic fungi. 58
3: Isolation and identification of Aspergillus ochraceus D2306 polyketide and other, metabolites. 59 3.1: Introduction. 59 3.2: Materials and methods. 59 3.2.1: Ochratoxin A. 59 3.2.2: Ochratoxin B. 60
3.2.3: Ochratoxin cx. 60
3.2.4: Ochratoxin 3. 61 3.2.5: 0-methyl, methylochratoxin A. 61
3.2.6: Diaporthin. 61 3.2.7: Orthosponn. 64
3.2.8: Mellein. 65
3.2.9: Hydroxymellein. 65
3.2.10: Cycloechinulin. 66
v 3.2.11: Aspergillic acids. 66 3.3: Results and Discussion. 67
3.3.1: The A. ochraceus polyketide UV absorption spectra. 67 3.3.2: Ochratoxins A and B. 67
3.3.3: Ochratoxin a. 70 3.3.4: Ocliratoxin . 74 3.3.5: 0-methyl, methylochratoxin A. 78
3.3.6:Diaporthin. 78
3.3.7: Orthosponn. 88 3.3.8: Mellein. 91 3.3.9: Hydroxymellein. 91 3.3.10: Cycloechinulin. 99
3.3.11: Aspergillic acids. 99
4: Feeding radiolabelled precursors of polyketide biosynthesis to, and the effect of ethionine on, Aspergillus ochraceus D2306 potato dextrose broth fermentations. 108 4.1: Introduction. 108
4.2: Materials and methods. 108 4.3: Results and discussion. 109
5: Feeding [14C1 mellein, u1O-14CJ ochratoxins a or 13 or I1O-14C,
phenylalanyl-3H1 ochratoxins A and B to Aspergillus ochraceus D2306 fermentations. 117 5.1: Introduction. 117
5.2: Materials and methods. 117
5.2.1: Feeding [ 14C] mellein to an Aspergillus ochraceus D2306 potato dextrose broth fermentation. 117
5.2.2: The course of intra- and extraceilular accumulation of mellein in
an Aspergi/lus ochraceus KBf potato dextrose broth fermentation. 118
5.2.3: Rate of incorporation of [1-' 4C] acetate into [14C] mellein in
v an Aspergillus ochraceus KBf potato dextrose broth fermentation. 119
5.2.4: Producing [14C] mellein of a higher specific radioactivity. 120 5.2.5: Establishing the optimal time of advanced intermediate addition
to an Aspergillus ochraceus D2306 solid substrate fermentation. 120
5.2.6: Feeding [ 14C] mellein or [1O- 14C] ochratoxins a or 13 to an Aspergillus ochraceus D2306 solid substrate fermentation. 121 of 5.2.7: The interconversion10- 14C, phenylalanyl-3H1 ochratoxins A and B into ochratoxins A and B. 122
5.3: Results and discussion. 123 5.3.1: Feeding [ 14C] mellein to an Aspergillus ochraceus D2306 potato dextrose broth fermentation. 123 5.3.2: The course of intra- and extracellular accumulation of mellein in an Aspergillus ochraceus KBf potato dextrose broth fermentation. 126 5.3.3: Rate of incorporation of[1- 14C] acetate into [ 14C] mellein in
an Aspergillus ochraceus KBf potato dextrose broth fermentation. 126 5.3.4: Producing [14C] mellein of a higher specific radioactivity. 127 5.3.5: Establishing the optimal time of advanced intermediate addition
to an Aspergillus ochraceus D2306 solid substrate fermentation. 127
5.3.6: Feeding [ 14C] mellein or [10-14C] ochratoxins a or 13 to an Aspergillus ochraceus D2306 solid substrate fermentation. 132 of 5.3.7: The interconversionA[10l4C, phenylalanyl-3H] ochratoxins A and B into ochratoxins A and B. 136
6: The chemical degradation of ochratoxin A. 141 6.1: Introduction. 141 6.2: Materials and methods. 144 6.2.1: Experiments involving ochratoxin A and its derivatives. 144
6.2.2: Experiments involving ochratoxin a and its derivatives. 145
6.3: Results and discussion. 145
6.3.1: Experiments involving ochratoxin A and its derivatives. 145
vu 6.2.2: Experiments involving ochratoxin a and its derivatives. 151
7: Feeding I'4C1 diaporthin, diaporthin and orthosporin to Penicillium citrinum lEa potato dextrose broth fermentations. 155
7.1: Introduction. 155
7.2: Materials and Methods. 155
7.3: Results and discussion. 158 8: Isolation and characterisation of fungi from unroasted coffee beans. 168 8.1: Introduction. 168
8.2: Materials and methods. 170 8.2.1: Mycological analysis. 170 8.2.2: Ochratoxins A and B analysis. 172 8.3: Results and discussion. 174
8.3.1: Mycological analysis. 174 8.3.2: Ochratoxins A and B analysis. 177
9: Isolation and characterisation of metabolite "429" of an Aspergilus ochraceus isolate from green coffee beans. 180 9.1: Introduction. 180 9.2: Materials and methods. 180 9.3: Results and discussion. 183 10: Summary. 196 Appendices.
I: DAD-HPLC calibration details of ochratoxin A. 202
II: DAD-HPLC calibration details of ochratoxin B. 203 ifi: DAD-HPLC calibration details of orthosporin. 204
IV: DAD-HPLC calibration details of diaporthin. 205
V: DAD-HPLC calibration details of mellein. 206 VI: DAD-HPLC calibration details of ergosterol. 207 VII: DAD-HPLC calibration details of citrinin. 208
VIII: DAD-HPLC calibration details of "429". 209
viii IX: The biosynthesis of citrinin. 210
References. 221
List of figures.
Figure 1.1: The structures of ochratoxin A (I), ochratoxin B (II), ochratoxin A methyl ester (1111), ochratoxin B methyl
ester (IV), ochratoxin A ethyl ester (V), ochratoxin B
ethyl ester (Vi) and 4-hydroxyochratoxin A (VII). 2 Figure 1.2: Decarboxylative condensation of a malonyl thioester with an acetyl thioester to form an acetoacetyl thioester, CO 2 and a
free thiol. 5 Figure 1.3: A schematic diagram of fatty acid biosynthesis. 5 Figure 1.4: Palmitic acid (VIII). 8
Figure 1.5: Monensin A (IX). 8 Figure 1.6: Erythromycin A (X). 8 Figure 1.7: 6-Deoxyerythronolide B (XI). 10 Figure 1.8: The organisation of the genes and enzymes of the erythromycin
producing PKS in S. erythraea. 10 Figure 1.9: 6-Methylsalicylic acid (XII). 11 Figure 1.10: Hypothetical scheme of the 6-methylsalicylic acid reaction
sequence (Beck eta!., 1990 from Dimroth eta!., 1970). 11
Figure 1.11: The folding pattern of the pentaketide fonning ochratoxin A. 14 Figure 1.12: The biosynthesis of ochratoxin A (Huff and Hamilton, 1979). 16 Figure 1.13: The structures of mellein (XIII), 6-methoxymellein (X1V), 3-
hydroxymellein (XV), 4-hydroxymellein (XVI), 6-methoxy-5-
chioromellein (XVII), 5-methylmellein (XVIII), 5-formylmellein (XIX), 5-carboxymellein (XX), 7-carboxymellein (XXI) and
7-carboxy-5-chloromellein (XXII). 18
x Figure 1.14: The folding pattern of the pentaketide forming mellein. 21
Figure 1.15: Aspyrone (XXIII) and alternariol (XXIV). 21 Figure 1.16: Routes to mellein aromatisation: a) via an enol b) via an
unflinctionalised double bond. 21 Figure 1.17: A possible early intermediate of mellein biosynthesis (XXV). 23 Figure 1.18: A schematic diagram of putative biosynthetic events on the
mellein PKS. 25 Figure 1.19: Reasoned post-polyketide chain biosynthesis events in mellein and 4-hydroxymellein biosynthesis. 26
Figure 1.20: Proposed first enzyme free intermediate of 6-methoxy-5- chloromellein biosynthesis (XXVI; Holker and Young, 1975). 27
Figure 1.21: The structures of diaporthin (XXVII) and orthosporin (also known as de-0-methyldiaporthin, XXVIII). 27 Figure 1.22: The folding pattern of the hexaketide forming diaporthin. 27
Figure 1.23: The putative biosynthesis of orthosporin and diaporthin. 30 Figure 2.1: DAD-HPLC chromatogram of a typical bicarbonate soluble
sample showing the resolution of ochratoxins A and B. 37 Figure 2.2: DAD-HPLC chromatogram of a typical bicarbonate insoluble sample showing the resolution of orthosporin, cycloechinulin, diaporthin and mellein. 37
Figure 2.3: Thin layer chromatography of ochratoxin A (OA), ochratoxin B
(OB), ochratoxin a (Oa), ochratoxin (03) and 0-methyl, methylochratoxin A (OA Me2) (developed in toluene-ethyl
acetate-formic acid, 50:40:10) and their appearence under UV
light (256 and 360 nm). 38
Figure 2.4: DAD-HPLC chromatogram of ergosterol. 43
Figure 2.5: UV absorbance spectrum of ergosterol. 43
Figure 2.6: DAD-HPLC chromatogram of citrinin. 44 Figure 2.7: UV absorbance spectrum of citrinin. 44
x Figure 2.8: Diy cell weight (DCW), ochratoxin A concentration ([OA]), ochratoxin B concentration ([OB]), orthosporin concentration ([Ortho.]), diaporthin concentration ([Diap.]) and mellein concentration ([Mellein]) versus time for a D2306 fermentation. 46
Figure 2.9: Extracellular ochratoxin A concentration ([OA]), diaporthin concentration ([Diap.]) and mellein concentration ([Mellein])
versus time for an Aspergillus ochraceus D2306 potato dextrose broth fermentation. 48
Figure 2.10: Intracellular ochratoxin A concentration ([OA]), diaporthin concentration (Diap.1) and mellein concentration ([Mellein])
versus time for an Aspergillus ochraceus D2306 potato dextrose broth fermentation. 48 Figure 2.11: Extracellular ochratoxin A concentration ([OA]) and diaporthin concentration ([Diap.]) versus time for an Aspergillus ochraceus D2306 yeast extract-sucrose fermentation. 50 Figure 2.12: Intracellular ochratoxin A concentration ([OA]) and diaporthin concentration ([Thap.]) versus time for an Aspergillus ochraceus D2306 yeast extract-sucrose fermentation. 50 Figure 2.13: Ergosterol concentration ([Ergo.]), ochratoxin A concentration ([OA]), ochratoxin B concentration ([OB]) and diaporthin concentration ([Diap.]) versus time for an Aspergillus ochraceus D2306 shaken shredded wheat fermentation. 56
Figure 3.1: 0-Methyl, methylochratoxin A (XXIX). 62
Figure 3.2: UV absorbance spectra of ochratoxins A, B, a and J3, 0-methyl, methylochratoxin A (OAMe2), diaporthin, orthosporin, mellein
and hydroxymellein. 68
Figure 3.3: Accurate mass electron impact spectrum of ochratoxin A. 69
Figure 3.4: Accurate mass electron impact spectrum of ochratoxin B. 71
Figure 3.5: DAD-HPLC chromatograins of ochratoxins a and 3 and 0-
xi methyl, methylochratoxin A (OAMe2). 72 Figure 3.6: Accurate mass electron impact spectrum of ochratoxin a. 73 Figure 3.7: Fragmentation pattern of ochratoxin a in electron impact mass
spectrometry. 75 Figure 3.8: Accurate mass electron impact spectrum of ochratoxin J3. 76 Figure 3.9: Fragmentation pattern of ochratoxin in electron impact mass
spectrometly. 77 Figure 3.10: Accurate mass electron impact spectrum of 0-methyl, methylochratoxin A. 79 Figure 3.11: Accurate mass electron impact spectrum of diaporthin. 80 Figure 3.12: Fragmentation pattern of diaporthin in electron impact mass
spectrometry. 81 Figure 3.13: 1H NMR spectrum of diaporthin in D6 acetone. 82 Figure 3.14: 13C NMR spectrum of diaporthin in D 6 acetone. 85
Figure 3.15: 13C NMR DEPT analysis of diaporthin in D6 acetone. 87
Figure 3.16: Accurate mass electron impact spectrum of orthosporin. 89
Figure 3.17: Reasoned fragmentation pattern of orthosporin in electron impact mass spectrometly. 90 Figure 3.18: Accurate mass electron impact spectrum of mellein. 92 Figure 3.19: Fragmentation pattern of meilein in electron impact mass
spectrometry. 93
Figure 3.20: DAD-HPLC chromatograms of hydroxymellein, aspergillic acid and deoxyaspergillic acid. 94 Figure 3.21: Accurate mass electron impact spectrum of hydroxymellein. 95
Figure 3.22: VG Autospec mass spectral library mass spectrum of a) 3- hydroxyniellein and b) 6-hydroxymellein. 96 Figure 3.23: a) VG Autospec mass spectral library mass spectrum and b) Cole and Cox (1981) mass spectrum of 4-hydroxymellein. 97
Figure 3.24: Fragmentation pattern of hydroxymellein in electron impact
XII mass spectrometry. 98 Figure 3.25: The structures of cycloechinulin (XXX), aspergillic acid
(XXX1), deoxyaspergillic acid (XXXII) and flavacol (XXXIII). 100
Figure 3.26: Accurate mass electron impact spectrum of cycloechinuim. 101 Figure 3.27: UV absorbance spectra of cycloechinulin, aspergillic acid and
deoxyaspergillic acid. 102 Figure 3.28: Accurate mass electron impact spectrum of aspergillic acid. 103 Figure 3.29: The structures of neoaspergillic acid (XXXIV) and
neohydroxyaspergiliic acid (XXXV). 105 Figure 3.30: Accurate mass electron impact spectrum of deoxyaspergilhic
acid. 106
Figure 4.1: Specific radioactivities of [ 14C] ochratoxin A and [14C] diaporthin isolated 168 hours post inoculation after the
additions of 3.7 iCi [1-'4C] acetate to D2306 PDB cultures at different times. 111
Figure 4.2: Specific radioactivities of [ 14C] ochratoxin A and [14C] diaporthin isolated 80 hours post inoculation after the
additions of 5 pCi [1 .-'4C] acetate to D2306 PDB cultures at different times. 111 Figure 4.3: Data of Lillehoj eta!. (1978) showing temporal separation
between the stages at which addition of[1- 14C] acetate most efficiently contributes to the radiolabelling of ochratoxin
A and the stages of maximum accumulation of ochratoxin A. 113 Figure 5.1: The course of accumulation of extracellular mellein in A.
ochraceus KBf potato dextrose broth fermentations at
two temperatures. 124 Figure 5.2:Changes in dry cell weight (DCW), intracellular mellein concentration ([Mellein] cells) and extracellular mellein
'Un concentration ([Mellein] broth) during an A. ochraceus KBf potato dextrose broth fermentation. 124
Figure 5.3: Percentage incorporation of 5 j.iCi [1 -14CJ acetate (Ace),
[methyl-14C] methionine (Met) or [U-' 4C] phenylalanine (Phe) into [ 14C] ochratoxin A by an Aspergillus ochraceus D23 206 shaken SW fermentation at various times post
inoculation. 131 Figure 5.4: Percentage incorporation of 5 tCi [1-' 4C1 acetate (Ace),
[methyl-14C] methiomne (Met) or [U-14C] phenylalanine
(Phe) into [14C] ochratoxin B by an Aspergillus ochraceus D23 206 shaken SW fermentation at various times post
inoculation. 131
Figure 5.5: An autoradiograph of a TLC plate of bicarbonate soluble and insoluble fractions of extracts of D2306 SW cultures fed radiolabelled putative advanced intermediates of ochratoxin A
biosynthesis. 135 Figure 6.1: The chemical degradation of ochratoxin A (Steyn eta!., 1970). 142 Figure 6.2: An alternative chemical degradation scheme of ochratoxin A. 143 Figure 6.3: Accurate mass electron impact spectrum of
0-methylochratoxin A. 147 Figure 6.4: The structure of 0-methylochratoxin A (XLII). 148
Figure 6.5: DAD-HPLC chromatogram of 0-methylochratoxin A. 149 Figure 6.6: The UV absorbance spectrum of 0-methylochratoxin A. 149 Figure 6.7: DAD-HPLC chromatograin of ochratoxin a, 0-methyl,
methylochratoxin a and unknown IV. 152 Figure 6.8: The UV absorbance spectra of ochratoxin a ("), 0-methyl,
methylochratoxin a (--) with unknown N (-). 152 Figure 6.9: Accurate mass electron impact spectrum of 0-methyl, methylochratoxin a. 153
xlv Figure 7.1: The structure of citnnin (XLIII). 156
Figure 7.2: The accumulation of extracellular citrinin in a P. citrinum
lEa potato dextrose broth fermentation. 156 Figure 7.3: DAD-HPLC chromatograms showing the resolution of acidic
'A' and 'B' and neutral 'A'. 160 Figure 7.4: UV absorption spectra of acidic 'A' and 'B' and neutral 'A'. 161
Figure 7.5: The accumulation of acidic 'A' and 'B' and neutral 'A' in a
P. citrinum potato dextrose broth fermentation. 162 Figure 7.6: An autoradiograph of a TLC plate of bicarbonate insoluble (-BiC) and soluble (+BiC) fractions of extracts of a lEa PDB
culture fed [ 14C] diaporthin with the corresponding TLC information including the appearence of the compounds of interest in daylight and under UV light (at 254 and 350 nm). 164 Figure 7.7: DAD-HPLC chromatogram of acidic orthosporin and citrinin. 166 Figure 7.8: Comparing the UV absorbance spectrum of acidic orthosporin
(-) with orthosponn (--) 166
Figure 8.1: DAD-HPLC chromatogram of acidic mellein. 178 Figure 8.2: Comparing the UV absorbance spectrum of acidic mellein (-) with mellein (--). 178
Figure 9.1: DAD-HPLC chromatogram of "429" and mellein. 181 Figure 9.2: Comparing the UV absorbance spectrum of "429" (-) with diaporthin (--). 181
Figure 9.3: The accumulation of "429" in an A. ochraceus Mocha Y-3 shredded wheat fermentation. 184
Figure 9.4: Accurate mass electron impact spectrum of "429". 185 Figure 9.5: The ammonia chemical ionisation mass spectrum of "429". 186
Figure 9.6: The fast atom bombardment mass spectrum of "429". 186 Figure 9.7: Fragmentation pattern of "429" in electron impact mass spectrometry. 188
xv Figure 9.8: 1H NMR spectrum of"429" in CD3OD. 189
Figure 9.9: Low field strength region of the 1H- 1H COSY spectrum of "429" in CD3OD. 190
Figure 9.10: High field strength region of the 1H- 1H COSY spectrum of "429" in CD3OD. 191
Figure 9.11: The 1H observed 1H- 13C one bond correlation spectrum of"429" in CD3OD. 192
Figure 9.12: The 1H observed 1H- 13C one bond correlation spectrum of "429" in CD3OD (high field strength expanded). 193
Figure A. 1: The structures of sclerotinin A (XL1V), dihydrocitrinone (XLV), the isocoumarin (XLVI) and the trimethyl
isocoumarin (XLVII). 212
Figure A.2: The structures of the lactone (XLVIII), the keto-aldehyde (XLIX), the aldehydo-alcohol (L), the keto-acid (LI) and the quinone methide (LII). 214
Figure A.3: The biosynthesis of citrinin. 217
Figure A.4: The origin of the citrinin atoms. 218
Figure A. 5: The resonance formulae of citrinin. 218 Figure A.6: The structures of the citrinin hydrates. 218 Figure A.7: A generalised substrate cycle between citrinin and acetate. 218
List of Tables.
Table 2.1: Selected data from stationary medium (control), small and large
scale A. ochraceus D2306 shredded wheat cultures. 52 Table 2.2: Selected data from shaken medium (control), small and large scale
A. ochraceus D2306 shredded wheat cultures. 54
Table 3.1: Published 1H NMIR diaporthin data (Ichihara el a!., 1989)
compared with measured A. ochraceus 'H NMR diaporthin
xvi data. 83
Table 3.2: 13C NMR data of orthosponn (Ichihara eta!., 1989) compared with that of A. ochraceus diaporthin. 86
Table 3.3: 13C NMR DEPT analysis of A. ochraceus diaporthin. 86
Table 4.1: First radiolabel experiment: 110 Table 4.2: Second radiolabel experiment: 110 Table 4.3: The extracellular concentration of ochratoxin A ([OA]), ochratoxin B ([OB]), orthosponn ([Ortho.]) and diaporthin ([Diap.]) 80 hours post inoculation in D2306 PDB fermentations fed with 50 mg ethionine at various times. The calculated ratio of
diaporthin to orthosporin is also shown. 115
Table 5.1: Radioactivity of[ 14C] mellein recovered 10 minutes, 1 and 5 hours after the addition of 2 .tCi [1- 14C] acetate to an Aspergillus ochraceus KBfPDB fermentation. 128 Table 5.2: Radioactivity (expressed as disintegrations per minute-dpm-
and pCi) of [14C] ochratoxin A recovered 5 hours after the addition of either 5 pCi [1- 14C] acetate, [methyl-14C] methionine ([methyl- 14C] met) or [U- 14C] phenylalanine ([U-'4C] phe) to an Aspergillus ochraceus D2306 shaken shredded wheat fermentation 3, 4, 5 or 6 days post inoculation. 129
Table 5.3: Radioactivity (expressed as disintegrations per minute-dpm-
and pCi) of [ 14C] ochratoxin B recovered 5 hours after the
addition of either 5 pCi [1 -14C] acetate, [methyl- 14C]
methionine ([methyl- 14C] met) or [U- 14C] phenylalanine ([U-14C] phe) to an Aspergillus ochraceus D2306
shaken shredded wheat fermentation 3, 4, 5 or 6 days post inoculation. 130
Table 5.4: Radioactivity (expressed as disintegrations per minute-dpm-
xvii and nCi) of[ 14C] ochratoxins A and B recovered 5 hours after the addition of either 0.13 tCi [10- 14C] ochratoxin a,
0.1 jCi [10-'4C] ochratoxin 13 or 0.096 tCi [U- 14C] mellein
to Aspergillus ochraceus D2306 shaken shredded wheat fermentations. 134
Table 5.5: The radioactivity (expressed as disintegrations per minute-dpm- and p.Ci) of dual labelled ochratoxins A and B recovered after
the addition of 5 tCi each of [methyl- 14C] methionine and
[2, 3, 4, 5, 6-3H] phenylalanine to a 4 day old A. ochraceus D2306 shaken SW culture. 137 Table 5.6: The radioactivity (expressed as dismtegrations per minute-dpm- and nCi) of dual labelled ochratoxins A and B recovered after
the addition of dual labelled ochratoxins A and B to 5 day old (1st OA and OB feeds) or 6 day old (2nd OA feed)
A. ochraceus D2306 shaken SW cultures. 139 Table 8.1: Details of fungi isolated from green coffee beans. 175 Table 9.1: 1H NMR data of "429" in CD3OD. 195
xviii Acknowledgements.
First and foremost, I would like to thank Professor Peter Mantle for his excellent supervision and providing me with the opportunity to work on this project. I sincerely hope many more will benefit from his considerable expertise, patience and enthusiasm, as I have done. I would also like to thank Mr Richard Sheppard and Mr John Barton for the first class NMR and El-MS analyses, Dr Nigel Lindner for his useful comments, Dr David Widdowson for his advice on chemical degradation and Dr Lucy Wigley, Miss Tina Naik and Miss Hanadi Hassan for providing an environment which was a pleasure to work in. I would also like to acknowledge the receipt of an A. F. R.
C. studentship. The extra special thanks are reserved for Mum and Barbara for all the love and
support you have given me throughout this study, Lucy and Waishey, for the hours of top-drawer entertainment, Nan, Pop, Paul and the rest of my family, Chris, King Louis (the funkiest thing), IC. Biochem. Dept. F.C. and, last and probably least, Bedford Town F.C., for the hours of bottom-drawer entertainment.
The mistakes, of course, are all mine.
xix 1: Introduction.
Ochratoxin A and other structurally related polyketide fungal metabolites.
1.1: The ochratoxins.
The discovery of toxic strains of the fungus Aspergillus ochraceus in the early 1960s in South Africa fuelled the search for toxic A. ochraceus metabolites. The mycotoxin ochratoxin A (figure 1.1, 1) was first isolated from an A. ochraceus isolate (C SIR
804) grown on sterilized maize meal (van der Merwe eta!., 1965 a). Chemically, ochratoxin A contains a 7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R- methylisocoumarin (the pentaketide-derived ochratoxin a, figure 1.13, XXII) which is linked through the carboxy group to L-3-phenylalanine. Less toxic structural analogues of ochratoxin A, i.e. de-chioro ochratoxin A or ochratoxin B (figure 1.1, H) and the ochratoxin A ethyl ester or ochratoxin C (figure 1.1, V), were also isolated from a CSIR 804 maize culture (van der Merwe eta!., 1965 b).
The structures of ochratoxins A and B were confirmed by their chemical synthesis (Steyn and Holzapfel, 1967 a). The natural occurrence of methyl esters of both of these acids and the ethyl ester of ochratoxin B (figure 1.1, III, IV and VI respectively) in maize meal cultures of CSIR 804 was reported in the same year
(Steyn and HoLzapfel, 1967 b).
Ochratoxin A was isolated from a stationary culture of Peniciiium viridicatum on YES medium (15% sucrose and 2% yeast extract) by van Walbeek et al. (1969). It was the same isolate grown under the same medium conditions that yielded 4- hydroxyochratoxin A (figure 1.1, VII), 7-carboxymellein (also known as ochratoxin
3, figure 1.13, XXI) and ochratoxin B (Hutchison eta!., 1971). The next year, Moore eta!. (1972) showed that an ochratoxinogenic A. ochraceus strain (NRRL 3174) produced mellein (figure 1.13, XIII) and 4-hydroxymellein (figure 1.13, XVI). COR
OCH2—H NH—CO 10
R R' R"
I Ochratoxin A OH Cl H
II Ochratoxin B OH H H
ifi OA Me ester OCH3 CI H
IV OB Me ester OCH3 H H
V OA Et ester t OCH2CH3 Cl H
VI OB Et ester OCH2CH3 H H
VII 4-OH OA OH Cl OH
Figure 1.1: The structures of ochratoxin A (I), ochratoxin B (II), ochratoxin A methyl ester (1ff), ochratoxin B methyl ester (1V), ochratoxin A ethyl ester (V), ochratoxin B ethyl ester (VI) and 4-hydroxyochratoxin A (VII). t Also known as ochratoxin C.
2 Pitt (1987) disputed the identification of some Penicillium species attributed to ochratoxin A production, suggesting that all ochratoxinogenic Penicillia be assigned to P. verrucosum. Hadidane et a!. (1992) isolated three new natural analogues of ochratoxin A, produced by an A. ochraceus isolate (NRRL 3174) grown on wheat, where the phenylalanine moiety of the compound was replaced by serine, hydroxyproline or lysine. Previously, chemically synthesized versions of these three analogues had caused lesions in animal cell cultures typical of ochratoxin A (Steyn et a!., 1975). Of the ochratoxins, only ochratoxin A and, very rarely, ochratoxin B have been found as a contaminant of foods and feeds. Although ochratoxin A is the most commonly found mycotoxin on stored cereal in the UK (Buckle, 1983), contamination of raw agricultural products is comparatively rare. The fungi capable of producing ochratoxin
A are frequently encountered on feed and foods. Yet, in a study in the U.S.A., the contamination rate of selected raw agricultural products was found to be approximately 3%, with contamination by ochratoxin A < 100 j.tgfKg. However, occurrence rate and amounts of ochratoxin A have been found to be lower in human foodstuffs than in raw agricultural products (Pohiand eta!., 1992). Ochratoxin A is a potent toxin in many different animals and has been shown to be the causal agent of mycotoxic porcine nephropathy (Krogh, 1987), which had a profound effect on the Danish bacon industry. Some believe ochratoxin A to be important in
human health since, in addition to being a nephrotoxin, ochratoxin A is a hepatotoxin, a teratogen, a very potent carcinogen, possibly a mutagen and an immunosuppressive
agent (Marquardt eta!., 1990), In order to understand the regulation of ochratoxin A biosynthesis by fungi in foods,
an intimate knowledge of the biosynthetic pathways involved is required. It is also
important to identi1' co-metabolites of an ochratoxinogenic flingal isolate. This is especially important with regard to other members of the ochratoxin family and other polyketides, which are biosynthesized via similar processes within the fungal cells and compete with each other for common primary metabolic intermediates such as acetate.
1.2: Polyketides.
Polyketides represent perhaps the largest family of secondary metabolites, especially abundant among fungi, actinomycete bacteria and higher plants, but also present in many other groups, including higher animals. Pnmaiy metabolites, by definition, are ubiquitous and vital for living, whereas secondary metabolites are often specific to one genus/species and are not considered essential. The polyketide biosynthetic pathway is very similar to fatty acid biosynthesis. In both cases acetate or malonate undergoes head-to-tail condensation reactions. The term polyketides originated from the belief that the polyketide biosynthetic enzymes did not reduce any of the ketone groups which arose from these head-to-tail condensation reactions, resulting in the fonnation of a poly-f3-keto methylene, or 'polyketide' chain. This is now known to be not strictly accurate (see 1.3). The favoured classification of polyketides is based on the number of C2 units used in their biosynthesis. For example, a metabolite derived from five acetate groups is termed a pentaketide whereas one derived from six is termed a hexaketide, etc (Turner, 1971).
1.3: Polyketide biosynthesis.
The enzyme system that catalyses the synthesis of fatty acids is termed the fatty acid synthase (FAS). The essence of fatty acid biosynthesis is a decarboxylative condensation in which simple carboxylic acids are joined in head-to-tail fashion to produce a chain of 6-50 carbon atoms, each building unit contributing two carbons to the backbone of the chain.
Figure 1.2 represents the simplest case, in which a malonyl 'extender unit' is condensed with an acetyl 'starter unit' to produce a four carbon chain and CO2. FASs
4
-s + -SI4 CH3 . -S1-4-4CH3 + CO2 + -S211
Figure 1.2: Decarboxylative condensation of a malonyl thioester with an acetyl thioester to form an acetoacetyl thioester, CO2 and a free thiol.
/ SKH AT /SKH AT ,SKCOCH3 CH3COSCoA + E E + CoASH E SAH SACOCH3 SAH
SKH AT ,SKCOCH2CH2CH3 E E 'SAil AT NADP TE rER NADPH / SKH SKH E + HOOC(CH2)CH3 ,SKCOCH3 E E 'SAC OCH=C HCH3 A 'SACOCH2CO2H KftS DH 1120 NADP NADPH Co2 ,SKH E 'SACOCH2COCH3
Figure 1.3: A schematic diagram of fatty acid biosynthesis. SKH and SAH denote the
KAS and ACP thiol groups, respectively, whilst AT, KAS, KR, OH, ER and TE represent the enzymes: acyltransferase, 3-ketoacy1-ACP synthase, 3-ketoacyl-ACP reductase, -hydroxyacyl-ACP dehydrase, enoyl-ACP reductase and thioesterase, respectively (based on Hopwood and Khosla, 1992).
5 require that the acetyl and malonyl groups taking part in the condensation are not free acids, but thioesters of them. The free acids are activated by attachment to the thiol of the 4'-phosphopantethine moiety of CoA, which acts as a carrier. Two essential thiols are found within the FAS, one provided by a 4'-phosphopantethine arm bound to a serine hydroxyl of a synthase component distinct from the condensing enzyme (the acyl carrier protein: ACP), and the other by an active site cysteine residue on the condensing enzyme (3-ketoacy1-ACP synthase: KAS) itself. An acyltransferase (AT) transfers thioesters between C0A, the ACP and the KAS. Each condensation brings a keto group to the growing chain which is removed during carbon chain assembly. This happens between successive condensations by a cycle of three reactions: i) reduction of the keto group to a hydroxyl group, catalysed by a - ketoacyl-ACP reductase: KR. ii) Elimination of water to form a C-C double bond in the carbon chain, catalysed by a -hydroxyacy1-ACP dehydrase: DH, and iii) saturation of this double bond, catalysed by an enoyl-ACP reductase: ER. The ACP is required to marshal the growing chain around the active centres of the enzyme complex.
Figure 1.3 illustrates the roles of the components of a typical FAS: The starting point is where acetyl CoA enters and is transferred first to the thiol of the ACP and then to the thiol of the KAS. Malonyl CoA is transferred to the now free ACP thiol and the decarboxylative condensation occurs with the acetyl thioester attaching to the malonyl thioester to form the acetoacetyl thioester, CO2 and the free KAS thiol. The
acetoacetyl thioester is reduced, dehydrated and then reduced again to form the butyryl thioester whilst still attached to the ACP. The thioester is transferred to the free KAS thiol, being replaced by another malonyl CoA moiety. Thus, another round
of decarboxylative condensation occurs. After the correct number of cycles, the completed carbon chain leaves the FAS, either by transfer back to CoA or by hydrolysis to the free acid by a thioesterase (TE). FASs have been classified into type I and type H categories. The vertebrate type I
enzymes consist of a single class of polypeptide carrying sites for all the catalytic
6 activities and the attachment point of the ACP prosthetic group. The type H FASs are found in certain bacteria and plants and consist of at least eight monofunctional
polypeptides, each responsible for one of the reaction steps of the FAS, with a separate ACP. Fungal FASs, although classified as type I, have the functionalities distributed between two large polypeptides, a and 3. Polyketide synthesis is reminiscent of fatty acid biosynthesis and there are probably no fundamental differences between the biosynthetic capabilities of FASs and polyketide synthases (PKSs): together, their repertoires cover a spectrum. At one extreme, the
FAS condenses eight acetyl residues, reducing every keto group to alkyl functionality, to form the straight 16 carbon chain of the simple saturated fatty acid palmitic acid (figure 1.4, YlIl). Near the opposite extreme, the highly programmed PKS for the
Streptomyces ionophore monensin (figure 1.5, LX) uses an acetyl starter unit, but the extender units vary and are chosen in a defined sequence. They include not only
acetate units but also malonate, propionate (from methylmalonate) and butyrate (from ethyhnalonate), the latter two introducing methyl and ethyl side chains into the molecule. Moreover, many of the side chains occur on a chiral carbon, so the PKS must make the correct choice of R or S conflguration.In addition is the handling of
the 12 keto groups: monensin has only two remaining keto groups, implying there is abortion of the entire reduction-dehydration-reduction cycle, whilst three are reduced to hydroxyls, by curtailment of the cycle before the dehydration step. There are three
double bonds, indicating ketoreduction and dehydrations but no enoyl reduction. These double bonds have specific stereochemistry, E rather than Z, and four keto groups are modified by a complete cycle to alkyl functionality. Thus, the monensin PKS is programmed with information regarding chain length, the choice of starter and
extender units, the handling of the 12 keto groups in a specific sequence and the induction of stereochemistry at nine chiral carbons and three double bonds (Hopwood
and Khosla, 1992).
Perhaps the best studied PKS is the one responsible for the biosynthesis of
erythromycin A (figure 1.6, X). Erythromycin A is a broad spectrum antibiotic
7
VIII
Figure 1.4: Palmitic acid (VIII).
HC
H3CO
NaO2C
Figure 1.5: Monensin A (IX).
0
Me NMe
HO I Mel" ,.. Me1, _J EV" 0 OH•r '
0"(
"OH x MIMe
Figure 1.6: Erythromycin A (X).
8 produced by the bacterium Saccharopolyspora erylhraea (formerly Streptomyces erythraeus). Leadlay eta!. (1993) described the genes involved in the biosynthesis of the polyketide 6-deoxyerythronolide B (figure 1.7, XI) which is the first identifiable product of the polyketide synthase that produces erythromycin A. The genes that encode antibiotic producing polyketide synthases are often clustered together, adjacent to at least one resistance gene that confers protection against the antibiotic. Erythromycin B genes are no exception. Genes thought to code for proteins involved in the late stages of the pathway, in which 6-deoxyerythronolide B is converted into the erythromycin A, were found on both sides of the erythromycin resistance gene.
Indeed, deletion of individual genes in these regions produced mutant organisms from which late intermediates have been isolated. Further from the resistance gene was revealed a region of DNA which contained sections corresponding to the 6- deoxyerythronolide B synthase (DEBS) gene cluster.
Initial DNA sequencing of the DEBS genes appeared to show that they would encode mono- or, more occasionally, bifunctional enzymes, clearly homologous to the components of a type H bacterial FAS. However, thorough DNA sequencing of the proximal end of the clustered DEBS genes then showed that nine successive active sites are encoded within a single multiflinctional polypeptide chain (now termed
DEBS 3). Subsequent DNA sequencing has lead to the current model of the DEBS genes and of the three multifunctional gene products: DEBS 1, DEBS 2 and DEBS 3 (figure 1.8), each of which apparently catalyses two of the six cycles of chain extension required to produce 6-deoxyerythronolide B. One of the most striking features of the DEBS polypeptides is that the order and spacing of the active sites in the primary structure exactly mirrors the order of the comparable active sites in a vertebrate fatty acid synthase. A eukaryotic PKS which has also been extensively studied is that involved with 6- methylsalicylic acid (figure 1.9, XII) biosynthesis. 6-Methylsalicylic acid is a tetraketide metabolite of the fungus Peniciiiumpatulum. It was pointed out by Dimroth eta!., (1970) that most of the 6-methylsalicylic acid synthase (MSAS)
9
,,Me
Me', ,,Me
Ft'11 "OH
XI Me
Figure 1.7: 6-Deoxyerythronolide B (Xl).
I Genel T Gene2 ) Gene3 )
I DEBS1J I DEBS2J I DEBS3I
Figure 1.8: The organisation of the genes and enzymes of the erythromycin producing
PKS in S. erythraea. ACP, KS, AT, DH, ER, KR and TE represent acyl carrier
protein, 13-ketoacyl-ACP synthase, acyltransferase (propionyl CoA or methylmalonyl
C0A:ACP), 3-hydroxyacy1 dehydrase, enoyl reductase, -ketoacyl-ACP reductase
and thioesterase or putative cyclase, respectively (Leadlay el a!., 1993)
10 COOH CH3.(LOH
xff
Figure 1.9: 6-Methylsalicylic acid (XI!).
[pcYLATION] ac. -CoA ____ EThANSFER] $20 F ICYCLIZATIo174v\-a. / E LMALONYL TRANSFERJ Co2 IC0NDENSAT1ON1 E( - _i ___ "srco-cNa--co-cH3 maloa-CoA [CONDENSATION] ( 0rCo-0 ,,Lon$-CoA SH
,nonyt-CoA IMAWNYL TRANSFER S-Co-CH2-t0OH __,_.-
\AINYL TRANSFER1 ESPIH3 \coh"o A-. co2 pENSAT1 REDUC ON E'y S-Co-CH2-CO-CH2-Co-a43
E\50 ___ ,SH E,
YDRATIÔi]
Figure 1.10: Hypothetical scheme of the 6-methylsalicylic acid reaction sequence
(Beck ci aL, 1990 from Dimroth eta!., 1970).
11 component enzymes are functionally very similar to those of the FAS complex. Beck el a!., (1990) characterised the 6-methylsalicylic acid PKS (or MSAS) further and they found it to be a homomultimer of a single, multiflinctional protein subunit
(resembling a fungal type I fatty acid synthase). When compared to other known PKSs, distinct amino acid sequence similarities of limited length were observed with some, although not all, of them. However, a comparatively low degree of similarity was detected between the MSAS and either the yeast or Penicillium FAS. In contrast, a significantly higher sequence similarity was found between the MSAS and the rat
FAS especially at their acyltransferase, 3-ketoacyl-ACP reductase, 3-ketoacy1-ACP synthase and acyl carrier protein domains. Since there is only one copy of each enzyme component in the complex1 the synthase is somehow 'progranuned' to modify the growing polyketide chain differently during each of the three cycles of chain extension (figure 1.10).
1.4: The biosynthesis of the ochratoxins.
The first investigation into ochratoxin A biosynthesis was by Searcy c/al. (1969). An
Aspergillus ochraceus (NRRL 3174) stationary culture was either fed [1-'4C] phenylalanine or [2-' 4C] acetate and the ensuing [ 14C] ochratoxin A was subjected to limited chemical degradation. [ 14C] Ochratoxin A derived from cultures fed with the radiolabelled primary metabolite precursors was initially acid hydrolysed to yield phenylalanine and ochratoxin a. The radioactivity of[ 14C] ochratoxin A from [1-'4C] phenylalamne feeds was found to be almost exclusively in the C-i position of the phenylalanine moiety. The radioactivity of [ 14C] ochratoxin A from [2-' 4C] acetate was almost exclusively found in the ochratoxin a moiety and, further chemical degradation of this showed significant labelling of only C atoms: 5, 7 and 8a (notably, C atoms 4 and 4a were unable to be analysed). It was concluded that phenylalanine is incorporated into ochratoxin A in 1010 via the ubiquitous shikiniic acid pathway, whereas the ochratoxin a is constructed, in part, by head-to-tail additions of acetate.
12 Ferreira and Pitout (1969) used an ochratoxinogenic A. ochraceus (C SIR 804)
submerged culture in a nitrogen free "resting" medium to show incorporation of[1-
14C] phenylalanine, [1-14C] acetate, [methyl- 14C] methionine and [ 14C] formate into ochratoxin A, albeit in rather small percentages. A crude cell-free enzyme preparation named "ochratoxin A synthetase" which was able to link ochratoxin a with phenylalanine was also described. The enzyme required ATP and Mg 2 ions to flinction. These results can be interpreted as either evidence for the last step in ochratoxin A biosynthesis or as an indication of the lack of specificity of the ochratoxin A synthetase enzyme.
Steyn et al. (1970) extended the work of Searcy el a!. (1969) by feeding a "resting"
A, ochraceus (CSIR 804) culture 14C labelled precursors, but treating the resultant
[14C] ochratoxin A to a more specific chemical degradation. DL-[1-14C] phenylalanine was incorporated solely into the phenylalanine moiety of ochratoxin A. A fifth of the radioactivity of[ 14C] ochratoxin A extracted from a culture fed with [1-
14C] acetate was found in C-3. C-b of ochratoxin A was only labelled when the culture was fed [methyl- 14C] methionine. From this evidence it was concluded that the ochratoxin a moiety of ochratoxin A is constructed from the head-to-tail additions of five acetate molecules with the C 1 addition at C-7 from methiomne, involving the
methyl donor S-adenosylniethionine (SAM or AdoMet). The folding pattern of the
pentaketide chain is shown in figure 1.11. Wei et a!. (1971) incorporated 36C1 directly into ochratoxin A by adding Na36C1 to a
submerged A. ochraceus (NRRL 3174) culture at different times. The highest specific
radioactivity of [36C1] ochratoxin A was achieved when Na36C1 was added at day three.
Yamazaki eta!. (1971) used an A. ochraceus strain isolated from mouldy rice grown as stationary cultures to be fed [U-'4C] phenylalanine and [2-' 4C] malonate. Both of
the radioactive compounds were incorporated into ochratoxin A, the [U-14C]
phenylalanine being localised in the phenylalanine moiety and the [2-' 4C] malonate
being localised in the ochratoxin a moiety. The methylation inhibitor ethionine was
13 U U U
Figure 1.11: The folding pattern of the pentaketide forming ochratoxin A.
* = C-I of acetate, U = C-2 of acetate.
14 added to some of the cultures and, at concentrations of 50 mgfL, ochratoxin A production was completely inhibited. There was no mention of any aromatic intermediates of ochratoxin A biosynthesis being accumulated or suppressed. In addition, 13C NMR spectroscopy was performed on ochratoxin A and the spectrum was partially assigned. [13C] Ochratoxin A isolated from A. ochraceus cultures that were fed [13C] formate showed a lone enriched signal in the 13C NMR spectrum which corresponded to C-10. A fuller report of this work was published a year later
(Maebayashi eta!., 1972). Huff and Hamilton (1979) authored a review article on the biosynthesis of the ochratoxins. The lack of research in this area was recognised although a full biosynthetic scheme of ochratoxin A biosynthesis was postulated (figure 1.12). The ochratoxin A PKS enzyme was reputed to produce mellein. The mechanism has now been shown to be wrong since there is no keto-enol tautomerism of the folded pentaketide chain (see mellein biosynthesis, 1.6). The methylation step was thought to occur alter the production of mellein for three reasons: i) mellein and ochratoxin A have been found together in the broth of some A. ochraceus cultures (Moore et a!., 1972). ii) Methylation of the polyketide chain was deemed to result in multiple methylations such as in citrinin biosynthesis. iii) The mellein hydroxyl group at position C-8 is ortho directing which is the position at which the carboxy group occurs (i.e. at C-7). 7-Methylmellein, 7-methoxylmellein and 7-formylmellein have never been isolated from an ochratoxinogenic fungal fermentation although the next intermediate, 7-carboxymellein (ochratoxin 3), has (Hutchison et aL, 1971). The next step proposed was chlorination of 7-carboxymellein to 5-chloro-7-
carboxymellein (also known as ochratoxin a) by a chioroperoxidase enzyme. It was
reasoned that the chlorination was the last substitution of the aromatic nucleus as it was para in the presence of a meta directing group such as the 7-carboxy, and that chlorination would also deactivate the ring. Joining the aromatic nucleus to the amino
acid phenylalanine by an amide bond was thought not to be via a typical GTP
requiring protein synthesis route, i.e. with the use of a special ochratoxin a tRNA
15
CH3CO2H PKS Ei 4X CH2(CO2H foldmg 0 0 0 0 0
OçCH3 Cl3 aldol condatxn ____mcthylatEn dehydration zeductioa
OH 0 n CI CH3 CH3 o,ádation ,çiixc chkiat,n
CH3/cIIII HO2C HO2C OH 0 OH 0 (11-1 fl 7-ca7-nithyhii box&u 7bo,,-5comnkmn (oclratoxm) (ocfratoxii a)
actMn CO2H CO2CH2CH3 S1icimic acid Cl pathway 2 CH
p1nyhimi plyh1u* etl4 est + tic;3 T T OH 0 A plsphoclitoxr CL synthetae Cl CO2CH2CH3,çJç(
OH 0 ocfratoxiiC
ctcrase C' CO2H CH3
oclratoxri A
Figure 1.12: The biosynthesis of ochratoxin A (Huff and Hamilton, 1979).
16 molecule, but via an acyl activated phospho-ochratoxin a reacting with phenylalanine which has had its carboxy group protected by its ethyl esterification. This reaction
would result in ochratoxin C, which would be trans-esterified to the ochratoxin A methyl ester or hydrolysed to ochratoxin A.
Ochratoxin C is rarely found as a natural contaminant of foodstuffs (WHO, 1979), although the toxicity of ochratoxin C is similar to that of ochratoxin A. It has been demonstrated that ochratoxin C can be directly converted into ochratoxin A in vivo after both oral and intravenous administration to rats. Ochratoxin C toxicity seemed
to be proportional to this conversion (Fuchs eta!., 1984). The 13C NMR spectrum of ochratoxin A was filly assigned by de Jesus eta!. (1980).
Stationary cultures of Penicillium viridicatum (P. verrucosum) were fed [1-'3C]
acetate and the 13C NMR spectrum of the [1-' 3C] acetate-derived ochratoxin A showed enhanced signals corresponding to C atoms: 1, 3, 4a, 6 and 8. Furthermore, when a stationary culture of A. suipliureus (NRRL 4077) was fed [1, 2- 13C2] acetate
and the resultant [ 13C] ochratoxin A was analysed using 13C NMR spectroscopy, there was - 13C coupling between C atoms: 9 and 3; 4 and 4a; 5 and 6; 7 and 8 and 8a and 1. This was unequivocal evidence to support a folding pattern of a
pentaketide chain in the biosynthesis of ochratoxin A similar to that which occurs in citrinin and mellein biosynthesis.
Reviews of ochratoxin A biosynthesis have also been written by Steyn (1971, 1984),
Harwig (1974), Vleggaar and Steyn (1980) and Betina (1989). However, the
biosynthesis of ochratoxin B has not been featured in the literature.
1.5: The melleins.
Mellein (figure 1.13, XIII) is a pentaketide metabolite found in many sources in
nature. Mellein was first isolated from a culture of Aspergillus melleus (Nishikawa,
1933) and in the same year was found to be identical to ochracin, a product of some
Aspergillus ochraceus cultures (Yabata and Sumiki, 1933). Mellein has been isolated
17 R
9 5
H
R1 R2 R3 R4 R5
Xm Mellem H H H H H XIV 6-MeO mel H CH3O H H H
Xv 3-OHmel H H H H OH xv' 4-OHmel H H H OH H XvLI 6-MeO 5-Cl mel H CH3O Cl H H xvLII 5-Me mel H H CH3 H H
xix 5-Form mel H H CHO H H
xx 5-Carb mel H H COOH H H XXI 7-Carb mel t COOH H H H H
XXII 7-Carb 5-Cl mel COOH H Cl H H
Figure 1.13: The structures of mellein (XIII), 6-methoxymellein (XIV), 3- hydroxymellein (XV), 4-hydroxymellein (XVI), 6-methoxy-5-chloromellem (XVII), 5-methylmellein (Xviii), 5-formylmellein (XIX), 5-carboxymellein (XX), 7- carboxymellein (XXI) and 7-carboxy-5-chloromellein (XXII). t Also known as ochratoxin f. Also known as ochratoxin a.
18 from phytopathogenic fungi such as Septoria nodorum (Sachse, 1992) and Botryosphaeria obtusa (Venkatasubbaiah and Chilton, 1990). Parisi eta!. (1993) believed (R)-(-)-mellein to be one of the phytotoxins produced by the parasitic fungus
Phoma tracheiphila which is responsible for the destructive wilt disease of citrus known as "mal secco". Mellem showed toxicity to tomato cuttings and cytotoxicity to brine shrimps. Nakamori ci a!. (1994) showed that mellein isolated from the fungus
Lasiodiplodia theobromae induced potato micro-tuber formation. Although meilein is not classed as a mycotoxin, toxicity to mice by mellein has been demonstrated (Sasaki eta!., 1970).
Mellein production, however, is not restricted just to fungi. Meilein was found to be a constituent of the mandibular secretions of carpenter ants (Brand eta!., 1973), one of the defence secretions of termites (Blum eta!., 1982), within the male hair pencils of the oriental fruit moth (Baker eta!., 1981) and has been shown to have pheremonal properties in the castes of Camponotuspennsylvanicus (Payne eta!., 1975). Despite its predominance in extracts of male hair pencils, mellein was itself inactive in electroantennogram bioassays. However, positive bioassay readings occurred when it was blended with ethyl trans cinnamate and methyl jasmonate, two other male hair pencil constituents.
Mellein has structural analogues: 6-methoxymellein (figure 1.13, XIV) was isolated from the culture broth of the fungus Sporormia bijxzrtis (Aue et a!., 1966). Mellein, 3-hydroxymellein (figure 1.13, XV) and 4-hydroxymellein (figure 1.13, XVI) were all isolated from anAspergillus oniki isolate (Sasaki eta!., 1970) and, a year later, 4- hydroxymellein was isolated from an Aspergillus ochraceus culture (Cole et a!.,
1971). 6-Methoxy-5-chloromellein (figure 1.13, XVU) was characterised by Holker and Young (1975) when it was extracted from a culture of the fungus Periconia macrospinosa. The fungus Nummulariella marginata is the causative agent of apple blister canker. 5-Methylmellein, 5-formylmellem and 5-carboxymeilein (figure 1.13, XVIII, XIX and XX, respectively) were all extracted from a submerged culture of N. marginata (Whalley and Edwards, 1985).
19 Recently, Schultz et aL (1995) extracted mellein from fermentations of the endophytic ascomycete Pezicula. Mellein was tested for antibacterial, flingicidal, algicidal and herbicidal activity. Marginal antibacterial and fungicidal activities were detected although mellein was found to be significantly algicidal especially toward Chiore/la fusca (inhibition of which usually correlates with broader herbicidal activity). In addition, mellein also inhibited the germination of Lepidium sativum and Medicago sativa, reinforcing previous reports of its phytotoxic nature.
1.6: The biosynthesis of the melleins.
Blair and Newbold (1955) confirmed, by chemical synthesis, the structure of mellein to be (-)-3-methyl-3,4-dihydro-8-hydroxyisocoumarin. The (+) optical antipode of mellein was isolated from an unidentified fungus by Patterson eta!. (1966). Holker and Simpson (1981) performed 13C enrichment studies using [1, 2- 13C2], [1-13C] and
[2-'3C] acetate fed to cultures of Aspergillus melleus. 13C-13C coupling in 13C enriched mellein resolved the folding pattern of a pentaketide chain identical to that of ochratoxin A (figure 1.14). Also discovered was aspyrone (3-(1, 2-epoxypropyl)-5,6- dihydro-5-hydroxy-6-methylpyran-2-one; figure 1.15, XXIII) the 13C-13C coupling of which from [1, 2-13C2] acetate enrichment indicated a pentaketide origin. Since both mellein and aspyrone lack the equivalent oxygen atom from the polyketide precursor and that, on repeated culture of A. melleus, yields of aspyrone decreased with concomitant increased mellein yields, it was suggested that both compounds were derived from a common deoxypentaketide which is diverted to mellein production when aspyrone production is inhibited.
Abell eta!. (1982) investigated the nature of the aromatisation in mellein biosynthesis.
It was reasoned that there were two possible routes to the loss of the C-6 hydroxyl group: either via an enol derivative or through an unfunctionalised double bond
(figure 1.16). Protons at positions C-5 and C-7 were replaced with deuterium by feeding A. melleus with [2-2H3J acetate and then isolating the labelled mellein.
20 ODD_ .4l.CH3 çJçJ OH 0
Figure 1.14: The folding pattern of the pentaketide forming mellein. * = C-i of acetate, U = C-2 of acetate.
0 HO, ..'1 13 0 OXXm OHO Figure 1.15: Aspyrone (XXIII) and alternariol (XXIV). cx: a) 0 0 HO R_____ LJL II b) 0 0 0 OH Figure 1.16: Routes to mellein aromatisation: a) via an enol b) via an unfunctionalised double bond. 21 Deutenum retention at these two points was measured directly using 2H NMR spectroscopy and the ratio of the signal intensities of deuterium at C-7 and C-5 was 2:1. The equivalent ratio for alternariol (figure 1.15, XX1V) produced by a culture of Alternaria alternata fed with [2-2H3] acetate was 0.3:1. It was presumed that altemariol was biosynthesized via an enol route as the C-6 hydroxyl group is retained. The enol route entails a keto-enol tautomerisation involving non-stereospecific reprotonation, and thus deutenum depletion. There was considerably less deuterium depletion in the mellein sample which was accounted for by there being no keto-enol interconversion. Hence, the irreversible dehydration route (b) was favoured. Abell et a!. (1983) continued the investigation of mellein biosynthesis with respect to an intermediate in common with aspyrone. In such a scheme, the epoxide in aspyrone would be derived from a double bond equivalent to a C-3 - C-4 double bond in a mellein biosynthetic intermediate. The C-4 protons would not be equivalent in mellein if one of the biosynthetic intermediates had been unsaturated in this position. [2-2H3] Acetate feeding experiments with A. melleus showed conclusively that the C-4 protons are equivalent and both are derived from acetate. If aspyrone and mellein have a common intermediate, it would have to be a very early one. Such an early intermediate was speculated (figure 1.17, XXV), later perpetuated by Brereton et a!. (1984). Abell et a!. (1987) fed [2-2H3] acetate, [170] acetate and 1702 to cultures of A. melleus and, through 170 and 2H NMR analysis of the resulting mellein and 4- hydroxymellein, determined the origin of the constituent oxygen atoms. In mellein, all of the oxygen atoms are derived from acetate. This is true for 4-hydroxymellein except for the 4-hydroxyl group which is formed from 02. Mellein was deuteriated at positions C-5 and C-i by treatment with trifluorodeutenoacetic acid and, after being fed to cultures of A. melleus, was found to be specifically incorporated into 4- hydroxyinellein. 4-Hydroxymellein was thought to be directly biosynthesized from mellein by a stereospecific hydroxylation at position C-4. 22 0 OH Enz xxv Figure 1.17: A possible early intermediate of mellein biosynthesis (XXV). 23 Sun and Toia (1993) reproduced earlier [1, 2- 13C2] acetate feeding experiments, but this time on mellein during its production by Australian povenne ants. Mellein biosynthesis may be re-assessed by applying modern views on polyketide biosynthesis. An assumed model of the biosynthetic events on the mellein PKS is shown in figure 1.18. An acetyl thioester and malonyl CoA undergo a decarboxylative condensation reaction to yield acetoacetyl thioester which is reduced (by a ketoreductase) to the corresponding alcohol. Two further decarboxylative condensations involving malonyl CoA occur, followed by a reduction (by a ketoreductase) and then a dehydration (by a dehydrase) to form the C-6 -C-7 unfunctionalised double bond. A final decarboxylative condensation involving malonyl CoA results in the finished polyketide chain. An E stereospecificity of the double bond will facilitate the polyketide chain folding prior to aldol condensation. The folded polyketide chain is the same as the proposed early mellein intermediate (figure 1.17, XXV) and, after cydisation, hydrolysis of the thioester to the free acid (by a thioesterase) enables the formation of the lactone mellein. A specific hydroxylation of mellein at C-4 would result in the formation of 4-hydroxymellein (figure 1.19). Holker and Young (1975) used [1-' 3C], [2-13C] and [1, 2-' 3C2] acetate fed to the fungus Periconia macrospinosa to elucidate the 6-methoxy-5-chloromellein (figure 1.13, XIV) biosynthetic pathway. The cumulative results indicated a pentaketide folding pattern identical to mellein. A first enzyme-free intermediate of 6-methoxy-5- chioromellein biosynthesis was proposed (figure 1.20, XXVI) which had retained the C-6 hydroxyl group in preparation for 0-methylation by SAM. The free hydroxyl group would result from a direct aldol condensation reaction of an enol intermediate when forming the ring. The ketone at the C-3 position would have to be reduced to the alcohol in order for the formation of the lactone after the thioester was hydrolysed. The chlorination, perhaps by a chloroperoxidase, at C-5 would result in 6- methoxy-5-chloromellein. 24 CH3COSEnz + HO2CCH2COSCoA AT/KAS CH3COCH2COSEnz ,,..-NADPH KR[ NADP CH3CH(OH)CH2CO SEflZ AT/KAS + HO2CCH2COSCoA CH3CH(OH)CH2COCH2CO SEnZ AT/KAS + HO2CCJ-I2COSCoA CH3CH(OH)CH2COCH2COCH2cosE ,ADPH KR[ NADP CH3CH(OH)CH2COCH2CH(OH)CH2COSEn DH(. HO CH3CH(OH)CH2C OCH2CH=CHC OSEnz AT/KAS + HO2CCH2COSCoA C}I3CH(OH)CH2COCH2CH=CHCOCH2COSEnZ Figure 1.18: A schematic diagram of putative biosynthetic events on the mellein PKS. AT, KAS, KR and DH denote the acyltransferase, 3-ketoacy1-ACP synthase, 3- ketoacyl-ACP reductase and -hydroxyacy1-ACP dehydrase activity of the PKS, respectively. 25 CH3CH(OH)CH2COCH2CH=CHCOCH2COSEnz fbldrng CIT3 O OH aldol OH S Enz S Enz condensation OH 0 0 0 -..CH3 LrJLOHCOOH OH esterification hydroIation OH 0 OH 0 nflein 4-hydroxymeflein Figure 1.19: Reasoned post-polyketide chain biosynthesis events in mellein and 4- hydroxymellein biosynthesis. 26 HO\(CH COOH OH XXVI Figure 1.20: Proposed first enzyme free intermediate of 6-methoxy-5-chloromellein biosynthesis (XXVI; Holker and Young, 1975). CH3 HOf&CH3 JL3ó OH XXVII ID XXVffl OH 0 OH 0 Figure 1.21: The structures of diaporthin (XXVII) and orthosporin (also known as de-O-methyldiaporthin, XXYffl). CH3O * * OH 0 Figure 1.22: The folding pattern of the hexaketide forming diaporthin. * = C-i of acetate, U = C-2 of acetate. 27 No biosynthetic studies have been carried out on the remaining mellein analogues, although 3-hydroxymellein biosynthesis can be envisaged as similar to 4- hydroxymellein i.e. by the direct hydroxylation of mellein. 5-Methylmellein could either be biosynthesised by the use of propionate (from methylmalonate) rather than acetate (from malonate) as the second extender unit addition in the biosynthesis of the polyketide chain or by the direct methylation of meilein (using SAM). Methylmalonate is biosynthesised from the carboxylation of propionyl CoA using ATP, HCO3 and the enzyme propionyl CoA carboxylase (Mathews and van Holde, 1990). Since the added carboxyl group is lost in the decarboxylative condensation reaction of polyketide biosynthesis, the methyl carbon is derived from the C-3 of propionate. Thus experimentation to determine whether the C-5 methyl group is derived from methionine (direct methylation using SAM) or the C-3 of propionate (methyl group incorporated in the polyketide chain construction) is required. The oxidation of the C-S methyl group would result in the formyl and carboxy analogues. 1.7: Diaporthin and orthosporin. Endothiaparasillca (Murr.) And. is the cause of canker in Chestnut trees. Epidemics of this fungus almost caused the extinction of Chestnut tree forests in North America. Diaporthin (figure 1.21, XXVII) was first described by Bazzigher (1953), who showed that when obtained from cultures of Endothiaparasitica, it could reproduce the symptoms of the canker in Chestnut trees. Boiler eta!. (1957) purified diaporthin and determined some of its chemical properties and also showed that diaporthin inhibited the growth of several bacterial species and yeasts. Further investigations into the structure of diaporthin were carried out by Hardegger et a!. (1966) who performed 'H NMR spectroscopy on diaporthinic acid, diaporthinic acid derivatives and chemically synthesized diaporthinic acid. Diaporthinic acid and acetaldehyde result from the anaerobic alkaline hydrolysis of diaporthin. 28 De-O-methyldiaporthin (figure 1.21, XXVIII) was isolated from shake flask cultures of the plant pathogenic fungus Drechslera siccans which causes irregular dark brown to reddish brown spots on the leaves of the host plants: oats, perennial ryegrass and Italian ryegrass (Hallock eta!., 1988). Published results included: accurate electron impact mass spectrometly (EI-MS) data for the molecular ion and a scheme of nominal El-MS fragmentation data, incomplete 1H NMR spectroscopic data, and a complete but unassigned 13C NMR spectrum for de-O-methyldiaporthin. The compound was found to produce necrotic areas when applied in solution to the leaves of maize, crabgrass, soyabean, barnyard grass and spiny amaranth, yet not to perennial ryegrass leaves. Ichihara eta!. (1989) isolated orthosporin from cultures of the plant parasitic fungus Rhynchosporium orthosporum which induces leaf scald on orchard grass. Accurate El-MS data on the molecular ion, UV and JR spectrum analysis and fully assigned 1H and 13C NMR spectra showed it to have the same structure as de-O- methyldiaporthin. Furthermore, orthosporin treated with diazomethane gave diaporthin and consequently the 1H NMR spectrum of diaporthin was assigned further. 1.8: The biosynthesis of diaporthin and orthosporin. No studies have been performed on diaporthin or orthosporin biosynthesis, yet diaporthin has been classified as a hexaketide (Turner, 1971). Principles of mellein biosynthesis can, however, be used to predict the mechanism of diaporthin biosynthesis. A folding pattern homologous to mellein would involve a hexaketide with the extra C2 extension attached to C-9 (figure 1.22). The reasoned steps of diaporthin and orthosporin biosynthesis can be summarised in figure 1.23. The first decarboxylative condensation reaction involving an acetyl thioester and malonyl CoA on the orthosporin PKS results in an acetoacetyl thioester which undergoes a ketoreduction by a ketoreductase. Further fatty acid biosynthetic steps are aborted. 29 AT/KAS CH3COSEnz + HOOCCH2COSCoA _ CH3COCH2COSEnz ,- NADPH KR NADP AT/KAS CH3CH(OHXCH2CO)5SFnz CH3CH(OH)CH2CO SEflZ + (HOOCCH2COSCoA)4 Ibiding 0 akiol colkiensation HOççfl( HOqç%( keto-enol tautonrLsm -' TE hydmlysis esterIfEati1n CH3O 3 HOçfrY nthykthon OHO OHO ort1isporin diaporthin Figure 1.23: The putative biosynthesis of orthosporin and diaporthin. AT, KAS, KR and TE denote the acyltransferase, 3-ketoacy1-ACP synthase, 3-ketoacy1-ACP reductase and thioesterase activity of the PKS, respectively. 30 Four decarboxylative condensations involving malonyl CoA occur without any reduction to produce the finished polyketide chain. The polyketide chain folds and an aldol condensation reaction results in the cycisation of the polyketide. Unlike mellein, there is no reduction and then subsequent dehydration of the keto group at position C-6, so a C-6 hydroxyl group results from the aldol condensation reaction. The keto group at position C-3 will undergo keto-enol tautomerism so the hydrolysis of the thioester to the free acid (by a thioesterase) enables the formation of the lactone orthosporin. 0-Methylation., using SAM, of the hydroxyl group at position C-6 results in diaporthin. 31 2: Dynamics of the biosynthesis of Aspergillus ochraceus D2306 polyketide metabolites in liquid and solid substrate fermentations. 2.1: Introduction. Aspergillus ochraceus has been grown on a variety of different media, both solid (rice, wheat, barley, oats, coffee beans, maize etc) and liquid (yeast extract-sucrose (YES) medium, Raulin-Thom medium, Ferreira medium, Yama.zaki medium, etc) yet not in potato dextrose broth. The purpose of these investigations has been primarily to assess or isolate ochratoxinogenic Aspergillus strains or for researching biosynthetic pathways. Literature concerning secondary metabolites of A. ochraceus generally concerns the production of only one or two compounds, e.g. ochratoxin A (Searcy eta!., 1969), ochratoxin B (Nesheim, 1969), mellein and 4-hydroxymellein (Cole eta!., 1971) or ochratoxin A and penidilic acid (Ciegler, 1972). Occasionally, more than two secondary metabolites have been considered, such as in the report by Delgadillo (1986) who studied the effect of the composition of a synthetic medium on the production of ochratoxins A, B and f3, mellein, 4-hydroxymellein and penicillic acid. However, only end point yield values were given, with no indication of the accumulation dynamics. Indeed, no published article has so far dealt with the dynamic relationship between the accumulation of structurally similar polyketides produced by A. ochraceus under standard culture conditions. This is unfortunate since these kinds of studies can provide clues as to the biosynthetic connectivity of various metabolites which may lead to an insight into reasons why ochratoxin A is, or perhaps more usually is not, produced by isolates of A. ochraceus. Thus, an investigation aimed to isolate and characterise polyketides structurally related to ochratoxin A and establish their production dynamics in a variety of media was instigated. In addition, attempts were made to optimise ochratoxin A production by A. ochraceus over a range of scales. In particular it was desirable to develop a 32 small scale system to facilitate the incorporation of fed 14C labelled advanced precursors of ochratoxin A biosynthesis, but also to optimise larger laboratory scale production of ochratoxin A, or to make foodstuffs highly contaminated with ochratoxin A, for future toxicological investigations. The A. ochraceus strain D2306 was originally isolated in Australia from soya meal and when it was grown on sterilized maize meal (100 g substrate in a I L flask was moistened to 40% (vlw) and incubated as a stationary culture at 28 °C for 21 days), ochratoxin A was produced at an astonishingly high yield of 20 mglg substrate (Connole eta!., 1981). However, Tapia and Seawright (1984) repeated the experiment using the same conditions but only reported an ochratoxin A yield of 2 mg/g substrate. Due to its obvious propensity for high yields, attempts were made in the present study to optimise ochratoxin A production by the same strain on the solid substrate shredded wheat (SW) over a range of scales: 1 to 1000 g. Two reports have been published regarding the optimisation of ochratoxin A production in solid state fermentations (a solid state fermentation may be defined as a fermentation on a substrate which is not liquid). Hesseltine (1972) reported the growth of A. ochraceus NRRL 3174 on rice, pearled wheat and cracked corn: 150 g (v/w) of substrate in a 2.8 L Fembach flask was moistened to 30-5O°j autoclaved and then inoculated with a spore suspension. Unusually, the flasks were incubated as shake flask cultures (at 200 rpm on a gump shaker at 28 °C for up to 12 days with two 3 ml water additions at 24 and 48 hours post inoculation) since traditionally the majority of solid substrate fermentations had always been stationaly. The shaken solid substrate fermentation technique had originally been developed to study afiatoxin production (Shotwell et a!., 1966). An ochratoxin A yield of 1.5 mglg substrate of shaken pearled wheat bettered previous stationary solid substrate culture values that were typically only about 35 j.tg/g substrate (Ciegler, 1972). Hesseltine (1972) also mentioned the (v/w) importance of maintaining: i) substrate moisture at approximately 28%, ii) a constant ratio of the volume of substrate : volume of culture vessel, which affects aeration, iii) 33 constant agitation, which inhibits sporulation and iv) a limited range of substrate particle size, to allow free air circulation. Lindenfelser and Ciegler (1975) constructed a baffled barrel-shaped 26 L solid state v/w fermenter in which 1 kg of cracked wheat (moistened to 30%2 was sterilized, inoculated with an A. ochraceus NRRL 3174 spore suspension and incubated at 25 °C whilst being continually rotated at 10 rpm. Yields of ochratoxin A were 3-4 mg/g substrate after 10-12 days, although production only commenced after 4 days. Better yields in the fermenter, as compared to shake flasks, was attributed to better aeration. The authors stressed the importance of agitation, which mixes the substrate, promotes aeration and also dissipates heat, and the correct initial substrate moisture, substrate moisture decreased markedly during the first few days, although it did eventually (v/w) reach approximately 4%, tor the production of ochratoxin A. A 2.2: Materials and methods. 2.2.1: Dynamics of the biosynthesis of Aspergillus ochraceus D2306 polyketide metabolites in liquid substrate fermentations. The fungus was maintained on potato dextrose agar (PDA) slopes. PDA slopes consist of 10 ml of Difco PDA medium autoclaved in 30 ml Universal bottles. PDA slopes were inoculated with D2306 and incubated at 25 °C for 1 to 2 weeks until the culture was sporulating fully and the agar had become highly fluorescent under UV light (350 nm) They were then stored at 4 °C. Spores from a PDA slope were used to inoculate a SW culture which in turn was the source of spores used to inoculate potato dextrose broth (PDB) cultures. The SW medium consisted of 20 g of shredded wheat (The Shredded Wheat Co., Weiwyn, UK) autoclaved in a 500 ml conical flask. Prior to inoculation, the shredded wheat was dampened thoroughly with 8 ml of v/w sterile distilled water (approximately 40% moisture; Mantle and McHugh, 1993). The inoculated SW cultures were incubated for up to 3 weeks at 28 °C before being 34 stored at 4 °C. Two g of extensively sporing D2306 SW culture was homogenised in 100 ml sterile distilled water using a Sorvall 0mm-Mixer. After allowing crude particulates to settle, the spore-suspension was used to inoculate each PDB flask so that the final spore concentration, as measured with a haemocytometer, was in the range 2-4 x 106 spores/nil. The PDB medium consisted of 100 ml Difco PDB medium autoclaved in each 500 ml conical flask. The D2306 PDB cultures were incubated at 28 °C, shaken at 200 rpm with a 10 cm eccentric throw At intervals, 10 ml of the D2306 PDB culture was removed for determination of dry cell weight (DCW), and the concentration of ochratoxins A and B, orthosporin, diaporthin and mellein in the broth. DCW was determined by filtering the broth through a pre-weighed sintered 10 ml syringe which was then freeze dried to constant weight. The filtered broth was acidified with conc. HC1 and extracted with an equal volume of ethyl acetate. The organic phase was then partitioned with an equal volume of 3% (w/v) sodium hydrogen carbonate. The organic phase was rotary evaporated to dryness to give a "bicarbonate insoluble" fraction. The bicarbonate phase was acidified with conc. HC1 before being extracted with an equal volume of ethyl acetate. The organic phase was rotary evaporated to dryness to give the "bicarbonate soluble" fraction. Bicarbonate soluble and insoluble samples were dissolved in 250 j.ti methanol and 20 l.Li of each sample was used for diode array detector (DAD) HPLC analysis. The DAD-HPLC system employed was an isocratic mobile phase of water-acetic acid- by volume acetonitrile (59.5:1 modified from Nesheim eta!., 1992) flowing at 1 mI/mm through a 150 x 5 mm Separon SGX C-18 column (Kolona Chromatogra.flcká) into a Hewlett Packard 1040M diode array detector controlled by HPLC3D ChemStation software on an IBM 486 personal computer. A calibration graph for each compound of interest was constructed using the appropriate ChemStation programme to enable quantitative analysis (see appendix I-V) and the spectral purity of the ensuing chromatograms was determined by the ChemStation "Peak Purity" facility. 35 The acidic ochratoxins A and B were resolved from the bicarbonate soluble samples as shown in figure 2.1. The monitoring wavelength was 332 mn. The bicarbonate insoluble samples contained orthosporin, diaporthin, mellein and cycloechinulin and each was resolved as shown in figure 2.2. The monitoring wavelength was 239 nm. 2.2.2: Intra- and extracellular polyketide concentrations versus time for Aspergillus ochraceus D2306 on either potato dextrose broth or yeast extract-sucrose. The relationship between the intra- and extracellular polyketide concentrations during a D2306 PDB fermentation was investigated. Approximately 5 g of extensively sporing D2306 SW culture was homogenised in 100 ml sterile distilled water to produce a spore suspension used to inoculate six 100 ml scale PDB flasks with a final spore concentration of 4 x 106 spores/mi. The shake flask cultures were incubated at 28 °C and at 0, 64, 88, 160, 208, and 256 hours post inoculation, 1 flask was removed for intra- and extracellular polyketide determination. Each sample flask was filtered, the cells were washed with distilled water and the broth was extracted for extracellular polyketides in the usual manner. The intracellular polyketides were extracted by refluxing 1 g of the water washed cells in 100 ml chloroform-methanol by' (2: 1, for 1 hour. The extraction mixture was cooled, filtered through anhydrous sodium sulphate and then rotaly evaporated to dryness. The residue was dissolved in 100 ml ethyl acetate which was partitioned with an equal volume of 3% (wlv) sodium hydrogen carbonate in order to yield bicarbonate soluble and insoluble fractions as described earlier. All fractions were analysed using DAD-HPLC. In addition, the fractions were analysed by thin layer chromatography (TLC). TLC was performed on 20 x 20 cm Polygram SIL G/UV254 TLC plates (0.25 mm silica gel with fluorescent indicator UV254 pre-coated on plastic) developed in toluene-ethyl acetate-formic acid by volume (50:40: 10. Ochratoxins A, B, a., 3 and the dimethyl ester were identified on the basis of Rf values and their appearance under UV light at 254 and 350 nm wavelength (figure 2.3). The stability of ochratoxins A and B, diaporthin and mellein in the cell 36 Figure 2.1: DAD-HPLC chromatogram of a typical bicarbonate soluble sample showing the resolution of ochratoxins A and B. Figure 2.2: DAD-HPLC chromatogram of a typical bicarbonate insoluble sample showing the resolution of orthosporin, cycloechinulin, diaporthin and mellein. 37 Compound Rf value UV256nm UV36Onm Ochratoxin A 0.76 light blue light blue Ochratoxin B 0.57 light blue light blue Ochratoxin alpha 0.63 dark blue dark blue Ochratoxin beta 0.53 dark blue dark blue Ochratoxin Me2 0.85 light blue light blue Figure 2.3: Thin layer chromatography of ochratoxin A (OA), ochratoxin B (OB), ochratoxin a (Oct), ochratoxin 3 (O3) and 0-methyl, methylochratoxin A (OA Me2) (developed in toluene-ethyl acetate-formic acid, 50:40:10) and their appearence under UV light (256 and 360 nm). 38 extraction procedure was tested by refluxing a known amount of ochratoxin A (0.95 mg), ochratoxin B (0.23 mg), diaporthin (0.23 mg) and mellein (1.4 mg) in 100 ml by volume chloroform-methanol (2: l for 1 hour and determining the degree of chemical degradation by DAD-HPLC analysis. The same inoculation procedure used for the PDB experiments was used to initiate six YES medium (2% yeast extract, 4% sucrose, w/v; 100 ml per 500 ml conical flask) shake flask cultures which were incubated at 28 °C. At 0, 24, 48, 72, 96 and 192 hours post inoculation, 1 flask was removed for intra- and extracellular polyketide determination as described above. All fractions were analysed using DAD-FIPLC and mc. 2.2.3: Aspergillus ochraceus D2306 solid substrate fermentations. Static SW cultures used 20 g shredded wheat in 500 ml conical flasks incubated at either 25 or 28 °C and sampled at regular intervals up to 3 weeks post inoculation Sampling involved, typically, removing 1 g of the culture and extracting the polyketides by soaking overnight in 100 ml ethyl acetate/0.01 M phosphoric acid by volume (9:12. The mixture was then filtered and the ifitrate partitioned against an equal volume of 3% (w/v) sodium hydrogen carbonate to yield bicarbonate soluble and insoluble fractions as described earlier. All fractions were analysed using DAD-HPLC. Small scale stationary SW culture investigations began by autoclaving 50 ml boiling tubes fitted with foam bungs and containing 1 g SW. The SW was moistened to 40% (v/w) using sterile water, inoculated, incubated at 28 °C and sampled regularly by extracting the contents of one tube. The experiment was repeated using 50 ml conical flasks. Further experiments involved 1 g per 5 ml conical flask SW cultures which were (v/w) autoclaved and moistened to either 40 or 60% with sterile water the latter to offset I' possible moisture deficit developing in such small fermentations. The flasks were inoculated and incubated at 25 °C in, or outside a high humidity chamber (the 60% , v/w, 39 moisture cultures within only). The humidity chamber was comprised of a 250 ml Schott screw top bottle containing approximately 100 ml of water-soaked cotton wool which provided a water saturated atmosphere. A loosely applied screw top allowed the necessaiy gaseous exchange. Samples were taken regularly up to 7 days post inoculation. The last stationary small scale D2306 SW experiment involved 1 or 2 g SW per 25 ml conical flask or 2 or 4 g SW per 50 ml conical flask being (v/w) autoclaved, moistened to either 40 or 60u/o*and incubated at 25 °C within a humidity chamber. Samples were taken 4, 10 or 13 days post inoculation. The first large scale stationary SW culture experiment employed a 10 L stoppered bottle containing 1 kg SW, to which 150 ml water was added. The vessel was weighed and then autoclaved, the added water ensuring heat penetration to the core (v/w) of substrate. The vessel was re-weighed and the SW moistened to 30%,with sterile water. The SW was inoculated with 5 g of heavily sporing D2306 SW culture and incubated stationary at 28 °C. The vessel contents was shaken daily and sampled by the removal of 1 g of the culture. The final large scale stationary SW culture experiment compared ochratoxin A production by D2306 grown either in 50 or 100 g SW per 1 L conical flask which had (v/w) been autoclaved, moistened to 4O%Aand then incubated at 28 °C. The cultures were sampled regularly during two weeks post inoculation. The initial shaken D2306 SW culture investigation compared 20 or 30 g SW per 500 (v/w) ml comcal flask scale cultures. Each flask was autoclaved, moistened to 3O°/oAwltn (approx. 0.25 g) sterile water, inoculated with a spatula fl1llof heavily sporing D2306 SW culture then incubated shaken at 200 rpm with a 10 cm eccentric throw at 28 °C. All of the flasks were moistened further with 1 and 0.5 ml sterile water per 10 g of SW additions at 24 and 48 hours post inoculation, respectively, and sampled 10 and 22 days post inoculation. A small scale shaken D2306 SW culture investigation was conducted with 1 or 2, 2 or 4, 4 or 8, 10 or 20 and 20 or 40 g SW per 25, 50, 100, 250 and 500 ml conical flask, (v/w) respectively. The flasks were autoclaved, moistened to either 30 or 40%1 with sterile 40 water, inoculated and incubated at 28 °C Conical flasks less than 500 ml were packed, using tissue, into 600 ml plastic beakers which fitted the shaker. All of the (viw) (v/w) 3O°/oAmolsture flasks and the 40%,,moisture flasks containing 10 g SW or more were moistened by the addition of 1 and 0.5 ml sterile water per 10 g of SW, 24 and 48 (v/w) hours post inoculation, respectively. The 4O%Amoisture flasks containing less than 10 g SW were moistened by the addition of 1 ml sterile water both 24 and 48 hours post inoculation. All of the flasks were sampled 8 days post inoculation; some were sampled also at 15 days. Large scale shaken D2306 SW cultures were investigated by using 160, 240 or 320 g (v/w) SW per 4 L conical flask. The substrate was autoclaved, moistened to 30%with sterile water, inoculated and then incubated at 28 °C. Each flask was moistened further at 24 and 48 hours post inoculation, as described previously, and sampled regularly up to 14 days post inoculation. 2.2.4: Dynamics of the biosynthesis of Aspergillus ochraceus D2306 polyketide metabolites in solid substrate fermentations. A 40 g scale shaken D2306 SW culture was initiated and incubated at 28 °C. At 0, 1, 2, 3, 5, 7, 9, 11 and 14 days post inoculation, 2 g of the culture was removed, 1 g for the extraction of the polyketides and 1 g for ergosterol determination. DAD-HPLC and TLC were used to identify and quantify polyketides of interest. Ergosterol was extracted by using the protocol described by Newell et aL (1988): 1 g of sample was placed in a 250 ml round bottomed flask and refluxed in 100 ml HPLC grade (w/v) methanol for 2 hours after which 20 ml of 4%,SKOH in 95% aqueous ethanol was added to the mixture and refluxed for a further 30 minutes. After cooling, 40 ml of water was added and the neutral lipids were partitioned from the base hydrolysis reagent into pentane. The series of three pentane additions (40, 20 and 20 ml) were pooled, dried, re-dissolved in 200 .d methanol of which 20 l.tl was injected into the DAD-HPLC system. The ergosterol DAD-HPLC system used was an isocratic mobile 41 (v/v) phase of 9S%aueous methanol flowing at I mi/mm through a 150 x 5 mm Separon SGX C-18 column (a modification of the system of Newell et a!., 1988). The diode array detector monitoring wavelength was 282 nm. The DAD-HPLC chromatogram and the UV absorbance spectrum for ergosterol is shown in figures 2.4 and 2.5. A calibration graph for quantitative ergosterol analysis was constructed using the appropriate ChemStation programme (see appendix Vi). 2.2.5: Effectiveness of shaken shredded wheat culture for other ochratoxinogenic fungi. SW culture, 20 g scale stationaly and 40 g scale shaken, was used to investigate whether the ochratoxinogenic firngi Aspergillus ochraceus NRRL 3174, Penicillium verrucosum IMI 260915 and Peniciiium verrucosum RS4 (Scudamore eta!., 1993) could give increased ochratoxin A production with agitation. The Penicillium cultures were incubated at 22 °C and the A. ochraceus NRRL 3174 cultures were incubated at 28 °C. After 14 days, all of the cultures were sampled. In addition to standard polyketide analysis, the RS4 extracts were analysed for citrinin. Citrinin., being acidic, would be found in the same extraction fractions as ochratoxins A and B. The DAD- HPLC system employed to detect citrinin was an isocratic mobile phase of water- by volume acetic acid-acetonitrile (59.5:1 :39.5,) containing 10 mM tetrabutylammonium dihydrogen phosphate flowing at 1 mI/mm through a 150 x 5 mm Separon SGX C-18 column (a modification of the system of Vail and Homann, 1990). The diode array detector monitoring wavelength was 327 nm. The DAD-HPLC chromatogram and UV absorbance spectrum of citrinin is shown in figures 2.6 and 2.7. A calibration graph for quantitative citrinin analysis was constructed using the appropriate ChemStation programme (see appendix VII). 42 Figure 2.4: DAD-HPLC chromatogram of ergosterol. Figure 2.5: UV absorbance spectrum of ergosterol. 43 Figure 2.6: DAD-HPLC chromatogram of citrinin. mAU 1000 800 600 400 200 Figure 2.7: UV absorbance spectrum of citrinin. 44 2.3: Results and discussion. 2.3.1: Dynamics of the biosynthesis of Aspergillus ochraceus polyketide metabolites in liquid substrate fermentations. Results from several D2306 PDB fermentations were combined to give the data shown in figure 2.8. The growth of D2306 in PDB took approximately 3 days. Concurrent with growth was an increase in ochratoxin A concentration, which reached a maximum also by about day 3. Production of ochratoxin A during growth has been observed before (Steyn eta!., 1970 and Wei eta!., 1971). Ochratoxin B production was on a smaller scale than ochratoxin A and reached a peak before maximum ochratoxin A accumulation (i.e. between 48 and 72 hours) although much of the production of ochratoxin B was concurrent with that of ochratoxin A. Inoculating the PDB medium with SW cultures caused cariy over of traces of ochratoxins A and B, which is why concentration values of both are not plotted starting from zero. There was a 24 hour lag between the start of growth and the appearance of diaporthin and orthosporin. There was no temporal separation between the production of these two compounds since initial rates of production were similar until 72 hours, after which orthosporin concentration declined. Diaporthin concentration seemed to peak at 96 hours before its eventual decline. Figure 2.2 shows that orthosponn was the least well resolved compound in the DAD-HPLC system so that later in the D2306 PDB fermentation, when the complexity of the bicarbonate insoluble extracts increased especially with regard to the more polar constituents, concealment of orthosporin in the chromatogram precluded its quantitative analysis. Mellein production occurred late in the D2306 fermentation, when other compounds of interest were in decline. This demonstrates that acetate, previously involved in the biosynthesis of ochratoxins A and B, diaporthin and orthosporin. was being diverted to the biosynthesis of mellein. The data are also consistent with mellein being an 45 0 00 I I-I • 0 ___ "0 .9 a) 0 0 0o ri: . a) 0 0 •G C) 0 I I .4 .' I 0 ,jI 0 0 : 0 'I:: • 0 0 p I- 0 ;.. / 0 a) 00 -- ' / <0 a, \ O F- .fl 00 0) ' H. E . .0 I- 0 0 I \ \ 0 .9 o a, 0 'I' a) 0 'I '4 00 0 a) a)cflQj a,00 00 C •J3 a) I- I a) ' '4 C su IS) IS) C) IS) C'l IS) - IS) 0 c) C..1 0 T&U 46 intermediate of the biosynthesis of the ochratoxins since the demise of ochratoxin biosynthesis would result in the accumulation of polyketide precursors. Mellein might also be an intermediate of orthosporin and/or diaporthin biosynthesis although this is unlikely since they are structurally less siniilar. None of the secondaiy metabolites observed in the time course experiments persisted, except for mellein for which only increased accumulation of end product was measured within this 7 day fermentation. This may have be due to recycling of the metabolites to acetate, a phenomenon which has been observed for the pentaketide citrmin (Barber eta!., 1988) or, alternatively but less likely, to mellein. 2.3.2: Intra- and extraceilular polyketide concentrations versus time for Aspergillus ochraceus D2306 on either potato dextrose broth or yeast extract-sucrose. The course of extra- and intracellular polyketide accumulation in a D2306 PDB fermentation is shown in figures 2.9 and 2.10. Sampling was over a more extended period than in the previous time course experiment in order to investigate mellein production further. Comparing extracellular polyketide production, (cf. figure 2.9 with 2.8) the profiles of diaporthin and mellein are very similar with regard to timing and magnitude. Mellein peaks at approximately 208 hours post inoculation before declining suggesting that mellein is also turned over. Post inoculation assay showed that the inoculating culture was ochratoxinogenic, yet no accumulation of ochratoxins A or B was observed in this experiment. However, a post inoculation occurrence of ochratoxin B (27 tgfL) did not persist. The reason for the absence of significant ochratoxin A production is unclear, yet the phenomenon has been observed before my inpublished data). Ochratoxin a was the only additional compound provisionally identified by TLC and its concentrations mirrored that of extracellular ochratoxin A. Detection of ochratoxin a in A. ochraceus fermentations has never been reported before, although the present identification by 1D TLC should not be considered definitive. Strikingly, there was no corresponding increase in structurally related 47 Figure 2.9: Extracellular ocbratoxin A concentration ([OA]), diaporthin concentration ([Diap]) and mellein concentration ([Mellem]) versus time for an Aspergillus ochraceus D2306 potato dextrose broth fermentation. 4 3.5 3 50 100 150 200 250 300 Time (hours) Figure 2.10: Intracellular ochratoxin A concentration ([OA]), diaporthin concentration ([Diap.]) and mellein concentration ([Mellein]) versus time for an Aspergillus ochraceus D2306 potato dextrose broth fermentation. 60 50 40 3D 20 10 0 0 50 100 150 200 250 300 Time (hours) [OA] -° [Diap.] - [Mellein] 48 polyketides (especially ochratoxin ) nor was there a premature occurrence of mellein associated with the lack of ochratoxin A biosynthesis. Unfortunately, orthosporin was too obscured in the DAD-HPLC chromatogram to be measured quantitatively. Comparing extra- and intracellular polyketide production (c.f. figure 2.9 with 2.10) illustrates that extracellular values are a close reflection of intracellular events. The occurrence of intracellular mellein coincided with its fleeting yet prominent appearance in the fermentation broth indicating that there is not a clandestine intracellular pool of mellein. The significance of the fluctuation of intracellular levels of ochratoxin A (ochratoxins c, and B were not detected) is not known. Perhaps ochratoxin A is being biosynthesized, but much later than previous experiments have shown, or it is an indication of the permeability of ochratoxin A across fungal cellular boundaries. Intra- and extracellular diaporthin concentration also rose in unison, although the maximum intracellular value occurred later than the extracellular one. Further experimentation is required to ascertain the significance of this. The authentic polyketide standards proved to be very stable in the chloroform- methanol extraction treatment with 0.90 mg ochratoxin A (equivalent to 5% degradation), 0.22 mg ochratoxin B (4% degradation), 0.19 mg diaporthin (17% degradation) and 1.35 mg mellein (4% degradation) being recovered. The dynamics of polyketide production in a D2306 YES fermentation is shown in figures 2.11 and 2.12. Once again, despite the inoculum being ochratoxinogenic, no ochratoxin was produced subsequently. However, this time extracellular ochratoxin A concentration decreased and there was a corresponding increase in intracellular ochratoxin A. The intracellular ochratoxin A concentration subsequently decreased. These results are consistent with metabolic turnover of ochratoxin A. Dianorthin was also produced, albeit in much smaller quantities than in previous PDB fermentations, and its dynamics showed a peak at approximately 24 hours post inoculation, earlier than in figure 2.8, but it appears that diaporthin production is linked to growth more in YES than in PDB. Orthosporin, mellein, ochratoxin ( and 0-methyl, methylochratoxin A were not detected. However, ochratoxin a was provisionally fc ( L) Figure 2.11: Extracellular ocbratoxin A concentration ([OA]) and diaporthin concentration ([Diap.]) versus time for an Asoerillus ochraceus D2306 yeast extract- sucrose fermentation. 2 1.8 1.6 1.4 1I 0 50 100 150 200 Time (hours) Figure 2.12: Intracellular ochratoxin A concentration ([OA]) and diaporthin concentration ([Diap.]) versus time for an Aspergillus ochraceus D2306 yeast extract- sucrose fermentation. 4.5 4 3.5 3 2.5 1.5 I 0.5 0 0 50 100 150 200 Time (hours) [OA] 0 iap. 50 identified by TLC only in the extracellular extracts and, similar to the PDB fermentation, its fate mirrored ochratoxin A. Ochratoxrn B was only detected extracellularly and its concentration declined to zero from 0.15 mgfL within the first 48 hours. Although the decline was not linked to an increase in intracellular ochratoxin B, it was evidence for an ochratoxin B turnover mechanism especially as ochratoxin A was not produced. In addition to the production of diaporthin, D2306 also produced aspergillic acid and another compound, which was provisionally identified as either deoxyaspergillic acid or flavacol, concurrently in YES media (see 3.2.11). Both of these substituted diketopiperazine molecules were produced early in the fermentation (from approximately 24 hours), peaking at approximately 72 hours then declining to zero approximately 192 hours post inoculation. 2.3.3: Aspergillus ochraceus D2306 solid state fermentations. The extraction procedure for SW samples does not differentiate intra- and extracellularly located polyketide metabolites. The results for the stationary D2306 SW culture experiments are summarised in table 2.1. Control stationary D2306 SW cultures proved to be very inconsistent at ochratoxin A production. For example, ochratoxin A produced after 10 days at 28 °C ranged from 0 5 to 2 1 mg/g substrate and ochratoxin A produced after 14 days at 25 O( ranged from 0 05 to 3 6 mglg substrate. The potential for D2306 to produce large amounts of ochratoxin A was apparent since the best yields of Lindenfelser and Ceigler (1975) were equalled. Low yields were usually associated with sponilation occurring within 7 days, which is consistent with the previous report that inhibition of sporulation increased ochratoxin A yield (Hesseltine, 1972). The small scale stationary D2306 SW culture experiments showed consistently poor yields of ochratoxin A. Water appears to be a critical factor in the production of ochratoxin A, with most of the cultures producing no ochratoxin A looking arid with 51 Experiment Scale (g) Volume (ml) % Water Temp oC High Day [OA] mg/g ______Humidity - ______ Control 20 500 41) 25 No 14 0.5 Control 20 500 40 25 No 14 3.6 Control 20 500 40 28 No 10 0.5 Control 20 500 40 28 No 10 2.1 Small 1 SO 40 28 No 11 0 Small 1 50 40 28 No 6 0 Small 1 5 40 25 Yes 6 0 Small 1 5 40 25 No 7 0 Small 1 5 60 25 Yes 6 0.31 Small 1 25 40 25 Yes 13 0 Small 2 25 40 25 Yes 13 0 Small 2 50 40 25 Yes 13 0 Small 4 50 40 25 Yes 13 0 Small 1 25 60 25 Yes 10 0.03 Small 2 25 60 25 Yes 4 0.28 Small 2 50 60 25 Yes 10 0.3 Small 4 50 60 25 Yes 4 0.06 Large 1000 10000 30 28 No 22 0.16 Large 50 1000 40 28 No 8 0.13 Table 2.1: Selected data from stationary medium (control), small and large scale A. ochraceus D2306 shredded wheat cultures. 52 no signs of fungal growth. The weight of substrate per volume of vessel ratio also proved to be important, with too low values leading to dehydration and too high values leading to poor aeration. Ratios between 40 and 80 mg substrateJml vessel volume appeared to facilitate higher ochratoxin A yields, especially in addition to higher initial moisture levels and the use of humidity chambers to maintain culture humidity. However, generally stationary solid substrate fermentation gave very poor yields of ochratoxin A. The 1 kg scale D2306 culture appeared to be adequately moist, so perhaps the substrate weight : vessel volume ratio was too high (100 mg substrate per ml flask) and impeded aeration. Despite regular agitation the ochratoxin A yields were poor, a phenomenon noted in the large scale fermentation performed by Lindenfelser and Ciegler (1975) in which a continuous rotation of at least 1 rpm was required to obtain satisfactory ochratoxin A yields. The erratic nature of ochratoxin A biosynthesis by D2306 in 50 and 100 g scale stationary cultures emphasizes unsuitability of the seemingly appropriate growth conditions. In contrast, the control shake flask D2306 SW cultures showed consistent ochratoxin A production leading to astonishingly high values of 8 to 10 mg ochratoxin A /g substrate after 22 days incubation (table 2.2). Ochratoxin B was usually only a minor component, typically less than 10% of the ochratoxin A concentration value. The small scale shaken cultures exhibited problems similar to the stationary ones in terms of dehydration. Cultures on 10 g or less of substrate benefited greatly from increased initial moisture levels and subsequent water additions to produce some excellent ochratoxin A yields exceeding 10 mg/g substrate after 15 days incubation. Generally, 40% initial moisture levels alone avoided dehydration problems although if the culture became too wet, the shreds would aggregate to form balls or even one solid mass of culture resulting in reduced ochratoxin A yields. Another problem encountered was that the dust formed from inter-shred abrasion impacted onto the area of water condensation near the neck of the flask to produce a cake, upto 5 mm thick, which typically contained only 20 to 60% of the ochratoxin A 53 Experiment Scale (g) Volume (ml) % Water LOAI mglg Control 20 500 30 10 1.47 Control 20 500 30 22 8.08 Control 30 500 30 10 1.25 Control 30 500 30 22 10.32 Small 1 25 30 8 0 Small 2 25 30 8 0 Small 2 50 30 8 0 Small 4 50 30 8 0 Small 4 100 30 8 0.1 Small 8 100 30 8 7.7 Small 8 100 30 15 10.81 Small 10 250 30 8 0.03 Small 10 250 30 15 0.06 Small 20 250 30 8 12 Small 20 250 30 15 4.45 Small 20 500 30 8 0.35 Small 20 500 30 15 0.63 Small 40 500 30 8 4.25 Small 40 500 30 15 10.99 Small 1 25 40 8 0 Small 2 25 40 8 3.2 Small 2 50 40 8 0.7 Small 4 50 40 8 6.9 Small 4 100 40 8 7.1 Small 8 100 40 8 6.4 Small 8 100 40 15 7.54 Small 10 250 40 8 4.9 Small 10 250 40 15 9.06 Small 20 250 40 8 5.3 Small 20 250 40 15 1135 Small 20 500 40 8 7.28 Small 20 500 40 15 12.18 Small 40 500 40 8 5.33 Small 40 500 40 15 8.8 Large 160 4000 30 16 6.47 Large 240 4000 30 16 4.72 Large 320 4000 30 12 2.57 Table 2.2: Selected data from shaken medium (control), small and large scale A. ochraceus D2306 shredded wheat cultures. 54 yield in the bulk of the shaken culture. This was especially prevalent within the flasks containing higher substrate content which leads to reduced ochratoxin A yields in the large scale flasks. More research is required to optimise scaling up of the shaken D2306 SW cultures to achieve consistent ochratoxin A yields approaching 10 mglg substrate, but the present experiment clearly demonstrated the superior potential of shaken solid substrate fermentations not only for toxin production but also as an experimental system in which to explore aspects of ochratoxin A biosynthesis. Despite potential decreases in ochratoxin A yields due to the formation of a cake near the neck of the flask, 40 g SW per 500 ml scale cultures with an initial moisture of 40%, in conjunction with further water additions, were used for further experiments since aliquots of colonised shredded wheat could be removed without disturbing the rest of the culture too greatly. 2.3.4: Dynamics of the biosynthesis of Aspergillus ochraceus D2306 polyketide metabolites in solid substrate fermentations. It is impossible to measure fungal cell growth directly within a solid substrate fermentation so a reasonably reliable biochemical marker indicating changes in fungal biomass is required. Such a marker is ergosterol which is the predominant sterol component of most fungi (including Aspergillus ochraceus) yet is either absent from, or is a minor component in, most higher plants (Seitz eta!., 1979) and should not occur in shredded wheat. Although there has been doubt cast on the validity of using ergosterol (Bermingham et a!., 1995), it still remains the most practical measure since the other favoured biochemical marker, glucosamine, is not restricted to fungi and persists after the death of the fungi. Polyketide and ergosterol production by shaken SW D2306 cultures is shown in figure 2.13. Growth, as indicated by ergosterol production, was complete within 8 days. Ochratoxins A and B and diaporthin were produced concurrently with growth. In the context of over production of ochratoxins, diaporthin was 1000 times less 55 Figure 2.13: Ergosterol concentration ([Ergo.]), ochratoxrn A concentration ([OA]), ochratoxin B concentration ([OB]) and diaporthin concentration ([Diap.]) versus time for an Asrergillus ochraceus D2306 shaken shredded wheat fermentation. 14 12 10 I- ti) 0 0 4 2 0 0 2 4 6 8 10 12 14 Time (days) • [Ergo.] mg/1O ° [OA] mg/g • [OB] mgI5 g ° [Diap.] ig/g g 56 abundant than in an equivalent PDB fermentation. Notably, ochratoxin production generally ceased as ergosterol reached a maximum indicating how closely the biosynthesis of these polyketides is linked with growth. Mellein, however, was not produced in these shaken solid substrate fermentations. In liquid fermentations, A. flavus produced approximately 4 mg ergosterol I g air dried ftingal mass and both A. jiavus and A. ochraceus are regarded as being strong producers of ergosterol (Seitz et al., 1979). Based on this evidence, at 8 days post inoculation approximately 30% of the sampled shredded wheat was flingal biomass and of this, 3% was ochratoxin A. TLC showed that i) ochratoxin 13 appeared briefly 3 days post inoculation ii) a compound provisionally identified as 0-methyl, methylochratoxin A was detected between 7 and 14 days post inoculation and iii) ochratoxins A and B as fluorescent metabolites increased as the DAD-HPLC data indicated. Ochratoxin (3 has been found in an A. ochraceus fermentation (Delgadillo, 1986) but 0-methyl, methylochratoxin A has not, although identification by ID TLC should not be considered definitive. To aid identification further, the TLC plate was sprayed with a 3% ferric chloride in butan-1-ol, followed by gentle heating. The blue fluorescent spot with an Rf value similar to 0-methyl, methyl ochratoxin A turned red indicating that it was not 0- methylated. The 0-methylation of the ochratoxin A phenolic hydroxyl group gives a negative ferric chloride reaction (van der Merwe eta!., 1965 b) so the compound was not 0-methyl, methylochratoxin A, although it could have been another ester of ochratoxin A. Standards of ochratoxins a, (3, A and B all gave a positive fernc chloride reaction (turning red, with the most intense colour from ochratoxins A and B) with authentic 0-methyl, methylochratoxin A giving a negative reaction. Ochratoxin A production was comparable with former shaken SW culture experiments (approaching 10 mg/g substrate after 14 days) although ochratoxin B levels were slightly higher than expected (approximately 18% of the ochratoxin A values). There seems to be no evidence of a temporal separation between the biosynthesis of ochratoxins A and B. The dynamics of ochratoxin A biosynthesis 57 shows that an opportune time for the addition of radiolabeiled putative intermediates of ochratoxin A biosynthesis would lie in the 3 to 5 days post inoculation. This model system would provide a better environment for biosynthetic intennediate studies since it is more reliable (with regards to ochratoxin A biosynthesis), the polyketide biosynthesis is predominantly ochratoxin A and it is quite homogeneous. 2.3.5: Effectiveness of shaken shredded wheat culture for other ochratoxinogenic fungi. There was only a marginal difference between the ochratoxin A yields produced in stationary and shaken SW cultures: 8 and 2, 6 and 5 and 26 and 19 j.g/g substrate for A. ochraceus NRRL 3174, P. verrucosum IMI 260915 and RS4 in stationary and shaken SW cultures, respectively. High yields of ochratoxin A have been achieved by shaken P. viridicatum (P. verrucosum) solid substrate fermentations (Hesseltine, 1972) which suggests that special conditions have to be perfected for each organism. Despite reports in the literature that RS4 produces citrinin, citrinin was not detected in any of the cultures. 58 3: Isolation and identification of Aspergillus ochraceus D2306 polyketide and other, metabolites. 3.1: Introduction. There are very few reference compounds commercially available as representatives of the vast array of fungal secondary metabolites. Thus, one is often reliant upon the purification and characterisation of unrecognised compounds isolated from fungi and comparing measured physico-chemical data with that published for potential candidate compounds before identification of known compounds can finnly be made. This approach has been necessary concerning several instances in the present study. 3.2: Materials and methods. 3.2.1: Ochratoxin A. Approximately 2 g of mature D2306 stationary SW culture was extracted to yield bicarbonate soluble and insoluble fractions. Bicarbonate soluble extracts contained ochratoxin A according to TLC using an ochratoxin A standard (figure 2.3). Two preparative HPLC systems were used to purify ochratoxin A. The first used an isocratic mobile phase of water-acetic acid-acetonitrile (59.5:1:39.5) flowing at 5.6 mi/mm through a 300 x 25 mm Dynamax Macro C-18 column. Eluate flowed first into a Perkin-Elmer I000M fluorescence spectrophotometer (fitted with a 340 nm excitation filter and a 400 nm emission filter) and then to a Pye Unicam PU4020 UV detector set at 332 nm. Ochratoxin A had a retention time of approximately 20 minutes. Enriched ochratoxin A fractions collected from this system were dried down and re-injected into the second HPLC system. The second system differed only with regard to the mobile phase which was methanol-water (70:30); ochratoxin A had a retention time of2l minutes. Subsequent DAD-HPLC confirmed the purity of any collected fractions as well as providing UV absorption spectra. Ochratoxin A was 59 confirmed by accurate mass electron impact spectrometly (EI-MS). El-MS was performed on a VG Autospec mass spectrometer with a source temperature of 210- 220 °C and an electron energy of 70 eV. Perfluorokerosene (PFK) was the internal reference and a resolution (MJAM) of approximately 5000 was used. 3.2.2: Ochratoxin B. Bicarbonate soluble extracts of D2306 stationary SW cultures revealed a second compound that was fluorescent on TLC plates under UV light, and also had a UV absorbance spectrum similar to ochratoxin A. Preparative HPLC, as descnbed for ochratoxin A, was employed to purify what was later identified as ochratoxrn B. Ochratoxin B had retention times of 15 and 16 minutes in the first and second HPLC systems, respectively. Ochratoxin B was confinned by accurate mass measurement El-MS. 3.2.3: Ochratoxina. Ochratoxin A (1 mg) was refluxed in 50 ml 6M HC1 in a 100 ml round bottomed flask for 18 hours. After cooling, the reaction mixture was extracted with an equal volume of ethyl acetate which was rotary evaporated to dryness prior to DAD-HPLC analysis (as for ochratoxin A). An unknown compound with a UV absorbance spectrum very similar to ochratoxin A, but having a much shorter retention time, was identified and preparative HPLC (water-acetic acid-acetonitrile, 59.5:1:39.5, flowing at 8.4 mI/mm, 323 nm, retention time 10 minutes) was used to purify it. The pure compound was subjected to El-MS. including linked scan and accurate mass measurement, which revealed it to be ochratoxin a. The linked scan El-MS used He as the collision gas in the first field-free region. Daughter ion (BIE) scans were performed on m/z 256 (M), 238, 223, 212 and 194 to interpret the electron impact fragmentation pattern of the mass spectrum of ochratoxin a. 60 3.2.4: Ochratoxin . Ochratoxin B (1 mg) was treated as for ochratoxin A, above. DAD-HPLC analysis revealed an unknown compound with a UV absorbance spectrum very similar to ochratoxin B and preparative HPLC (water-acetic acid-acetonitrile, 59.5:1:39.5, flowing at 84 mi/mm, retention time 6 minutes) was used to purify the compound. El-MS analyses (accurate mass measurement and linked scan) enabled recognition of ochratoxin 3. B/E scans were performed on m/z 222 (Mt ), 204, 189 and 160 and a parent ion (B2IE) scans were performed on m/z 204 and 189 to interpret the electron impact fragmentation pattern of the mass spectrum of ochratoxin 3. 3.2.5: 0-methyl, methylochratoxin A. Ochratoxin A (1 mg in 1 ml methanol) was methylated by the addition, dropwise, of diazomethane (1 ml of a 60% ethereal solution). Diazomethane was prepared by reacting 0.7 mg 1-methyl-3-nitro-1-nitrosoguanidine with a few drops of 5 M NaOH for 45 minutes. The ensuing diazomethane gas was collected in 1 ml diethyl ether, using a dia.zomethane generator apparatus (Pierce, UK), cooled in ice. The methylation reaction mixture was allowed to evaporate to dryness and the resulting solid was re-dissolved in methanol and analysed by DAD-HPLC. An unknown compound, with a UV absorbance spectrum similar to ochratoxin A but with a longer HPLC retention time, was identified. Preparative HPLC (water-acetic acid- acetonitrile, 59.5:1:39.5, flowing at 8.4 mI/mm 1 ? 209 nm, retention time 37 minutes) was used to purify the compound which was identified by accurate mass measurement El-MS to be 0-methyl, methylochratoxin A (figure 3.1, XXIX). 3.2.6: Diaporthin. Radiolabelling experiments with [1 - 14C] acetate and [methyl- 14C] methionine fed to D2306 PDB cultures showed strong incorporation of these early precursors of ochratoxin A biosynthesis into an unknown compound found in certain bicarbonate insoluble samples as shown by TLC and autoradiography (see 4.2). The unknown 61 C' Figure 3.1: 0-Methyl, methylochratoxin A (XXIX). 62 compound was visualised on a TLC plate as it quenched the fluorescent indicator. Autoradiography involved, under safe light conditions, sandwiching the dried TLC plate with Fuji RX X-ray film between two sheets of glass which was then wrapped in black plastic to make it light proof Exposure lasted from 2 to 4 weeks at -70 °C. 2D TLC and autoradiography led to the purification of a small amount of this labelled unknown compound. For 2D TLC, 10 x 10 cm Polygram SIL G1UV254 TLC plates were used with xylene-methanol-acetic acid (90:5:5) as the mobile phase for the first direction and toluene-ethyl acetate-formic acid (50:40:10) for the second direction. Radioactivity on the TLC plate was confined to the area of the unknown compound, which was scraped off the plate and eluted from the silica with propan-2-ol. Preliminary low resolution El-MS and chemical ionisation mass spectrometry (CI- MS), using ammonia as the reagent gas, showed the M+. of the unknown compound to be 250. The El mass spectrum did not match any of the 42,262 mass spectra contained on the School of Pharmacy mass spectral library (Carter, 1993). To prepare enough unknown compound for further analysis, twenty 100 ml scale flasks ofD2306 PDB culture were incubated for 5 days before being extracted. Preparative HPLC using two solvent systems was used to purify the unknown compound from the bicarbonate insoluble organic fraction. The preparative HPLC system was similar to that used for ochratoxins A and B except detection was only by UV absorbance at wavelength 239 nm. The retention times for the compound were 23 and 15 minutes for the first and second HPLC systems, respectively. 'H NMR, 13C NMR and 13C NMR DEPT analyses in D6 acetone revealed the compound to be diaporthin. NMR spectroscopy was performed on a Bruker NMR spectrometer, at 500 MHz for 1H and at 125.8 MHz for 13C. The 1H NMR results were compared with published diaporthin 1H NMR data and the C NMR results were compared with published 1 C NMR data of orthosporin (Ichihara eta!., 1989). 13C NMR DEPT analysis was used to resolve methyl, methylene, methine and quaternary C signals in the 13C NMR spectrum. In addition, accurate mass measurement and linked scan El- MS analyses were performed on diaporthin. BIE ion scans were achieved for m/z 250 63 (M), 235, 206, 191 and 178 to interpret the electron impact fragmentation pattern of the mass spectrum of diaporthin. Diaporthin was not produced by any other A. ochraceus SW or PDB culture tested. These A. ochraceus isolates included KKb, KB!', KBd (non-ochratoxinogenic strains isolated from American Kidney and Butter beans by Mantle and McHugh, 1993), 19DB' (a non-ochratoxinogenic strain isolated from Bulgaria by Mantle and McHugh, 1993) and NRRL 3174. Approximately 2 g of SW culture of each isolate was extracted and the bicarbonate soluble and insoluble fractions analysed by DAD- HPLC. Ochratoxins A and B were only found in a control sample of isolate D2306. The SW cultures were used to inoculate corresponding PDB shake flask cultures which were incubated for 1 week prior to the standard extraction procedure and DAD-HPLC analysis. Ochratoxin A was detected in NRRL 3174 SW cultures and ochratoxins A and B and diaporthin were found in D2306 extracts. Although the A. ochraceus isolate NRRL 3174 has been reported to produce ochratoxins A and B, mellein and 4-hydroxymellein, only mellein and a hydroxymellein were positively identified in NRRL 3174 PDB cultures. 3.2.7: Orthosporin. DAD-HPLC of some D2306 PDB bicarbonate insoluble samples showed a chromatographic peak which had a UV absorbance spectrum identical to that of diaporthin, but it had a much shorter retention time. Twenty flasks of 100 ml scale PDB medium were inoculated with D2306 spores and incubated for 72 hours before being extracted in the usual way. The diaporthinesque compound was purified by preparative HPLC using two solvent systems in the same way as diaporthin. Orthosporin had retention times of 14 and 13 minutes in the first and second HPLC systems, respectively. The purified compound was subjected to accurate El-MS which matched with the published El-MS data of orthosporin (Hallock eta!., 1988) and was used in conjunction with the diaporthin data to interpret the fragmentation pattern of orthosporin in El-MS. 64 3.2.8: Mellein. The search for diaporthin in other A. ochraceus isolates led to the recognition of a bicarbonate insoluble compound common to both D2306 and KBf when grown in PDB. However, the KBf culture in PDB produced much more of the compound than D2306. Thus, a I week old KBfPDB flask was extracted and the bicarbonate insoluble fraction was subjected to preparative HPLC (water-acetic acid-acetonitrile, 59.5:1:39.5, flowing at 5.6 mI/mm, 239 nm, retention time 30 minutes). The eluate collected corresponding to the unknown compound was doubled in volume with water and purified fi.irther by solid-phase extraction. A C 18 Sep-Pak Classic cartridge (Milhipore, Watford, UK) was pre-equilibrated with 20 ml of methanol then 20 ml of water. The sample was then introduced, followed by a 20 ml water wash. The unknown compound was eluted with ethanol, and corresponded to the 2nd and 3rd 0.5 ml fraction collected. The purity of the compound was checked by DAD-ITPLC. The nominal El mass spectrum of the compound matched with the VG Autospec mass spectral library El mass spectrum of mellein and accurate mass measurement El- MS confirmed this. BIE ion scans of m/z 178 (M), 160, 149 and 134 provided rationalisation of the fragmentation pattern of mellein in El-MS. 3.2.9: Hydroxymellein. The search for diaporthin in other A. ochraceus isolates led to the recognition of a bicarbonate insoluble compound similar to mellein, but with a shorter DAD-HPLC retention time, produced by A. ochraceus NRRL 3174 in PDB. The bicarbonate insoluble extraction from a week old NRRL 3174 PDB culture was subjected to preparative HPLC (initially water-acetic acid-acetonitrile, 59.5:1:39.5, flowing at 8.4 mi/mm, ? 239 nm, was used to enrich the compound, retention time 9 minutes, then 70% aqueous methanol flowing at 5.6 mI/mm, X 239 nm, was used for the final purification, retention time 12 minutes). The purified compound was analysed by accurate mass measurement and linked scan (BIE ion scans of m/z 194 (Mt ), 176 and 150) El-MS. 65 3.2.10: Cycloechinulin. Stationaiy D2306 SW cultures did not produce diaporthin but instead produced a large amount of another compound, present also in D2306 PDB cultures, which was likewise bicarbonate insoluble. One stationary 20 g scale D2306 SW culture was extracted and the bicarbonate insoluble fraction was subjected to preparative HPLC (water-acetic acid-acetonitrile, 59.5:1:39.5, flowing at 5.6 mI/mm, 235 nm, retention time 16 minutes). The eluate corresponding to the unknown compound was collected and needed no further purification prior to accurate mass measurement EL- MS analysis. The compound was identified as cycloechinulin on the basis of a strong correlation between its El-MS data and the published cycloechinulin El-MS data (De Guzman eta!., 1992). 3.2.11: Aspergillic acids. A. ochraceus D2306 grown on YES medium produced two compounds with similar UV absorbance spectra yet having different bicarbonate solubility (see 2.2.2). Two 100 ml scale D2306 YES cultures 48 and 96 hours post inoculation, were extracted and the bicarbonate soluble fractions combined. Preparative HPLC purified the unknown acid (water-acetic acid-acetonitrile, 59.5:1:39.5, flowing at 8.4 mI/mm, X 325 nm, retention time 15.5 minutes). The bicarbonate insoluble fractions of the 72 and 96 hour post inoculation 100 ml scale D2306 YES culture were also combined and preparative FIPLC was used to puri1r the unknown neutral compound (water- acetic acid-acetonitrile, 59.5:1:39.5, flowing at 8.4 mI/mm, 325 nm, retention time 14.5 minutes). Accurate mass measurement El-MS revealed the unknown acid to be aspergillic acid and the unknown neutral compound to be 16 mass units lighter which would correspond either to deoxyaspergillic acid or flavacol. Aspergillic acid and the neutral compound were analysed in a Optical Activity AA-10 polarimeter (approximately 0.4 mg of each compound was dissolved in 10 ml HPLC grade methanol). 66 3.3: Results and Discussion. 3.3.1: The A. ochraceus polyketide UV absorption spectra. The UV absorption spectra for the ochratoxins (A, B, a, 3 and A 0-methyl, methyl derivative), diaporthin, orthosporin, mellein and hydroxymellein are shown in figure 3.2. All of the spectra were measured in the DAD-I-IPLC solvent (water-acetic acid- acetonitrile, 59.5:1:39.5) which makes comparisons with published data in the chemical literature inadvisable. The UV absorption spectra for ochratoxins A and B are very similar except for the general shift of the second UV absorbance maximum to a lower wavelength for ochratoxin B (the two UV absorbance maxima being at 218 and 332 nm and 222 and 318 nm for ochratoxins A and B, respectively). This shift was noted by Cole and Cox (1981) and is also reflected in the ochratoxins a and J3 UV absorbance spectra where the two maxima are 222 and 334 nm and 230 and 322 nm for ochratoxins a and 3, respectively. Methylation of ochratoxin A drastically altered its UV absorption, more significantly than removing the phenylalanine moiety, resulting in a more squashed spectrum (UV absorbance maxima at 241 and 309 nm). The UV absorption spectra of diaporthin and orthosporin are almost identical, with both sharing the maxima of 244 and 237 nm. The UV spectra of ochratoxins a and 3, 0-methyl, methylochratoxin A, orthosporin and diaporthin have never been published. The spectra of mellein and hydroxymellein are very similar above 230 nm with two maxima being shared (246 and 313 mm). However, below 230 mn, hydroxymellein has an enhanced UV absorbance maximum of216 nm compared to the much reduced 222 mm maximum of mellein. Cole and Cox (1981) and Cole et aL (1971) show the UV spectra of mellein and 4-hydroxymellein to be essentially identical. 3.3.2: Ochratoxins A and B. The El mass spectrum of [35C11 ochratoxin A showed the most intense peaks at ni/z 239 and 255 with the corresponding 37C1 isotope giving peaks at m/z 241 and 257 (figure 3.3). This is in agreement with Phillips eta!. (1983) and Gallagher and Stahr 67 1 Ochratoxin A 4 Ochratoxln beta MU MU 140 700 eo 40 400 JL 1200 000 eoo eco 400 'I \ 0 30 300 400 5(0 Figure 3.2: UV absorbance spectra of ochratoxins A, B, a and J3, 0-methyl, methylocbratoxin A (OAMe2), diaporthin, orthosporin, mellein and hydroxymellein. 68 m/z Formula 403 C20H13N06C1 385 C20H16N05C1 359 C19H18N04C1 255 C11H10N04C1 239 C11H804C1 Figure 3.3: Accurate mass electron impact spectrum of ochratoxin A. 69 (1981) who proposed that previous ochratoxin A El mass spectra giving the main peaks as 240/242 and 256/25 8 were incorrect. One such deficient publication is Cole and Cox (1981) but the error is also in the VG Autospec mass spectral library. Ochratoxin B exhibits an El mass spectrum analogous to that of ochratoxin A but without the chlorine isotope pattern and shifted down 34 mass units (-Cl, +H; figure 3.4). Once again, this agrees with Gallagher and Stahr (1981) rather than Cole and Cox (1981), but this time does agree with the VG Autospec mass spectral library. Accurate mass measurement fragmentations have not been published for either ochratoxins A or B. The predominant El-MS fragmentation ions of ochratoxins A and B were thought to arise from the loss of the phenylalanine moiety via cleavage of a N-C bond either side of the N atom in the amide bond to yield the C 11H11(10)N04(Cl), m/z 221 (255/257), and the C11H9(8)N04(C1). m/z 205 (239/241), fragments (Gallagher and Stahr, 1981). This is in complete agreement with the measured El-MS data. 3.3.3: Ochratoxina. Ochratoxin a has never been found as a metabolite of A. ochraceus, only as a product of ochratoxin A chemical degradation (Steyn and Holzapfel, 1967 a; Searcy eta!., 1969; Steyn eta!., 1970). The DAD-HPLC chromatogram of ochratoxin a is shown in figure 3.5. The compound has a very short retention time and is usually obscured by other polar constituents of bicarbonate soluble extracts. Hence, TLC is the best method of ochratoxin a detection, albeit qualitatively. The accurate mass spectrum of ochratoxin a is shown in figure 3.6. Neither the accurate or nominal mass spectrum has been published, although the first four fragmentation ions (m/z 256 [Mt ], 238, 223 and 212) matched with the data of Delgadillo (1986). There are two signals at m/z 194: 193.9767 is the major signal and corresponds to C 9H3O3C1, whereas 194.0 137 corresponds to C10H702C1 and has only 10% of the former's relative abundance. Linked scan El-MS showed that mlz 256 [M] fragmented to 238, 223, 212 and 193.9767, m/z 238 fragmented to 223 and 193.9767, m/z 223 had no clear 70 m/z Formula 369 C20H19N06 351 C20H17N05 325 C19H19N04 221 C11H11N04 205 C11H904 Figure 3.4: Accurate mass electron impact spectrum of ochratoxin B. 71 DADI A, Sig=332,4 Ref=450,80 of HARRIS)AO501 .D iMU- 200O- )chratoxmalpha 1500- 1000- ___ 1 1'5 DADI A, S1g332,4 Ref450,80 of HARRSOA0502.D mAU. chratoxin beta 2000- 1500- 1000- ___ DAD1 A, Sig310,4 Ref=450,80 of HARRIS\0A0444.D inAU- OAMe2 2000- 1500- 1 - I I • o 5 10 15 ml Figure 3.5: DAD-HPLC chromatograms of ochratoxins a and 3 and 0-methyl, methylochratoxin A (OAMe2). 72 m/z Formula 256 C11H905C1 238 C11H704C1 223 C10H404C1 212 C10H903C1 194 C9H303C1 194 C10H702C1 166 C9H7OC1 149 C8HSO3 138 C7HC1 Figure 3.6: Accurate mass electron impact spectrum of ochratoxin a. 73 daughter ions, mlz 212 fragmented to 194.0137, 166 and 149 and m/z 193.9767 fragmented to 166 and 138. The fragmentation pattern of ochratoxin a in El-MS is summarised in figure 3.7. The mlz 256 [M] loses 44 (CO2) to become 212 (C 10H903C1) which is 5-chioromellein. The effect of the chlorine atom upon the fragmentation is illustrated by the daughter ions of m/z 212. Only one of the three measured daughter ions is analogous to mellein (194.0137 = 160.0525 + 34) compared to all three for ochratoxin 13(134, 149 and 160.0525). 3.3.4: Ochratoxin (3. The DAD-HPLC chromatogram of ochratoxin (3 is shown in figure 3.5. Ochratoxin (3 has a shorter retention time than ochratoxin a and is similarly obscured. The accurate mass measurement El mass spectrum is shown in figure 3.8. Ochratoxin (3 exhibits an El mass spectrum analogous to that of ochratoxin a but without the chlorine isotope pattern and shifted down 34 mass units (rather like the El mass spectra of ochratoxins A and B). There are two signals at m/z 160: the major 160.0 144 corresponds to C9H403 whereas 160.0525 corresponds to C 10H80 and has a relative abundance of approximately lO% of the former. These are analogous to the m/z 194 signals in the ochratoxin a El mass spectrum. Neither the accurate nor nominal mass spectrum has been published, although the first five fragmentation ions (222 [M], 204, 189, 178 and 160) matched with ochratoxin (3 El mass spectral peaks published by Delgadillo (1986). BIE linked scan El-MS showed that m/z 222 [M] fragmented to 178, m/z 204 fragmented to 160.0144, m/z 189 to 105 and m/z 160.0144 to 105 and 115. The fragmentation pattern of ochratoxin (3 in El-MS is summarised in figure 3.9 and is very similar to, if less complete than, ochratoxin a (figure 3.6). B2IE scans showed that, although m/z 189 was weakly linked to 222, 204 was not, which suggests that the water loss was a thermal rather than an electron impact effect. The ochratoxin a linked scan analysis, however, showed strong linkage between the M and the daughter ions that had lost H20 or CH5O. 74 256 (C 1 1H9O5CI 212 H5O 238 (C10H903C1) I (C11H704C1) (Chioro) mellein fragments 223 (C10H4O4C1) 194 m1z 149, 166, 194? (C9H303C1) /\ 138 166 Figure 3.7: Fragmentation pattern of ochratoxin a in electron impact mass spectrometry. 75 mlz Formula 222 C11H1005 204 C11H804 189 C1aH5O4 178 C10H1003 160 C9H403 160 C10H802 149 C8HSO3 134 C8H602 Figure 3.8: Accurate mass electron impact spectrum of ochratoxin f. 76 222 (C11H1005) 178 LI LI 2U4 (C 10H1003) (C 1 111804) 189 (C 1011504) QH4O 160 Mellein fragments m/z: 134, 149, 160? (C9H403) 105 /\ 105 115 Figure 3.9: Fragmentation pattern of ochratoxin 3 in electron impact mass spectrometry. 77 Since the 178 fragment is equivalent to mellein (C 1 H1003) and the accurate mass measurement of m/z 134, 149 and 160.0525 corresponded to the mellein El-MS fragments C8H602, C8HSO3 and C 10H802, respectively, an El-MS fragmentation leading to the m/z 178 ion of mellein was assumed. 3.3.5: 0-Methyl, methylochratoxin A. The DAD-HPLC chromatograin of 0-methyl, methylochratoxin A is shown in figure 3.5 and, as expected, it has a much longer retention time than the more polar ochratoxin A. Ochratoxin A has previously been shown to react readily with diazomethane to produce the 0-methyl, methyl derivative (van der Merwe et aL, 1965 b; Phillips eta!., 1983) with methylochratoxin A apparently never being a by- product. The accurate mass measurement El mass spectrum of 0-methyl, methylochratoxin A (figure 3.10) is very similar to ochratoxin A with there being many analogous fragmentation ions, for example: m/z 239, 253 (239 + 14), 269 (255 + 14) and the M 431(403 (M) + 14 + 14). The 0-methyl, methylochratoxin A accurate mass measurement El mass spectrum matched very well with the nominal one published by Phillips eta!. (1983). 3.3.6: Diaporthin. Neither nominal nor accurate mass data for diaporthin (figure 3.11) has been published. The El mass spectrum of diaporthin was, however, analogous to that of orthosporin (figure 3.16). The main difference between them was a shift up by 14 mass units (+CH3, -H). The linked scan El-MS of diaporthin indicated a complex fragmentation pattern (figure 3.12). The ion m/z 250 (M t ) fragmented directly to m/z 206 but not to 235, suggesting that CH3 loss may have been a thermal effect. The 1H NMR spectrum of diaporthin is shown in figure 3 13. Table 3.1 shows this 1H NMIR data compared with published data of diaporthin that had been chemically synthesized by treating orthosporin with diazomethane (Ichihara eta!., 1989). The Ichihara eta!. (1989) diaporthin data was from a sample in an unnamed solvent run in a 100 MHz 78 m/z Formula 431 C22H22N06C1 269 C12H12N04C1 253 C12H1004C1 239 C11H804C1 Figure 3.10: Accurate mass electron impact spectrum of 0-methyl, methylochratoxin A. 79 m/z Formula 250 C13H1405 235 C12H1105 206 C11H1004 191 C10H704 188 C11H803 178 C10H1003 177 C10H.903 163 C9H703 160 C10H802 149 C9H902 135 C8H702 Figure 3.11: Accurate mass electron impact spectrum of diaporthin. 80 250 (C 13H1405) 40 3 ______CO ______HO 178 206 235 (C 10H1003) (CHO) (C12H1105) 177 188 1OH3o jCH3 (C11H803)) 160 163 (C9H7O'I— 191 ICHO (C QH7O4) 149 (C9H902) 1C2o2 135 (C 811702) Figure 3.12: Fragmentation pattern of diaporthin in electron impact mass spectrometry. 81 : I I 82 -a .- (_ 0 9 ¶ —o\ o\ '0 1 — — N 'r rn—. 'r c, '0'N c'i ei — — I I I cq I ,— -a -a -a-a -a '_, ' ' , e'S Cl C 0flOO c . o Q% O —e1 c- '0 OOIrNoO — — I I I I .-' .-' -a.-' -a.-' - — N 0 00 o0enN 00 C1C (O —c C O0 83 instrument which was unable to resolve the two unequivalent C-9 protons. Unlike the Ichihara eta!. (1989) data, the diaporthin 1H NMR data showed no signals for the C- 10 and the C-8 OH protons. It is very difficult to visualise hydroxyl protons as they will exchange with 2H2O. 2H20 results from there being 1H20 in the sample which has exchanged H atoms with the D 6 acetone. 1H11H decoupling of the signal at ö 1.23 simplified the ö 4.16 region signals and 1H11H decoupling at 64.16 simplified the signals at both 6 1.23 and 62.6. Finally, 1H11H decoupling at 62.6 only simplified the 6 4.16 region of the 1H NMR spectrum. These results are consistent with the assignments given. The aromatic protons at C-5 and C-7 are meta coupled, and correspond to signals at 6 6.45 and 6.52, although it is very difficult to determine which is which. Ichihara et al. (1989) had assigned the three diaporthin aromatic protons as the C-7 proton having the lowest field strength and C-4 having the highest. The 1H NMR results of diaporthin contradict this by showing C-5/C-7 to have the lowest and highest field strengths. The 13C NMR spectrum of diaporthin (figure 3.14) has never been published, however, it can be compared with 13C NMR data of orthosporin (Ichihara eta!., 1989) as shown in table 3.2. There is a great deal of similarity between the 13C NMR spectra of both compounds although orthosporin does not have the signal for C-12. 13C NIMR DEPT experiment data (figure 3.15 and table 3.3) corresponds with the assignments of the diaporthin 13C NMR signals from comparison with the orthosporin 13C NMR data. There was a small signal at 6 56.23 in the CH enhanced spectrum of the 13C NMR DEPT experiment which was regarded as spurious since the relative intensity compared with 6 65.45 in this spectrum had altered from the original 13C NMR spectrum and from the CHICH 3 +ve, CH2 -ye spectrum of the 13C NMR DEPT experiment. Diaporthin production has never previously been reported in any Aspergillus or Penicillium species. Diaporthin is regarded as being a phytotoxin and its production has only been reported from plant pathogenic fungi (Bazzigher, 1953; Boiler eta!., 1957; Hardegger eta!., 1966; Hallock et a!., 1988). A. ochraceus D2306 appears 84 ft 0 C.) 0 I- I- 85 Orthosporin A. ochraceus diaporthin Assignment o Assignment 23.6 11-CH3 23.61 11-CH3 43.9 9-CH2 43.86 9-CH2 56.23 1 2-OCH3 65.5 10-CH 65.45 10-CH 99.7 8a-C 100.62 8a-C 102.2 7-CH 100.93 7-CH 103.2 5-CH 101.86 5-CH 106.3 4-CH 106.48 4-CH 140.8 4a-C 140.75 4a-C 156.3 3-C 156.71 3-C 164.5 8-C 164.23 8-C 166.2 6-C 167.05 6-C 166.9 1-C 167.88 1-C Table 3 2: 13C NMR data of orthosponn (Ichihara eta!, 1989) compared with that of A. ochraceus diaporthin. o CHICH3 +ve CH2 -ye CH enhanced Assignment 23.61 + CH3 43.86 + CH2 56.23 + '/2 OCH3? 65.45 + + CH 100.62 C 100.93 + + CH 101.86 + + CH 106.48 + + CH 140.75 C 156.71 C 164.23 C 167.05 C 167.88 C Table 3.3: 13C NMR DEPT analysis of A. ochraceus diaporthin. 86 1: I I 87 only to produce diaporthin in PDB, and to a much lesser extent in SW shake flask cultures. 3.3.7: Orthosporin. An accurate mass data for orthosporin (figure 3.16) has never been published but the nominal El mass spectrum of the present orthosporin compared very favourably with published data (Hallock eta!., 1988). Since diaporthin and orthosporin are so similar, a fragmentation pattern of orthosporin in El-MS was constructed without the benefit of linked scans (figure 3.17). There is one spurious signal at m/z 222 which appears to be the expected 221 signal but a proton in excess. All of the fragmentations associated with m/z 222 are consistent with the diaporthin model but one mass unit greater. It can be envisaged that this fragment is derived from a loss of CH2 from the molecular ion by a thermal effect (the equivalent loss in the diaporthin model is CH 3). The ion m/z 222 fragments to m/z 192 by a CH 2O loss (the equivalent loss in the diaporthin model is CHO) and fragments to mlz 177 by a loss of C 2HSO (the equivalent loss in the diaporthin model is C2H40). Hallock eta!. (1988) proposed a more limited scheme of orthosporin El mass spectral fragmentation which agreed very well with figure 3.16. The major differences between the two schemes were that the Hallock et a!. (1988) version omitted ions of m/z 222, 177, 174 and 121 and included the mlz 150 as a direct fragment of m/z 192 through a loss of CH 2CO. The accurate mass data of orthosporin showed that the fragmentation losses proposed by Hallock et a!. (1988) and common to figure 3.17 were all correct. Orthosporin production has never been reported in any Aspergillus or Peniciiium species. Orthosporin is regarded as being a phytotoxin and its production has only been reported from plant pathogenic fungi (Hallock eta!., 1988; Ichihara et a!., 1989). A. ochraceus D2306 appears only to produce orthosporin in PDB shake flask cultures. As orthosporin and diaporthin are so structurally similar, the 0-methylation of orthosponn could easily be envisaged as the last step of diaporthin biosynthesis. 88 mlz Formula 236 C12H1205 222 C11H1005 192 C10H804 177 C9HSO4 174 C10H603 164 C9H803 163 C9H703 121 C7HSO2 Figure 3.16: Accurate mass eLectron impact spectrum of orthosporin. 89 236 (C 12H1205) 02H40 % H2 C, (H2O 164 192 --- 222 (C9H803) (C 1oH8O4) (C 11H1005) N 163 I I 174 (C9H703) JC 3 (C10H603) ) 177 C-2HSO (C9HSO4) 121 (C7HSO2) Figure 3.17: Reasoned fragmentation pattern of orthosporin in electron impact mass spectrometry. 90 3.3.8: Mellein. The nominal El mass spectrum of mellein matched the VG Autospec mass spectral library data although both Cole and Cox (1981) and Delgadillo (1986) quoted the m/z 160 peak as at 161. The accurate mass data of mellein (figure 3.18) has never been published. BIE linked scan of mellein showed that m/z 178 (M) fragmented to 134, 149 and 160, m/z 160 fragmented to 132, m/z 149 to 121 and m/z 134 to 106. The fragmentation pattern of mellein in El-MS is summarised in figure 3.19. 3.3.9: Hydroxymellein. The DAD-HPLC chromatogram of hydroxymellein is shown in figure 3.20. The retention time is very short, leading to the obscurity problems suffered by ochratoxins a and and orthosponn. The accurate mass data of hydroxymellein is shown in figure 3.21. AVG Autospec spectral library search revealed strong similarities between the El mass spectrum obtained and those of 3-hydroxymellein and 6-hydroxymellein (figure 3.22). 6-Hydroxymellein has never been found in fungal fermentations, whereas 3-hydroxymellein was isolated from cultures of A. oniki (Sasaki eta!., 1970). The El mass spectrum was markedly different from the VG Autospec spectral library data and the Cole and Cox (1981) spectrum for 4-hydroxymellein (figure 3.23). However, Delgadillo (1986) isolated 4-hydroxymeilein from cultures of A. ochraceus NRRL 3174 and quoted the three major fragmentation ions as m/z 194 (M), 150 and 121. 4-Hydroxymellein has also been isolated from NRRL 3174 by Cole et a!. (1971) and Moore eta!. (1972). BIE ion linked scans showed that m/z 194 fragmented to 176, 150, 121 and 122, m/z 176 fragmented very wealdy to 161 and 134 and m/z 150 to 122. The fragmentation pattern of hydroxymellein in El-MS is summarised in figure 3.24. Cole eta!. (1971) deduced that the 44 mass unit loss from m/z 194 (M) to 150 was due to cleavage of the C-1/O-2 and C-3/C-4 bonds to give a fragment that was C2H40 deficient. This fragment then loses a further 28 mass units (the CO group at C-i) to give the 91 m/z Formula 178 C10H1003 160 C10H802 149 C8HSO3 134 C8H602 132 C9J180 121 C7HSO2 106 C7H60 Figure 3.18: Accurate mass electron impact spectrum of mellein. 92 178 (C10H10C 134 149 160 (C8H602) (C8HSO3) (C10H802) Co co Co 106 121 132 (C7H60) (C7HSO2) (C9H80) Figure 3.19: Fragmentation pattern of mellein in electron impact mass spectrometry. 93 Figure 3.20: DAD-HPLC chromatograzns of hydroxymellein, aspergilhic acid and deoxyaspergillic acid. 94 m/z Formula 194 C10H1004 176 C10H803 150 C8H603 122 C7H602 121 C7HSO2 Figure 3 2 1: Accurate mass electron impact spectrum of hydroxymellein. 95 a) b) Figure 3.22: VG Autospec mass spectral library mass spectrum of a) 3- hydroxymellein and b) 6-hydroxymellein. 96 a) b) I,' z III z w > - MASS TO CHARGE RATIO Figure 3.23: a) VG Autospec mass spectral library mass spectrum and b) Cole and Cox (1981) mass spectrum of 4-hydroxymellein. 97 C ioH1o0 (194) s*b C8H603 C 10H803 (150) (176) CJ-f5O C31-/402 C7H602 (122) C7HSO2 (12!) mi'z Formula 194 C10H1004 176 C10H803 150 C8H603 122 C7H602 121 C7HSO2 Figure 3.24: Fragmentation pattern of hydroxymellein in electron impact mass spectrometry. 98 fragment ion mfz 122. This fragmentation pathway is apparent in mellein El-MS (figure 3.19). Thus, in the case of 3-hydroxymellein, the initial cleavage would result in a loss of 60 mass units (C2H402) to give a fragment ion m/z 134. The second cleavage would be identical to that of mellein, with a 28 mass unit loss of CO to 106. Figure 3.21 clearly shows this is not the case which suggests that the hydroxyl group is either in the 4 or 6 position. However, figure 3.22 shows that, according to the VG Autospec mass spectral library, 3-hydroxymellem does not follow this fragmentation paftern predicted by Cole eta!. (1971). Without further investigation or authentic standards, it is very difficult to determine the exact position of the hydroxyl group in the presently isolated A. ochraceus metabolite. 3.3.10: Cycloechinulin. Cycloechinulin (figure 3.25, XXX) was first isolated from the sclerotia of A. ochraceus and was structurally determined by El-MS and NMR analyses (De Guzman eta!., 1992). The nominal El mass spectrum of cycloechinulin compared very well with the published data, with all thirteen fragments corresponding. The accurate mass data of cycloechinulin (figure 3.26) has never been published. The UV absorbance spectrum of cycloechinulin is shown in figure 3.27. Cycloechinulin is not a polyketide but a diketopiperazine metabolite derived from tryptophan, alanine and an isoprene subunit. 3.3.11: Aspergillic acids The El mass spectrum of an acidic metabolite of A. ochraceus (figure 3.28) matched veiy well with the VG Autospec mass spectral library data for aspergillic acid (figure 3.25, XXXI) which was first isolated from the culture filtrate of anAspergillusfiavus fermentation and was shown to have antibacterial activity (White and Hill, 1942). The acidity was due not to a carboxylic acid but to a hydroxamic acid group (- N(OH)C(0)-). Dutcher (1947) partially determined the structure and recorded the UV absorbance maxima to be approximately 230 and 325 nm (in ethanol). The DAD- 99 H K>-CHTCH3 H H3CO CH3CH2CH(CH3)—'LL0 OH XXXI CH3CH2}CH3 H XXXII (CH3)2CHCH2__ç H XXXffl Figure 3.25: The structures of cycloechinulin (XXX), aspergillic acid (XXX!), deoxyaspergillic acid (XXXII) and flavacol (XXXIII). * Denotes a chiral carbon. 100 m/z Formula 351 C20H21N303 336 C19H18N303 308 C18H18N302 296 C16H14N303 293 C18H17N202 280 C17H16N202 265 C16H13N202 252 C16H16N20 251 C16H15N20 237 C15H13N20 225 C13H9N202 222 C14H10N20 197 C12H9N20 Figure 3.26: Accurate mass electron impact spectrum of cycloechinulin. 101 Figure 3.27: UV absorbance spectra of cycloechinulin, aspergiffic acid and deoxyaspergillic acid. 102 m/z Formula 224 C12H20N202 207 C12H19N20 193 C11H17N20 182 C9H14N202 166 C9H14N20 153 C8H13N20 137 C8H13N2 123 C6H7N20 Figure 3.28: Accurate mass electron impact spectrum of aspergillic acid. 103 HPLC chromatogram and UV absorbance spectrum of presently isolated aspergilhic acid (figures 3.20 and 3.27, respectively) show the UV absorbance maxima at 238 and 325 nm. One of the ahiphatic side chains of aspergilhic acid is a secondary butyl with a chiral C atom giving it an optical activity of [a]D25 +12 (±3) in ethanol (Dutcher, 1947). The unknown acid, as measured in a polarimeter, gave no optical activity, which may be an indication of a lack of material rather than a lack of chiral centre. Aspergillic acid is believed to be biosynthesised from leucine and isoleucine (MacDonald, 1961). Aspergihlic acid can easily be reduced by either hydriodic acid in acetic acid solution or by treatment with hydra.zine at elevated temperatures to yield deoxyaspergillic acid (Dutcher, 1947). Deoxyaspergillic acid (figure 3.25, XXXII) has a similar UV absorbance spectrum to aspergilhic acid, but it has never been isolated from flingal fermentations. However, Micetich and MacDonald (1964) observed the specific incorporation of radiolabelled deoxyaspergillic acid into aspergiliic acid by washed mycelia of Aspergillus scierotiorum and thus concluded that deoxyaspergilhic acid may be an advanced intermediate in aspergillic acid biosynthesis. Flavacol (figure 3.25, XXXIII) is an isomer of deoxaspergillic acid and was first isolated from the culture filtrate of an A. flavus fermentation (Dunn eta!., 1949), and then later from an A. ochraceus culture (Yamazaki et a!., 1972). Its structure was determined after chemical synthesis (Dunn eta!., 1949) and showed it to have no chiral centres. Micetich and MacDonald (1965) made the observation that radiolabelled flavacol was specifically incorporated into neoaspergiffic acid (figure 3.29, XXXIV) but also neohydroxyaspergihlic acid (figure 3.29, XXXV) when fed to cultures of A. scierotiorum. It was concluded that flavacol was probably an immediate precursor of neoaspergillic acid, which was itself an immediate precursor of neohydroxyaspergillic acid. The unknown neutral compound had a UV absorbance spectrum almost identical to aspergillic acid (figure 3.20) and its accurate mass spectrum (figure 3.30) was also 104 N Jr —CH2CH(CH3)2 (CH3>CHCH2__lLNAo OH XXXIV CH2CH(CH3 (CH3CHCH(O OH XXXV Figure 3.29: The structures of neoaspergilhic acid (XXX1V) and neohydroxyaspergiliic acid (XXXV). 105 m/z Formula 208 C12H20N20 193 C11H17N20 166 C9H14N20 123 C6H7N20 Figure 3.30: Accurate mass electron impact spectrum of deoxyaspergilhic acid. 106 similar to aspergillic acid, there being many fragmentation ions either shared or analogous, for example: m/z 123, 166, 193 and 208 (= 224-16). Unfortunately, the El mass spectra of deoxyaspergillic acid and flavacol have not been published. The unknown neutral compound also showed no optical activity which also may be an indication of the lack of material. Although the unknown neutral compound is likely to be deoxyaspergillic acid, further experimentation is required for positive identification. 107 4: Feeding radiolabelled precursors of polyketide biosynthesis to, and the effect of ethionine on, Aspergillus ochraceus D2306 potato dextrose broth fermentations. 4.1: Introduction. The feeding of radiolabelled precursors of polyketide biosynthesis to ochratoxinogenic cultures can be used to identify temporal separation of steps in the biosynthesis of the dihydroisocoumarin moiety of ochratoxin A, and thus elucidate the biosynthetic pathway of ochratoxin A fi.irther. Ethionine is a methylation inhibitor which can be added to ochratoxinogenic cultures and, in conjunction with the radiolabel studies, be used to charactense the methylation events in ochratoxin A biosynthesis. 4.2: Materials and methods. The first experiment, in which radioactive precursors of polyketide biosynthesis were used, involved five 100 ml scale culture flasks. [1-' 4C] Acetate (3.7 RCi) was added to a flask 32, 50 or 72 hours post inoculation, whilst [methyl- 14C] methionine (2 pCi) was added to other flasks 32 or 72 hours post inoculation. All flasks were incubated until 168 hours post inoculation, when they were extracted in the usual manner (see section 2.2.1). TLC and autoradiography of the bicarbonate soluble and insoluble extract fractions revealed a neutral compound which was heavily labelled by both radiolabelled precursors and was later identified as diaporthin (see section 3.2.6). A portion (10-25%) of each of the extracts was injected into the DAD-HPLC system and the eluate corresponding either to ochratoxin A or diaporthin was collected in scintillation tubes. Concentration and purity of the collected samples were calculated and assessed, respectively, using the appropriate ChemStation programmes and their radioactivity was measured by scintifiation counting. Scintillation counting involved 108 adding to the samples in the scintillation tubes 8 ml of EcoLume scintillant (ICN Biomedicals, California, USA) and then counting each sample three times for 1 minute in an Intertechnique SL 30 liquid scintillation counter with a measured counting efficiency of the order of 9O%. Scintillation counting was repeated after the samples had been refrigerated overnight and until the values, above background, became reproducible. Specific radioactivity of the [' 4C1J ochratoxin A and [14C] diaporthin were calculated. A second experiment involved four 100 ml scale flasks. [1-'4C] Acetate (5 tCi) was added to a flask at 8, 22.5, 32 or 50 hours post inoculation. All flasks were incubated until 80 hours post inoculation when they were extracted. Ochratoxin A and diaporthin were isolated from the extracts by preparative HPLC and aliquots of the purified [ 14C] ochratoxin A and [14C] diaporthin samples were used for DAD-HPLC analysis to determine purity and concentration in order to calculate specific radioactivities. The methylation inhibitor ethionine (50 mg) was administered (10 ml of a 5 mg/ml sterile solution) to each of five 100 ml scale flasks at post inoculation stages of 32, 50, 56 or 72 hours. Sterile water (10 ml) was added to a fifth flask as a control. The flasks were incubated until 80 hours post inoculation when the broth was extracted. DAD-HPLC was used to determine, quantitatively, the yield of ochratoxins A and B, orthosporin and diaporthin in each experimental culture flask. 4.3: Results and discussion. In the first radioactivity experiment (the essence of the data in table 4.1 is shown in figure 4.1) both acetate and methionine were incorporated into diaporthin and ochratoxin A, albeit in rather low amounts. The later either [1-' 4C] acetate or [methyl-14C] methionine was fed, the lower was the specific radioactivity of [14C] ochratoxin A isolated 168 hours post inoculation. This would be consistent with a declining rate of ochratoxin A biosynthesis during the phase of radiolabel addition. 109 [1-' 4C] Acetate feed: [ ' 4C] OA [14C] Diaporthin Time Amount DPM Sp.Rad. Amount DPM Sp.Rad. (hours) (jig) (dpm/mg) (jig) (dpm/mg) 32 4 60 15000 4 150 37500 50 43 70 1630 9 530 58890 72 47 50 1060 10 730 73000 [methyl- 14C] Methionine feed: [14C] OA [ 14C] Diaporthin Time Amount DPM Sp.Rad. Amount DPM Sp.Rad. (hours) (jig) (dpm/mg) (jig) (dpm/mg) 32 29 160 5515 6 120 20000 72 17 50 2940 4 610 152500 Table 4.1: First radiolabel experiment: Addition of 3.7 jiCi [1-' 4C] acetate or 2 jiCi of [methyl-'4C] methionine to D2306 PDB cultures at the given times post inoculation, with flasks being harvested after 168 hours post inoculation. [ 14C] OA [14C] Diaporthin Time Amount DPM Sp.Rad. Amount DPM Sp.Rad. (hours) (jig) (dpm/mg) (jig) (dpm/mg) 8 23 132 5740 155 431 2780 22.5 143 5327 37250 63 2214 35140 32 143 2620 18320 113 7933 70205 50 152 2250 14805 52 4840 93075 Table 4.2: Second radiolabel experiment: Addition of 5 j.tCi [1- 14C] acetate to D2306 PDB cultures at the given times post inoculation, with flasks being harvested after 80 hours post inoculation. 110 Figure 4.1: Specific radioactivities of[14C] ochratoxin A and [14C] diaporthin isolated 168 hours post inoculation after the additions of 3.7 pCi [1-14C] acetate to D2306 PDB cultures at different times. go - 70 60 -o 50 : 40 0 o 30 0 . 0 10 20 30 40 50 60 70 80 Addition times (hours post inoculation) Figure 4.2: Specific radioactivities of[14C] ochratoxin A and [14C] diaporthin isolated 80 hours post inoculation after the additions of 5 pCi [1-14C] acetate to D2306 PDB cultures at different times. 100 90 80 70 0 60 5° - .9 40 0 '-0 30 0 20 '3) (#D 10 0 0 10 20 30 40 50 60 70 80 Addition times (hours post inoculation) . Ochratoxin A Diaporthin 111 However, figure 2.8 clearly shows that between 32 and 72 hours post inoculation in a D2306 PDB model fermentation, ochratoxin A in the broth was not in decline but was steadily increasing in concentration. It was assumed that a similar situation had occurred in the present experimental fermentation. Therefore, the decrease in specific radioactivity could just reflect decline in the rate of the biosynthesis of the dihydroisocoumarin moiety of ochratoxin A. Perhaps most of the pentaketide chains destined for ochratoxin are produced early in the fermentation but are not incorporated into ochratoxin A immediately. In the second radioactivity experiment (table 4.2 and figure 4.2) the specific radioactivities of [ 14C] ochratoxin A isolated 80 hours post inoculation decreased after the feed with [1-'4C] acetate at 22.5 hours post inoculation, which is consistent with the results of the first radioactivity experiment. However, the specific radioactivity increased up to 22.5 hours post inoculation. This seems to suggest that polyketide chain biosynthesis mostly occurs between 10 and 30 hours post inoculation, and there is a lag of approximately 50 hours between the apparent end of polyketide synthesis and the time of maximum ochratoxin A yield in the broth. Lillehoj eta!. (1978) used a high ochratoxin A yielding (up to 350 mgfL) stationary culture of Aspergillus suiphureus (NRRL 4077) to produce [ 14C] ochratoxin A of a high specific radioactivity. [1- 14C] Acetate was fed to the culture at different times to establish the optimum way of producing the highest specific radioactivity at the time of maximum yield (11 days). The highest specific activity of labelled toxin obtained was 70 .tCi4tg and the maximum incorporation of[1-'4C] acetate was 5.3%. The dynamics of ochratoxin A accumulation in the medium was also described. If both sets of data are plotted on one graph (figure 4.3), that Lillehoj et al. (1978) did not do, it seems that there is a 7 day lag between the addition time ultimately producing the highest specific radioactivity of[ 14C] ochratoxin A and the stage of maximum ochratoxin A yield. Lillehoj et aL (1978) neither recognised nor discussed this aspect of their data but re-interpretation clearly shows temporal separation of biosynthetic events. 112 '1J LU [y uIxoJipo] 0 0 0 0 0 0 t) 0 U) 0 U) 0 0 r) C) C'1 CJ - i— U) 0 c..J 1 U 0 0) co 0 U 0 0 .E 0 ;- —e 0 CD .=0QQC 0 I 0l) .- c-) I +- CN 0 0 0 0 0 0 0 0 0 0 0 F— (0 it) C) C1 w (1,!DT1) zjiood 113 Diaporthin has been classified as a hexaketide (Turner, 1971) and, assuming one methylation, could be derived solely from the polyketide pathway (see 1.8). The incorporation of both [1-' 4C] acetate and [methyl-14C] methionine into [14C] diaporthin in the present study is the first piece of expenmental evidence indicating its metabolic origin and is in agreement with the proposed biosynthetic scheme (figure 1.23). In contrast to the experimental results concerning ochratoxin A, the specific radioactivity of[ 14C] diaporthin isolated either 80 or 168 hours post inoculation steadily increased with lateness of feeding either [1 - 14C} acetate or [methyl-14C] methionine. Further, the addition time of [1- 14C] acetate producing the highest specific radioactivity of [14C] diaporthin appears to be close to the time of maximum diaporthin yield. For diaporthin biosynthesis occurring in the later part of the fermentation, there is less competition for acetate and methionine from the primary metabolic processes of growth. There is no apparent delay between the production of the polyketide and the accumulation of diaporthin in the broth. The production of ochratoxins A and B, orthosporin and diaporthin by each of the cultures fed ethionine is shown in table 4.3. The control values were typical for a D2306 PDB shake flask fermentation (cf. figure 2.8). Ethionine effectively inhibited ochratoxin A biosynthesis, by nearly 60%, only when it was added at the earliest stage post inoculation. This seems to indicate that methylation, like the biosynthesis of the polyketide chain, is an early event and there is a lag between the end of methylation and the time of maximum ochratoxin A concentration in the culture broth. Ochratoxin B appeared to be less affected by ethionine than ochratoxin A. Perhaps ochratoxin B production commenced before ocbratoxin A. If ochratoxin f3 was a common intermediate in the biosynthetic pathways of ochratoxins A and B, then perhaps ethionine hindered ochratoxin a production by inhibiting the chlorination of ochratoxin 3. Orthosporin production was as affected by ethionine as was diaporthin. Indeed, table 4.3 shows that the ratio between diaporthin and orthosporin yields after ethionine treatment at various stages is nearly constant (range 1.6 - 2.2). Diaporthin has at least 114 Addition time [OA] [OB] [Ortho.] [Diap.] [Diap.]/ (hours post inoc.) mgfL mgfL [Ortho.] 32 2.1 1.2 0.3 0.5 1.7 50 4.2 1.3 0.9 1.4 1.6 56 3.7 1.3 0.9 1.8 2 72 4.5 1.3 1.4 2.9 2.2 Control 4.8 1.4 1.2 2.3 1.9 Table 4.3: The extracellular concentration of ochratoxin A ([OA]), ochratoxrn B ([OB]), orthosporin ([Ortho.]) and diaporthin ([Diap.]) 80 hours post inoculation in D2306 PDB fermentations fed with 50 mg ethionine at various times. The calculated ratio of diaporthin to orthosporin is also shown. 115 one methylation in its biosynthetic pathway (the 0-methylation of the hydroxyl group at C-6, figure 1.23), yet one can envisage orthosporin being derived from acetate alone. May it be that even if diaporthin is biosynthesized in the flingal cells via the 0- methylation of orthosporin, the orthosporin found in the culture broth is derived by some subsequent de-methylation of diaporthin? Another plausible explanation is that ethionine could be having a rather more general effect on polyketide biosynthesis. If this was true, then ochratoxin A production would only be inhibited if the ethionine was administered when its polyketide synthesis was in operation, i.e. early in the fermentation, which was the case. Yamazaki et a!. (1971) grew an ochratoxinogenic Aspergillus ochraceus isolate (IFM 4443) in a medium containing ethionine and ochratoxin production was completely inhibited. Unfortunately, no attempt was made to add ethionine at any other time. Ethionine does not seem to inhibit any of the later (J)ost polyketide) enzymes in ochratoxin A biosynthesis. The diaporthin concentration values obtained from the experiment were consistent with a complete inhibition of methylation by ethionine, i.e. diaporthin concentration values measured 80 hours post inoculation corresponded with diaporthin concentration values expected at the time of ethionine addition (cf. figure 2.8). The orthosporin concentration values are consistently lower than the expected residual values. However, recycling of orthosporin may occur between 72 and 80 hours post inoculation within the fermentation (see section 2.3.1). If this is true, then the orthosporin degradation enzymes are not sensitive to ethionine unlike the enzymes of citrinin degradation (Barber eta!., 1988). Huff and Hamilton (1979) included methylation of mellein as a step in the biosynthesis of ochratoxin A. Thus, one would expect a increase in the accumulation of mellein if methylation were to be inhibited. Significantly, mellein was not detected in any extracts of the cultures fed ethionine, which would not be consistent with the Huff and Hamilton (1979) hypothesis. 116 5: Feeding [14CJ mellein, I1O-' 4CJ ochratoxins a or 13 or 11O14(2, phenylalanyl- 3j ochratoxins A and B to Aspergillus ochraceus D2306 fermentations. 5.1: Introduction. The feeding of radiolabelled putative advanced intermediates to cultures of Penicillium citrinum formed the basis of the elucidation of the citrinin biosynthetic pathway (Carter eta!., 1979; Barber and Staunton, 1980 a; Colombo eta!., 1981). Analogous methodology has not so far been applied to ochratoxin A biosynthesis. Huff and Hamilton (1979) suggested that mellein and ochratoxins a and 13 were advanced intermediates of ochratoxin A biosynthesis (see 1.4), and so experiments were devised to see whether [14C] mellein, [10-14C] ochratoxins a and 13 or [10-14C, phenylalanyl-3H} ochratoxins A and B could be incorporated into ochratoxins A and B in A. ochraceus fermentations. 5.2: Materials and methods. 5.2.1: Feeding [ 14C] mellein to an Aspergillus ochraceus D2306 potato dextrose broth fermentation. [ 14C] Mellein was produced by feeding [1-' 4C] acetate to Aspergillus ochraceus KBf PDB shake flask cultures and then isolating the ensuing [ 14C] mellein from the broth. Two g of extensively sporing KBf SW culture was homogenised in 100 ml sterile distilled water using a Sorvall Omni-Mixer. After allowing crude particulates to settle, the spore suspension was used to inoculate 100 ml scale PDB flasks so that the initial spore concentration, as measured with a haemocytometer, was in the range 2-4 x 106 spores/ml. The KBfPDB cultures were incubated at 28 °C and shaken at 200 rpm with a 10 cm eccentric throw. At intervals, 10 or 25 ml of the culture was removed for the measurement of mellein concentration. To one flask, 10 j.tCi of[l-' 4C] acetate was fed 96, 120 and 144 hours 117 post inoculation, spanning the period of mellein accumulation. The flask was harvested 168 hours post inoculation and the [ 14C} mellein was purified. Two percent of the purified [ 14C] mellein was injected into the DAD-HPLC system for quantitative analysis and 5% was scintillation counted. The remaining [ 14C] mellein was dissolved in 2 ml ethanol prior to feeding to a D2306 PDB shake flask culture. A 100 ml scale D2306 PDB shake flask culture with an initial spore density of approximately 4 x 106 spores/mI was fed one half of the purified [ 14C1 mellein at 17 hours post inoculation and the other half at 25 hours in an attempt not to miss the most appropriate stage. Half of the fed flask was harvested at 44 hours post inoculation and half at 68 hours partly to guard against losses by product turn-over. The broth of the harvested flask was extracted in the usual manner to yield bicarbonate soluble and insoluble fractions. Five percent of all of the extracts were chromatographed on TLC plates using toluene-ethyl acetate-formic acid (50:40:10) as the mobile phase. The developed TLC plate was autoradiographed for three months. The remaining 95% of the samples was injected into the DAD-HPLC system and the eluate conesponding to either ochratoxin A or diaporthin was collected and scintillation counted. The purity and specific radioactivity of the collected compounds were calculated using the appropriate ChemStation programmes. In addition, the intracellular polyketides of a weighed amount of the filtered cells of the fed D2306 culture were extracted by refluxing in 100 ml chloroform-methanol (2:1) for 1 hour. Eight percent of all each fraction was analysed using the relevant DAD-HPLC system, 20% was scintillation counted and 20% was loaded onto TLC plates and developed in toluene-ethyl acetate-formic acid (50:40:10) prior to autoradiography for 1 month. 5.2.2: The course of intra- and extracellular accumulation of mellein in an Aspergillus ochraceus KBf potato dextrose broth fermentation. The relationship between the intra- and extracellular mellein concentrations during a KBfPDB fermentation was investigated. Approximately 5 g of extensively sporing 118 KBf SW culture was homogenised in 100 ml sterile distilled water. The homogenate was filtered through muslin prior to centrifugation for 20 minutes at 10000 rpm at 4 °C (using a Sorvall RC-5 Superspeed refrigerated centrifuge and Sorvall GSA rotor). The pellet was washed in 100 ml sterile water, re-centrifuged, re-suspended in 10 ml sterile water and the spore concentration determined using a haemocytometer. The spore suspension was used to inoculate eight 100 ml scale PDB flasks to give cultures that were mellein-free at the start, with an initial spore concentration of 4 x 106 spores/mi. The cultures were incubated at 22 °C and shaken at 200 rpm with a 10 cm eccentric throw. Intra- and extracellular mellein concentration values and growth measurements were taken at 24 hour intervals up to 96 hours post inoculation. Growth was measured as dry cell weight by filtering the contents of one of the sample flasks through a pre-weighed 9 cm Whatman No. 50 filter paper which was then dried in a 40 °C oven to constant weight. A duplicate sample flask was filtered, the cells were washed with water and the broth was extracted for extracellular mellein. Intracellular mellein was determined after refluxing 2 g of the water-washed cells in 100 ml chloroform-methanol (2:1) for 1 hour. All samples were analysed by DAD- HPLC. 5.2.3: Rate of incorporation of[1- 14C] acetate into [14C] mellein in anAspergillus ochraceus KBf potato dextrose broth fermentation. The first feeding experiment giving [1-' 4C] acetate to a KBfPDB fermentation to produce [14C] mellein made no attempt to establish the optimum stage for [1-14C] acetate feeding, or the optimum [14C] mellein harvesting time in order to obtain [14C] mellein of the highest specific radioactivity. In an attempt to rectify this, two 100 ml scale PDB flasks were inoculated with KBf spores ensuring an initial mellein-free spore concentration of 4 x 106 spores/mi. The KBfPDB cultures were incubated shaken at 22 °C, and at 24, 41, 48 and 64 hours post inoculation, 10 ml samples were removed from alternate flasks for extracellular mellein quantification. At 65 hours post inoculation, the volume of culture in one flask was estimated and 2 RCi of [1- 119 14C] acetate was added. At 10 minutes, 1 and 5 hours after the radiolabel was added, 10 ml samples were removed from the fed flask. The samples were extracted, 17% of the resulting bicarbonate insoluble fractions was injected into the DAD-HPLC system and the eluate corresponding to mellein was collected enabling the purification and quantification of [14C] mellein. The pure [ 14C] mellein was scintillation counted and the percentage incorporation of the fed [1 - 14C] acetate into [ 14C] mellein was calculated for each recovery time. 5.2.4: Producing [ 14C] mellein of a higher specific radioactivity. A 100 ml scale KBfPDB culture was initiated to give a mellein-free spore concentration of 4 x 106 /int. The flask was shaken at 22 °C and, at 35.5 hours post inoculation, 10 ml of culture was removed for rapid quantification of extracellular mellein to ensure the feeding of the radiolabel would correspond to the start of mellein biosynthesis. At 36.5 hours post inoculation, 10 .tCi [1-'4C] acetate was added. The fed culture was left to incubate for a further 1 hour after which the volume of the broth was noted prior to being extracted for mellein. Eight percent of the bicarbonate insoluble extract of the fed flask was injected into the appropriate DAD-HPLC system and the eluate corresponding to mellein was collected and scintillation counted. Peak purity and quantitative mellein analysis enabled calculation of the percentage incorporation of [1- 14C] acetate into, as well as the specific radioactivity of, the [ 14C] mellein collected. Portions of the remaining [ 14C] mellein were purified from the bicarbonate insoluble extract of the fed flask using HPLC and Cl 8 Sep-Pak techniques. 5.2.5: Establishing the optimal time of advanced intermediate addition to an Aspergillus ochraceus D2306 solid substrate fermentation. The addition of advanced intermediates to an Aspergillus ochraceus D2306 SW culture must coincide rather closely with the biosynthesis of ochratoxin A in order to obtain evidence of any direct incorporation. The percentage incorporation of[1-14C] 120 acetate, [U-'4C] phenylalanine and [methyl- 14C] methionine into ochratoxin A provides an excellent guide to the state of ochratoxin A biosynthesis during a SW fermentation. Thus, a 40 g scale shaken D2306 SW culture was initiated and incubated at 28 °C. After 3, 4, 5 and 6 days 2 g of SW culture was transferred to a sterile 50 ml conical flask. To one 2 g aliquot was added 5 LLCi [1-14C] acetate, to another 5 itCi [U- 14C] phenylalanine and to the third 5 pCi [methyl-' 4C1 methionine. All of the radiolabels were fed as a sterile 200 p1 aqueous solution distributed widely as droplets on the SW substrate. The fed flasks were incubated, shaken, for a further 5 hours before being extracted for ochratoxins A and B. Eight percent of each of the resulting bicarbonate soluble fractions was injected into the DAD-HPLC and the eluate corresponding to ochratoxins A and B was collected enabling the purifi cation and quantification of the [ 14C] ochratoxins A and B. The pure [ 14C] ochratoxins A and B were scintillation counted and the percentage incorporation of the fed [14C] labelled compounds into them was calculated for each experimental condition. 5.2.6: Feeding [ 14C] mellein or [10-'4C] ochratoxins a or 3 to anAspergillus ochraceus D2306 solid substrate fermentation. Limited chemical degradation of ochratoxins A and B can result in the cleavage of the amide bond to cede the dihydroisocoumarin moieties ochratoxins a and 3, respectively (see 3.2.3-4). [10-' 4C] Ochratoxins A (1.16 mg) and B (1.44 mg), purified from the 2 g D2306 shaken SW culture fed 10 pCi [methyl- 14C] methionine 4 days post inoculation (see 5.2.5), were each acid hydrolysed by refluxing in 50 ml 6M HC1 overnight. The dihydroisocoumarins were partitioned from the cooled reaction mixture into an equal volume of ethyl acetate which was then rotary evaporated to dryness. Eight percent of both [10-' 4C] dihydroisocoumarins were scintillation counted before and after a DAD-HPLC clean up step to ensure all of the radioactivity was attributed to the relevant [10-' 4C] dihydroisocoumarin. A 40 g scale shaken D2306 SW culture was initiated and, at 4 days post inoculation, three 2 g D2306 SW culture aliquots were transferred into 3 sterile 50 ml conical 121 flasks. To one 2 g aliquot was added 0.13 tCi [10-'4C] ochratoxin a, to another 0.1 pCi [10-'4C] ochratoxin and to the last 0.096 pCi [ 14C] mellein. All of the radiolabels were fed as a sterile 200 p1 aqueous solution. The fed flasks were incubated for a further 5 hours before being extracted for polyketides. Eight percent of each of the resulting bicarbonate soluble fractions was used for [ 14C] ochratoxins A and B purification and quantification by DAD-HPLC. The pure [ 14C] ochratoxins A and B were scintillation counted and the percentage incorporation of the fed [14CJ labelled compounds into them was calculated. Eight percent of each of the resulting bicarbonate insoluble fractions was used for analysis by DAD-HPLC and 5% of all the fractions was loaded onto a TLC plate which was developed in toluene-ethyl acetate- formic acid (50:40:10) prior to autoradiography for 1 month. 5.2.7: The interconversion,1014C, phenylalanyl-3H] ochratoxins A and B into ochratoxins A and B. The [10-'4C] ochratoxins a and feeding experiment (5.2.6) was unable to determine whether ochratoxin was incorporated into ochratoxin A via ochratoxin B or ochratoxin a. Indeed, there have been no studies regarding the presence, or extent, of any inter-conversion between ochratoxins A and B. Dual labelled ochratoxins (3H in the phenylalanine moiety and 14C in the dihydroisocoumarin moiety) can be fed to D2306 cultures in order to determine any inter-conversion between the two ochratoxins. Thus, a 40 g scale shaken D2306 SW culture was initiated and incubated at 28 °C. After 4 days, 2 g of SW culture was transferred to a sterile 50 ml conical flask and then fed 5 pCi [methyl-14C] methionine and 5 pCi [2, 3, 4, 5, 6-3H] phenylalanine. The culture was incubated, shaken, for a further 5 hours before being extracted for ochratoxins A and B. The dual labelled ochratoxins A and B were purified using preparative HPLC, quantified using DAD-FIPLC and a 1O% sample of each was scintillation counted. 122 An initial 'pilot' dual labelled ochratoxins A and B feeding experiment was performed using 2 g aliquots of a 5 days old 40 g scale D2306 shaken SW culture. To one aliquot was added 0.231 mg dual labelled ochratoxin A (equivalent to 10.2 and 5 nCi H and 14C, respectively) and to another 1.018 mg dual labelled ochratoxin B (equivalent to 200 and 78 nCi 3H and 14C, respectively). All of the radiolabels were fed as a sterile 200 tl aqueous solution. The fed cultures were incubated, shaken, for a further 5 hours before being extracted for ochratoxins A and B. In a second similar experiment, to a 2 g aliquot of a 6 day old 40 g scale D2306 shaken SW culture was added 1.28 1 mg of dual labelled ochratoxin A (equivalent to 57 and 28 nCi 3H and 14C, respectively). On this occasion the radiolabel was added as a sterile 200 p.1 solution in 3% aqueous sodium hydrogen carbonate. This fed flask was incubated, shaken, overnight before being extracted for ochratoxins A and B. Eight percent of all of the resulting bicarbonate soluble fractions was injected into the DAD-HPLC and the eluate corresponding to ochratoxins A and B was collected and scintillation counted. A further lO% of each of the bicarbonate soluble and, in addition, insoluble fractions was loaded onto a TLC plate, developed in toluene-ethyl acetate-formic acid (50:40:10) and then autoradiographed for 1 week. 5.3: Results and discussion. 5.3.1: Feeding [ 14C] mellein to anAspergillus ochraceus D2306 potato dextrose broth fermentation. The concentration of mellein in the broth versus time for the KBfPDB fermentation at 28 °C is shown in figure 5.1. Mellein appeared much earlier in the KBfPDB fermentation (shortly after 2 days post inoculation) than in an equivalent D2306 PDB culture (after 6 days post inoculation) and the yields were over 10 times greater (50 mgfL compared to 3.5 mg(L from D2306). Extracellular mellein concentration decreased after 96 hours post inoculation indicating a mechanism that can catabolise mellein perhaps ultimately to acetate. Although only the first of the three 10 p.Ci [1- 123 Figure 5.1: The course of accumulation of extracellular mellein in A. ochraceus KBf potato dextrose broth fermentations at two temperatures. 50 45 40 35 E 30 .E 25 20 a15 10 5 0 0 20 40 60 80 100 120 140 160 180 200 Time (hours) 28°C 22°C Figure 5.2:Changes in dry cell weight (DCW), intracellular mellein concentration ([Mellein] cells) and extracellular mellein concentration ([Mellein] broth) during an A. ochraceus KBf potato dextrose broth fermentation. 80 70 60 0 .50 40 30 20 10 0 0 20 40 60 80 100 120 140 160 180 200 Time (hours) DCW (mg/100 ° [Mellein] cells • [Mellein] broth ml) (ig/g) (mgfL) 124 14C] acetate feeds to the KBfPDB culture coincided with the apparent production phase (between 50 and 100 hours post inoculation), [ 14C] mellein was successfully isolated for the subsequent feeding experiment (225 .tg with a total radioactivity of 30000 dpm, i.e. a specific radioactivity of 130 dpm/p.g). [ 14C] Mellein produced by the [1- 14C] acetate fed culture had exceeded 2 mg, but much of it was used for perfecting the purification techniques. Seventy-five .tg of ochratoxin A and 10 j.tg of diaporthin were purified from the half of the D2306 PDB culture fed [ 14C] mellein and harvested 44 hours post inoculation; 31 j.tg of ochratoxin A and 13 jig of diaporthin were purified from the half harvested 68 hours post inoculation. All of these samples registered only background radiation when scintillation counted. in addition, the autoradiograph revealed no detectable radiation on the TLC plate. Notably also, no residual [ 14C] mellein was detected in the bicarbonate insoluble fractions. Cells from the fed D2306 culture, 0.4 and 0.25 g harvested 44 and 68 hours post inoculation, respectively, were extracted for bicarbonate soluble and insoluble intracellular constituents. The DAD-HPLC bicarbonate soluble metabolic profile showed the absence of ochratoxins A and B in the fed cells harvested at both 44 and 68 hours post inoculation, In the DAD-HPLC bicarbonate insoluble metabolic profile, only cycloechinulin and diaporthin were positively identified. There was no trace of mellein. A total radioactivity of 600 and 690 dpm from the bicarbonate soluble and insoluble fractions of the cells harvested 44 hours post inoculation and 675 and 780 dpm from the bicarbonate soluble and insoluble fractions of the cells harvested 68 hours post inoculation, respectively, was detected. This suggests that although mellein was not detected intracellularly intact, it did become cell-associated, perhaps via a catabolic route prior to distribution amongst bicarbonate soluble and insoluble metabolite biosynthesis. 125 This experiment was therefore unable to provide evidence that mellein was incorporated directly into ochratoxin A when it is fed, apparently in an advantageous way, to an ochratoxinogenic fungal liquid culture. 5.3.2: The course of intra- and extracellular accumulation of mellein in anAspergillus ochraceus KBf potato dextrose broth fermentation. The intra- and extracellular mellein concentration and dry cell weight values are shown in figure 5.2. Intra- and extracellular mellein concentrations increased concurrently with time showing that extracellular mellein is a faithful indication of the state of mellein biosynthesis. These results are also consistent with free movement of mellein across the cell boundary. The growth of A. ochraceus KBf in PDB is very similar to A. ochraceus D2306 (figure 2.8) especially, as might be expected, with regard to magnitude (maximum dry cell weight approaching 5 g/L). Mellein production in the KBf fermentation appears to be linked with growth (unlike the D2306 fermentation) which suggests that there are either two mellein biosynthesis control systems in Aspergillus ochraceus or that mellein produced early in D2306 fermentations is metabolised further (e.g. to ochratoxins A and B). There is a great deal of similarity between figures 5.1 and 5.2 with regard to extracellular mellein biosynthesis. Unfortunately, the picture in figure 5.2 is slightly distorted due to the lack of a 48 hour reading. Mellein probably did not start to accumulate in the broth until after approximately 40 hours post inoculation, rather than after 24 hours as the dotted line suggests. 5.3.3: Rate of incorporation of[1- 14C] acetate into [ 14C] mellein in anAspergillus ochraceus KBf potato dextrose broth fermentation. Mellein concentration versus time for the KBfPDB fermentation at 22 °C is shown in figure 5.1. The results are very compatible with previous results even though the cultures were incubated at different temperatures, although it was found that the fungus produced mellein more consistently at 22°C. Unlike in the first [1- 14C] acetate 126 feeding experiment, the radiolabel was fed during the steepest part of the mellem production curve. The volume of the fed KBfPDB culture was 60 ml and the radioactivity calculations for the [ 14C] mellein recovered are shown in table 5.1. At 65 hours post inoculation, [1-'4C] acetate was incorporated significantly into [14C] meilein even after only 10 minutes (0.25%), although a higher incorporation was achieved after 1 hour (O.9%). Interestingly, the incorporation of [1-'4C] acetate into [ 14C] mellein decreased between I and 5 hours (0.4%) which suggests there was a significant turnover of mellein even during the apparent maximum mellein production phase. 5.3.4: Producing [ 14C] mellein of a higher specific radioactivity. The extracellular mellein concentration measured at 35.5 hours post inoculation was already approximately 23 mgfL which implies that there is some variability in the earliness of mellein production between KBfPDB culture batches. Nevertheless, the addition time of 10 .tCi [l-' 4C] acetate still corresponded to an active phase of mellein production. The extraceilular mellein concentration measured 37.5 hours post inoculation was approximately 37.5 mgfL which suggested a mellein production rate of 7 mgfLihour during the incubation period. Eight percent of the total extracted [14C] mellein (178 mg) corresponded to 0.133 pCi radioactivity which is equivalent to a specific radioactivity of 1658 dpm/mgand a 16.6% incorporation of[l-14C] acetate, a significant improvement on previous results. 5.3.5: Establishing the optimal time of advanced intermediate addition to an Aspergillus ochraceus D2306 solid substrate fermentation. The radioactivity calculations leading to the percentage incorporation values of[1- 14C] acetate, [methyl- 14C] methionine and [U-' 4C] phenylalanine added 3, 4, 5 or 6 days post inoculation into [ 14C] ochratoxins A and B are shown in tables 5.2 and 5.3, respectively. The percentage incorporation data is also displayed graphically in figures 5.3 and 5.4. All three radiolabels were successfully incorporated into [14C] 127 Time Measured ______Calculated ______dpm dpm nCi % inc. ______(17 percent) (100 percent) ______ 10 minutes 1890 11118 5 0.25 1 hour 6745 39676 17 9 0.9 5 hours 3035 17853 8 0.4 Table 5.1; Radioactivity of [ 14C] mellein recovered 10 minutes, 1 and 5 hours after the addition of 2 pCi [1- 14C] acetate to an Aspergillus ochraceus KBfPDB fermentation. % inc. indicates the percentage incorporation of the 2 pCi [1-'4C] acetate into the recovered [ 14C] mellein. 128 Measured { Calculated I dpm dpm l.tCi %inc. (8%) (100%) Day 3: [1-14C] acetate 5529 69113 0.031 0.62 [methyl-14C] met 10887 136088 0.061 1.22 [U-14C] phe 17456 218200 0.098 1.96 Day 4: [1-14C] acetate 50760 634500 0.29 5.80 [methyl- 14C] met 102486 1281075 0.58 11.60 [U-14C] phe 211440 2643000 1.19 23.80 Day 5: [1-'4C] acetate 39351 491888 0.22 4.40 [methyl-' 4C] met 55962 699525 0.32 6.40 [U-14C] phe 233729 2921613 1.32 26.40 Day 6: [1-'4C] acetate 16159 201988 0.09 1.80 [methyl-14C] met 24172 302150 0.14 2.80 [U-14C] phe 132799 1659988 0.75 15.00 Table 5.2: Radioactivity (expressed as disintegrations per minute-dpm- and tCi) of [14C] ochratoxin A recovered 5 hours after the addition of either 5 iCi [1-'4C] acetate, [methyl- 14C] methionine ([methyl- 14C] met) or [U-14C] phenylalanine ([U- 14C] phe) to anAspergillus ochraceus D2306 shaken shredded wheat fermentation 3, 4, 5 or 6 days post inoculation. % inc. indicates the percentage incorporation of the radiolabels into the recovered [ 14C] ochratoxin A. 129 Measured { Calculated } dpm dpm %mc. (8%) (100%) Day 3: [1-'4C] acetate 900 11250 0.005 0.10 [methyl-'4C] met 2992 37400 0.020 0.40 [U-'4C] phe 4918 61475 0.030 0.60 Day 4: [1-14C] acetate 25829 322863 0.15 3.00 [methyl-'4C] met 50819 635238 0.29 5.80 [U-14C] phe 104988 1312350 0.59 11.80 Day 5: [1-14C] acetate 34393 429913 0.19 3.80 [methyl- 14C] met 49340 616750 0.28 5.60 [U-14C] phe 210835 2635438 1.19 23.80 Day 6: [1-'4C] acetate 22093 276163 0.12 2.40 [methyl- 14C] met 40162 502025 0.23 4.60 [TJ...l4C] phe 204397 2554963 1.15 23.00 Table 5.3: Radioactivity (expressed as disintegrations per minute-dpm- and i.tCi) of [14C] ochratoxin B recovered 5 hours after the addition of either 5 RCi [1-'4C] acetate, [methyl- 14C] methionine ([methyl- 14C] met) or [U- 14C] phenylalanine ([U- 14C] phe) to an Aspergillus ochraceus D2306 shaken shredded wheat fermentation 3, 4, 5 or 6 days post inoculation. % inc. indicates the percentage incorporation of the radiolabels into the recovered [ 14C] ochratoxin B. 130 Figure 5.3: Percentage incorporation of 5 tCi [1-14C] acetate (Ace), [methyl-14C] methionine (Met) or [U-14C] phenylalanine (Phe) into [14C] ochratoxin A by an Aspergillus ochraceus D23206 shaken SW fermentation at various times post inoculation. 30 25 0 .. o 20 0 U • 15 10 U 0 3 3.3 4 4.5 5 5.5 6 Time post inoculation (days) Figure 5.4: Percentage incorporation of 5 tCi [1-14C] acetate (Ace), [methyl-14CJ methionine (Met) or [t.T-14C} phenylalanine (Phe) into [14CJ ochratoxin B by an Aspergillus ochraceus D23206 shaken SW fermentation at various times post inoculation. 25 0 20 I- 0 o 15 C.) V 10 V C.) I- V 0 3 3.5 4 4.5 5 5.5 6 Time post inoculation (days) Ace -°-- Met • Phe 131 ochratoxins A and B which not only confirmed earlier biosynthetic studies by Searcy et a!. (1969), Ferreira and Pitout (1969) and Steyn et a!. (1970) but also set a rather unusual precedent for the use of solid substrate fermentation in incorporation studies. Indeed, the relatively large percentage incorporation values obtained for the radiolabels suggests that a solid substrate fermentation feeding system is as good as, if not superior to, the liquid fermentations used previously. [U- 14C] Phenylalanine was incorporated into both [ 14C] ochratoxins A and B to the highest extent (26.4%), which may be due to it being incorporated directly and entirely, whilst acetate is incorporated the least (5.8 %, which is still higher than the 5.3% reported by Lillehoj et a!., 1978) since it is utiised in a multitude of competing metabolic pathways. Maximum incorporation of[1- 14C] acetate and [methyl-14C] methionine (closely linked dihydroisocoumarin precursors) into [ 14C] ochratoxin A occurred at 4 days post inoculation whereas maximum incorporation of [U- 14C] phenylalanine was at 5 days post inoculation, reinforcing previous observations of a temporal separation of biosynthetic events in ochratoxin A biosynthesis. This may also be true for ochratoxin B biosynthesis which occurred concurrently. The percentage incorporation of each of radiolabelled primary metabolites into both ochratoxins A and B are additive so, for example, an astonishing 50.20% (26.40 + 23.80) of the [U- 14C] phenylalanine added at 5 days post inoculation was incorporated into the two ochratoxins. This gives an indication of the extent of ochratoxin biosynthesis at this stage of the D2306 fermentation. Therefore, the compromised optimum time for the addition of putative advanced intermediates of ochratoxins A and B biosynthesis appears to be 4 days post inoculation. 5.3.6: Feeding [ 14C] mellein or [10-'4C] ochratoxins a or 3 to anAspergillus ochraceus D2306 solid substrate fermentation. According to the literature, [methyl- 14C1 methionine labels ochratoxin A at C-10 (Steyn eta!., 1970) and thus should label ochratoxin B similarly. Acid hydrolysis of [10-14C] ochratoxins A (461311 dpm) and B (300829 dpm) resulted in [10-'4C] 132 ochratoxins a (334963 dpm) and 13 (249250 dpm) in yields of 73 and 83%, respectively, assuming all of the label is in the dihydroisocoumarin moiety. The radioactivity calculations leading to the percentage incorporation values of[10-14C] ochratoxin a, [1O-'4C] ochratoxin 13 and [ 14C] mellein into [ 14C] ochratoxins A and B are shown in table 5.4. [10-'4C] Ochratoxin a was significantly incorporated into [10-'4C] ochratoxin A (4.85%) but much less so into [10-' 4C] ochratoxin B (0.47%). Ochratoxin 13, however, was significantly incorporated into both (14.32 and 18.98% was incorporated into [10- 14C] ochratoxins A and B, respectively). [14C] Mellem was incorporated into both [ 14C] ochratoxins A and B but to a much lower extent (0.56 and 0.68%, respectively). DAD-HPLC revealed that there was no trace of mellem in any extracts. Interestingly, an enhanced peak in the bicarbonate insoluble DAD-HPLC metabolic profile of the [ 14C] mellein fed culture corresponded (with regard to retention time and UV absorbance spectrum) to the hydroxyinellein metabolite isolated from Aspergillus ochraceus NRRL 3174 (see 3.2.9). Abell eta!. (1987) showed that [2H] mellein was specifically incorporated into [2H1 4-hydroxymellein by a culture of Aspergillus melleus. It should be noted that the percentage incorporation values for each feeding experiment are additive. Thus, the total percentage incorporation into both [ 14C] ochratoxins A and B was 5.32, 33.30 and 1.24 for ['4C] ochratoxins a, 13 and mellein, respectively. An autoradiograph of the developed TLC plate of bicarbonate soluble and insoluble fractions of the fed culture extracts is shown in figure 5.5. Lane 1 shows clearly the incorporation of[10-'4C] ochratoxin a into [10-'4C] ochratoxin A, since the majority of the radioactivity is found in these two compounds. There is some metabolism of the [10-'4C] ochratoxin a into neutral compounds (lane 2) especially a very non-polar one which runs to the solvent front. Perhaps this lipophilic compound could be a fungal sterol constructed partially from [ 14C] acetate released from the catabolism of [10-'4C] ochratoxin a. If indeed there was recycling of ochratoxin a, then perhaps the low incorporation of[l0- 14C] ochratoxin a into [10J C} ochratoxin B (0.47%) was due to the indirect incorporation of[10J4C] ochratoxin a-derived [ 14C] acetate. 133 Measured { Calculated } dpm dpm nCi % Inc. (8%) (100%) [10-14C] Ochratoxin a: [ 14C]ochratoxinA 1120 14000 6.31 4.85 [ 14C]ochratoxinB 109 1363 0.61 0.47 [10- 14C] Ochratoxin 13: [ 14C]ochratoxinA 2543 31788 14.32 14.32 [ 14C]ochratoxinB 3370 42125 18.98 18.98 [14C] Mellem: [ 14C]ochratoxinA 95 1188 0.54 0.56 [ 14C]ochratoxinB 115 1438 0.65 0.68 Table 5.4: Radioactivity (expressed as disintegrations per minute-dpm- and nCi) of [14C] ochratoxins A and B recovered 5 hours after the addition of either 0.13 tCi [10-14C} ochratoxin a, 0.1 pCi [1 0- 14C] ochratoxin 13 or 0.096 pCi [U- 14CJ mellein to Aspergillus ochraceus D2306 shaken shredded wheat fermentations. % Inc. Indicates the percentage Incorporation of the radiolabels Into the recovered [14C] ochratoxins A and B. 134 I IN oA._, 1 2 3 4 5 6 Figure 5.5: An autoradiograph of a TLC plate of bicarbonate soluble and insoluble fractions of extracts of D2306 SW cultures fed radiolabelled putative advanced intermediates of ochratoxin A biosynthesis. Lanes 1, 3 and 5 correspond to bicarbonate soluble fractions of extracts of cultures fed [1O-' 4C] ochratoxin a, [10- 14C] ochratoxin f3 or [ 14C] mellein, respectively. Lanes 2, 4 and 6 correspond to the equivalent bicarbonate insoluble fractions. OA, Oa, OB and 013 indicate the positions of ochratoxins A, a, B and 13 on the TLC plate, respectively. These coincided exactly with the revealed radiolabel. 135 Lane 3 shows the incorporation of[10-' 4C1J ochratoxin 13 directly into [10-14C] ochratoxins A and B. There appears to be less metabolism of[10- 14C] ochratoxin 13 to neutral non-polar compounds than [1O- 14C] ochratoxin cx (lane 4). Lane 5 shows the lack of any strongly radiolabelled acidic compounds from the cultures fed [14C] mellein and the neutral radiolabelled compounds from [ 14C] mellein are also comparatively weak (lane 6). Radioactivity added to the [14C] mellein fed culture was equivalent in amount to that fed to the other two cultures, so why was there so little label detected by the autoradiograph? Perhaps the [14C] mellein-derived compounds were strongly cell bound and remained undetectable using the current extraction procedure. The lack of mellein and any specific [ 14C] mellem-derived compounds in the extracts suggests that the low incorporation of [14C] mellein into [ 14C] ochratoxins A and B (0.56 and 0.68%, respectively) may be due only to the indirect incorporation of [14C] mellein-derived [ 14C] acetate. Thus, there is compelling evidence to suggest that the latter events in the biosynthesis of ochratoxin A could be: the chlorination of ochratoxin 13 to produce ochratoxin a, which is then converted directly to ochratoxin A by condensation with phenylalanine. In addition, ochratoxin 13 is directly converted to ochratoxin B. However, ochratoxin 13 could be incorporated into ochratoxin A via the direct chlorination of ochratoxin B. No experimental evidence to support the inclusion of mellein has been forthcoming. of 5.3.7: The interconversion [10- 14C, phenylalanyl-3H] ochratoxins A and B into ochratoxins A and B. The radioactivity of the dual labelled ochratoxins A and B recovered after the addition of both 5 pCi [methyl- 14C] methionine and [2, 3, 4, 5, 6-3H1 phenylalanine is shown in table 5.5. Both precursors were incorporated into the ochratoxins, although with lower efficiency than expected. Perhaps the additions were a little premature with regard to ochratoxins A and B production. The ratio of 3H: 14C was 1:0.49 and 1:0.39 for the dual labelled ochratoxins A and B, respectively. This is consistent with previous investigations which have shown that phenylalanine is more readily 136 Compound Isotope Measured Calculated ______dpm (10%) dpm (100%) pCi % inc. OchratoxinA 3H 33880 338800 0.153 3.1 14C 16481 164810 0.074 1.5 Ochratoxin B 3H 65020 650200 0.293 5.9 ______14C 25412 254120 0.114 2.3 Table 5.5: The radioactivity (expressed as disintegrations per minute-dpm- and pCi) of dual labelled ochratoxins A and B recovered after the addition of 5 pCi each of [methyl-14C] methionine and [2, 3, 4, 5, 6-3H] phenylalanine to a 4 day old A. ochraceus D2306 shaken SW culture. % inc. indicates the percentage incorporation of the fed radiolabels into the recovered dual labelled ochratoxins A and B. 137 incorporated into the ochratoxins than is a methyl group from methionine. The radioactivity of the dual labelled ochratoxins A and B recovered after the addition of either dual labelled ochratoxins A or B is shown in table 5.6. Approximately 43% of the added dual labelled ochratoxin A was recovered after the 5 hour incubation and its 3H: 14C ratio was vely similar to that of the fed material (1:0.47 compared to 1:0.49). The fed dual labelled ochratoxin A was also incorporated into ochratoxin B, but to a lesser degree (approximately 10%) and also in part non-specifically, since the 3H: 14C ratio had changed to 1:0.33. Apparently the phenylalanine moiety of ochratoxin A can be transferred to ochratoxin B more readily than the dihydroisocoumarin moiety, which has to undergo an additional de-chiorination step. A very low percentage of the added dual labelled ochratoxin B was recovered (approximately 6.5%) after the 5 hour incubation and its 3H:' 4C ratio was quite close to that of the material added (1:0.37 compared to 1:0.39). The low recovery, and also possibly the slight change in 3H: 14C ratio, suggests there was a lot of metabolic activity involving ochratoxin B. An even smaller proportion of ochratoxin B was incorporated into ochratoxin A, and once again the 3H: 14C ratio was similar (1:0.43 compared to 1:0.39). It is very difficult to determine whether these changes in 3H: 14C ratio are significant or not. In the absence of treatment replicates, the observed changes may be within experimental error, especially with respect to differentiating energy signals in the scintillation counting of 3H and 14C mixtures. The first feeding experiment used very little of the dual labelled ochratoxins A and B due to their poor solubility in water. However, in the second experiment, the dual labelled ochratoxin A was administered much more efficiently in a bicarbonate solution. In this second feeding experiment, approximately 58% of the added dual labelled ochratoxin A was recovered after an overnight incubation. This increase in recovery from the first experiment may be an indication of the decrease in ochratoxin A turnover at this later feeding time in the fermentation. Once again, the 3H: 14C ratio of the recovered dual labelled ochratoxin A was very similar to that of the fed material (1:0.48 compared to 1:0.49). The fed dual labelled ochratoxin A was also less 138 Isotope Measured Calculated 3H:14C ______dpm ( 8%J dpm (100%) nCi % Inc. ______ 1st OA feed: Ochratoxrn A 3H 782 9775 4.4 43.2 1:0.47 14C 368 4600 2.07 41.5 Ochratoxin B 3H 219 2738 1.23 12.1 1:0.33 14C 73 913 0.41 8.2 1st OB feed: Ochratoxrn A 3H 487 6088 2.74 1.4 1:0.43 14C 212 2650 1.19 1.5 Ochratoxin B 3H 2394 29925 13.48 6.7 1:0.37 14C 895 11188 5.04 6.4 2nd OA feed: OchratoxinA 3H 5871 73388 33.06 58.3 1:0.48 14C 2827 35338 15.92 57.5 OchratoxinB 3H 337 4213 1.9 3.3 1:0.29 14C 98 1225 0.55 2 Table 5.6: The radioactivity (expressed as disintegrations per minute-dpm- and nCi) of dual labelled ochratoxins A and B recovered after the addition of dual labelled ochratoxins A and B to 5 day old (1St OA and OB feeds) or 6 day old (2nd OA feed) A. ochraceus D2306 shaken SW cultures. % inc. Indicates the percentage Incorporation of the fed radiolabels into the recovered dual labelled ochratoxins A and B. 139 specifically incorporated into ochratoxin B (having a 3H: 14C ratio of 1:0.29) and with rather low efficiency (approximately 3%). The autoradiograph of the extracts of the cultures fed the dual labelled ochratoxins A and B showed the absence of any label in the bicarbonate insoluble fractions. This is in contrast to the results from feeding [ 14C] ochratoxins a, 1 and mellein to D2306 fermentations. The radioactivity of the bicarbonate soluble extracts of the cultures fed dual labelled ochratoxin A was restricted to the areas on the TLC plate corresponding to ochratoxin A, whereas the radioactivity of the bicarbonate soluble extracts of the culture fed ochratoxin B was restricted to the areas corresponding to ochratoxin B and, to a lesser extent, ochratoxin A. The low percentage recovery of the added dual labelled ochratoxins fed indicates that there was some metabolism of them. However, they probably were not broken down to acetate, since acetate would almost inevitably be incorporated into compounds extractable by ethyl acetate and detectable by autoradiography. Therefore, the breakdown products of both added ochratoxins A and B were resistant to the ethyl acetate extraction procedure, perhaps because they were bound tightly within the flingal cells. Thus, it was shown that although ochratoxin A can be converted to ochratoxin B non- specifically, there was no convincing evidence to show whether or not specific incorporation of ochratoxin B into ochratoxin A had occurred. 140 6: The chemical degradation of ochratoxin A. 6.1: Introduction. In order to make the results of studies on the incorporation of radiolabelled advanced intermediates more compelling, it is necessaiy to show that any incorporations were specific. The ['4C1 ochratoxins A and B purified from shaken solid substrate D2306 cultures fed [methyl-'4C1 methionine (see 5.2.5) were theoretically labelled in position C-b which can be isolated from the rest of the molecule. Steyn eta!. (1970) demonstrated that the C-10 carbon of ochratoxin A was derived from methionine by acid hydrolysis of [b0-'4C1 ochratoxin A (from [methyl- 14C1 methionine) to yield [10-'4C] ochratoxin a and phenylalanine. The [10-'4C] ochratoxin a was then methylated to produce the [10-' 4C] 0-methyl, methyl derivative (figure 6.1, XXXVI) which was subsequently saponified to yield [10-' 4C] 0-methylochratoxin a (figure 6.1, XXXVII). [10-14C] 0-Methylochratoxin a was converted to the [10-' 4C] acid chloride (figure 6.1, XXXVIII) and then to the [10-' 4C] azide (figure 6.1, XXXIX). Conc. H2SO4 was added to the [10- 14C] azide derivative to release the 14C-10 carbon as 14CO2, which was collected and scintillation counted, leaving the 0- methylochratoxin a amine (figure 6.1, XL). There is, however, an alternative degradation pathway. Ochratoxin A has been shown readily to become methylated to produce the 0-methyl, methyl derivative (section 3.3.5; van der Merwe eta!., 1965 b; Phillips eta!., 1983; figure 6.2, XXIX) which then could be hydrolysed directly to 0-methylochratoxin a (figure 6.2, XXXVII). The acid could then be converted to the azide (figure 6.2, XXXIX) or the isocyanate (figure 6.2, XLI) using diphenyiphosphoryl azide (DPPA; Ninomiya et aL, 1974; Denholm et aL, 1995). Both the azide and isocyanate derivatives will yield the C-10 as CO2. when treated with conc. H2SO4, to leave the 0-methylochratoxin a amine (figure 6.2, XL). Both chemical degradation schemes are shown in figures 6.1 and 6.2. 141 A Jr * r.r__I_CH3 Phenylalaniie + HOOC Ociatoxm a H 1 0 DAnnetharie b 24 )x,in * CH3O0C— .)L,0 XXXVI 0-MetI mek,ctiatoxx a IVt.113 IU Jrsn HOOzIçxXVll 0-Me ybdiatoxin a OCH3 0 Reed in SOC fbr 2 hours CH3 0-Meth4ochatoxm a acid chloride JrNaN3 N3O_IJ1X00d1X 0-Me cwatoxin a thie OCH3 0 I Conc. H2SO4 added to azide v CkH=CCl2 at 35 O( 4,ovet.lOniIles CH3 * CO2 + XL (coected as BaCO3) 0-MeiIbcliatoxi a anine OCH3 0 Figure 6.1: The chemical degradation of ochratoxin A (Steyn ci aL, 1970). 142 Co Q—CH2H ratoxin A Excess d )nthax in nct11 C' COOCH3 O_CH2H _I%f1I3 NH-CO 0-Methyl, nd'kc1ratoxn A Hydro * PhotyIaIanhie + XXXVII O-Methybclratoxhi a /IS\ tori in &y THF, sthed fr 18 hours * N IX OCN O-Methybclwatoxhi a aziie 0-MetIk)clratoxhi a ocyanate Coi. H2SO4 added to azkIe1ocyanate in CH–CC at 35 °C over 10 munites * CO2+ * (colcted as BaCO3) 0-Methylochratoxina aninc H3 Figure 6.2: An alternative chemical degradation scheme of ochratoxin A. '43 6.2: Materials and methods. 6.2.1: Experiments involving ochratoxin A and its derivatives. Ochratoxin A has been demonstrated to produce the 0-methyl, methyl derivative when treated with diazomethane, arid ochratoxin a when refluxed in 6M HCI overnight. However, the conversion of 0-methyl, methylochratoxin A to 0- methylochratoxin a has, so far, not been shown. Thus, 0-methyl, methylochratoxin A (approximately 1 mg) was refluxed overnight in 50 ml 6M HCI. After cooling, the reaction mixture was extracted with an equal volume of ethyl acetate, and the organic phase was analysed by DAD-HPLC. Another 1 mg of 0-methyl, methylochratoxin A was added to 50 ml 6M HC1 but was hydrolysed by limited refluxing under nitrogen (for approximately 15 minutes) followed by standing overnight. The reaction mixture was extracted and analysed as above. A further 1 mg of 0-methyl, methylochratoxin A was added to 25 ml 0. 1M NaCI I 0.02M Tris buffer (pH 7.5) to which 2 mg carboxypeptidase A was added. The mixture was incubated, stirring, at room temperature overnight. After incubation, the reaction mixture was made up to 100 ml using slightly acidic water which was then partitioned against an equal volume of ethyl acetate. The organic phase was analysed by DAD-HPLC. Another 1 mg of 0-methyl, methylochratoxin A was added to 10 ml 50 mM ammonium bicarbonate buffer (pH 8.5) containing approximately 2 mg of a chymotrypsin which was incubated at 37 °C for 3 hours. After incubation, the reaction mixture was extracted and analysed in the manner used in the carboxypeptidase A experiment. The a chymotrypsin digestion investigation was repeated with 2.5 mg of ochratoxin A. Ochratoxin A (approximately 5.5 mg) was treated with 2 mg carboxypeptidase A, using the conditions described above, and after 0.5, 1.5 and 20 hours, 20 .d of the reaction mixture was removed and injected directly into the DAD-HPLC in order to monitor the reaction. 0-Methylochratoxin A (approximately 0.5 mg) was refluxed in 50 ml 6M HC1 under nitrogen overnight. After cooling, the reaction mixture was extracted with an equal 144 volume of ethyl acetate which was analysed by DAD-HPLC. Another 0.5 mg of 0- methylochratoxin A was refluxed in 100 ml 6M HC1 under nitrogen. Ten ml aliquots were removed from the reaction mixture hourly and, after cooling, were extracted with an equal volume of ethyl acetate. All of the samples were analysed by DAD- HPLC. A further 0.5 mg of 0-methylochratoxin A (in I ml methanol) was methylated by the addition of diazomethane (1 ml of a 60% ethereal solution). The solvent was allowed to evaporate overnight and the ensuing solid was dissolved in methanol prior to analysis by DAD-HPLC. 6.2.2: Experiments involving ochratoxin a and its derivatives. Ochratoxin a (approximately 1 mg in 1 ml methanol) was methylated with diazomethane (1 ml of a 60% ethereal solution was added dropwise). The reaction mixture was kept overnight in a stoppered 3 ml glass vial before being dried, re- dissolved in methanol and then analysed by DAD-HPLC. The remaining sample was subjected to preparative HPLC using the same conditions used to puri& ochratoxin a. 0-Methyl, methylochratoxin a (approximately 0.2 mg in 1 ml methanol) was treated with diazomethane in the same manner as ochratoxin a. The reaction mixture was also left overnight in a stoppered 3 ml glass vial, dried, re-dissolved in methanol and then analysed by DAD-HPLC. 6.3: Results and discussion. 6.3.1: Experiments involving ochratoxin A and its derivatives. 0-Methyl, methylochratoxin A refluxed overnight in HCI yielded a compound which was identified (by its DAD-HPLC retention time, UV absorbance spectrum and El- MS) as ochratoxin a. Apparently, the methyl ether bond is susceptible to hydrolysis by a strong acid under harsh conditions. The limited acid hydrolysis of 0-methyl, methylochratoxin A resulted in the detection of three compounds: ochratoxin a, unknown compound I and unknown compound II. Treatment of 0-methyl, 145 methylochratoxin A with carboxypeptidase A revealed two compounds detectable by DAD-HPLC: ochratoxin a and another compound which had the same retention time and UV absorbance spectrum as unknown compound I. Treatment of 0-methyl, methylochratoxin A with a chymotrypsin revealed two compounds detectable by DAD-HPLC: a compound which had the same retention time and UV absorbance spectrum as unknown compound I and another compound adjudged to be 0-methyl, methylochratoxin A (also based on its same retention time and UV absorbance spectrum). The unknown compound I was purified by preparative HPLC using the same conditions used to purify ochratoxin a. The eluate corresponding to the unknown compound 1 was collected, dried and then analysed by El accurate mass measurement spectrometry. The ensuing accurate mass El spectrum (figure 6.3) was very similar to the ochratoxin A El mass spectrum (figure 3.2) but had the ochratoxin A fragmentation ions m/z 403, 255 and 239, all 14 mass units (+CH3, -H) higher. The El mass spectrum also shared many ions with the 0-methyl, methylochratoxin A El mass spectrum (figure 3.9). The fragmentation pattern and molecular formulae of the fragmentation ions were consistent with that of 0-methylochratoxin A (figure 6.4, XLII). Unfortunately, there is no published 0-methylochratoxin A El mass spectrum available for companson. The DAD-HPLC chromatogram and UV absorbance spectrum of 0- methylochratoxin A is shown in figures 6.5 and 6.6. 0-Methylochratoxin A shares its UV absorbance maxima with 0-methyl, methylochratoxin A (241 and 310 nm) which suggests that the observed shift from the ochratoxin A maxima (218 and 332 nm) was due to the methylation of the C-8 hydroxyl group rather than of the phenylalanine moiety carboxylic acid. * No attempt was made to identify unknown compound II, although its UV absorbance spectrum was very similar to that of ochratoxin a (absorbance maxima at 226 and 334 tim compared with 222 and 334 tim for ochratoxin a), although its DAD-HPLC retention time was very much longer (10.09 compared with 1.56 minutes for * due to time limitation 146 mlz Formula 417 C21H20N06C1 400 C21H19N05C1 372 C20H19N04C1 269 C12H12N04C1 253 C12H1004C1 239 C11H804C1 Figure 6.3: Accurate mass electron impact spectrum of O-methylochratoxin A. 147 CI CH3 COOH II NH—C II I II 0 OCH3O Figure 6.4: The structure of O-methylochratoxin A (XLL1). 148 Figure 6.5: DAD-HPLC chromatogram of O-methylochratoxin A. Figure 6.6: The UV absorbance spectrum of O-methylochratoxin A. 149 ochratoxin a). The lack of shift of the observed UV absorbance maxima from that of ochratoxin a suggests that there was no 0-methylation. Ochratoxin A was unchanged by treatment with a chyinotrypsm, whereas it was hydrolysed to ochratoxin a in the presence of carboxypeptidase A. Ochratoxin a was detected after 0.5 hours and the reaction was complete by 20 hours. These results are consistent with Pitout (1969) who showed that carboxypeptidase A catalysed the hydrolysis of ochratoxin A to ochratoxin a and phenylalanine, whereas a chymotrypsin showed only low activity and any activity that it did have may be attributed to carboxypeptidase A contamination. It was also stated that carboxypeptidase A was stereospecific for the L form and required a free carboxyl group to function. The products of the treatment of 0-methyl, methylochratoxin A with carboxypeptidase A seemed to contradict reports stating the requirement of a free carboxylic acid group. However, perhaps it is the methylation of the carboxylic acid which caused the alteration in the enzymes activity. Instead of cleaving the ainide bond to yield 0-methylochratoxin a, the enzyme removed the methyl groups, whether ester or ether linked. It is not clear whether the de-methylation steps are required prior to the cleaving of the amide bond, although the absence of 0-methylochratoxin a in the reaction products suggests that perhaps they are, a Chymotrypsin was also * shown to cleave the methyl ester link, although not the methyl ether bond. Overnight acid hydrolysis (under nitrogen) of 0-methylochratoxin A resulted in the production of ochratoxin a. In the time course experiment, the conversion appeared to be direct with the reaction being completed within 3 hours. Another less harsh method is required to cleave only the phenylalanine moiety to leave 0- methylochratoxin a. 0-Methylochratoxin A treated with diazomethane resulted in the production of 0- methyl, methylochratoxin A (continued by its DAD-HPLC retention time and UV absorbance spectrum). * which could easily have been an effect of the reaction nuxture 150 6.3.2: Experiments involving ochratoxin a and its derivatives. Ochratoxin a treated with diazomethane resulted in three compounds detectable by DAD-HPLC (figure 6.7). The first compound to be eluted was identified as ochratoxin a based on its DAD-HPLC retention time, UV absorbance spectrum and El-MS data. The compounds eluted second and third were named unknown compounds Ill and IV, respectively. The UV absorbance spectrum of the unknown compound ifi showed a shift in UV maxima characteristic of an 0-methylation (229 and 310 nm compared to 222 and 334 nm of ochratoxin a), whereas the UV absorbance maxima of the unknown compound IV was the same as ochratoxin a (figure 6.8). Preparative HPLC using the same conditions used to purify ochratoxin a was used to purify unknown compound III. The eluate corresponding to the unknown compound (which had retention times of 19.5 minutes) was collected, dried and analysed by El accurate mass measurement spectrometry. Unknown compound ifi was identified as 0-methyl, methylochratoxin a (figure 6.1, XXXVI) based on its El mass spectrum (figure 6.9) in addition to the UV absorbance spectrum evidence. The 0-methyl, methylochratoxin a El mass spectrum is quite dissimilar to that of ochratoxin a. The mass deficient ions in the spectrum (297.9828, 250.9880, 166.9893 etc) are markers used in accurate mass measurement calculations. The measured fragmentation ions of 0-methyl, methylochratoxin a are not simply ochratoxin a ions which are 14 (CH3 - H) or 28 (C2H6 - 2H) mass units higher, with the exception of the molecular ion m/z 284 (256 + 28) and mlz 208 (194 + 14). The ion m/z 253 appears to be the molecular ion which has lost a C 2H3 of which there is no corresponding fragmentation in ochratoxin a El mass spectrometry. The remaining two measured ions of m/z 224 and 239 may correspond to ochratoxin a ions m/z 194 and 223, respectively, which are 14 mass units plus an additional 2H greater in mass. * No attempt was made to identify unknown compound IV, although its lack of shift of the observed UV absorbance maxima from that of ochratoxin a suggests that there was no 0-methylation. It may be significant that methylation of both ochratoxins A * due to time limitation 151 Figure 6.7: DAD-HPLC chromatogram of ochratoxin a, 0-methyl, methylochratoxin a and unknown IV. L] DADI, 1.530(953 mAU,Apx) of 0A0504.D No 17501 1"1% 'i 1500 Ii 12501 I 1o00- IJit ' 1' \' 750j I, ' Ii 500 350 Figure 6.8: The UV absorbance spectra of ochratoxrn a ("), 0-methyl, methylochratoxin a (-) with unknown IV (-). 152 m/z Formula 284 C13H1305C1 253 C11H705C1 239 C11H804C1 224 C11H903C1 208 C11H902C1 Figure 6.9: Accurate mass electron impact spectrum of 0-methyl, methylocbratoxin cL 153 and a resulted in compounds being produced that had longer DAD-HPLC retention times than the di-methylated products, yet were apparently not 0-methylated. There was no change to 0-methyl, methylochratoxin a when it was treated with diazomethane, according to DAD-HPLC and UV absorbance spectrum analyses. Limited time precluded further investigation into the chemical degradation of ochratoxin A. 154 7: Feeding I'4C1 diaporthin diaporthin and orthosporin to Penicillium citrinum lEa potato dextrose broth fermentations. 7.1: Introduction. Diaporthin is a dihydroisocoumann-Iike polyketide but which lacks a C 1 group attached at position C-7 that is found in ochratoxins A and B (figures 1.1, I and II and 1.21, XXVII). Citrinin (figure 7.1, XLffl), however, is not only a polyketide with similar dihydroisocoumarin ring structure, but is methylated at C-7 and is produced in relatively large amounts by some P. citrinum isolates. Notwithstanding the current thinking on citrinin biosynthesis (see appendix IX), an investigation to see whether a citrininogenic P. citrinum isolate could methylate diaporthin at position C-7 was devised. This experiment might also discern some biological activity of diaporthin and/or indicate its metabolic fate in another fungus. Various isolates of P. citrinum grown on PDA slopes were tested for yellow fluorescence in the medium under UV light (350 nm) as this is a good indicator of citrinin. Three isolates with the brightest fluorescence were selected for further investigation (lEa, l4Aa and l8Ha: Mantle and McHugh, 1993). lEa was ultimately chosen as the experimental organism as it was found to produce the most citrinin in either PDB or Bhattacharya medium (Bhattacharya and Majumdar, 1984) shake flask fermentations. 7.2: Materials and Methods. Production of[ 14C1 diaporthin was achieved by feeding [1- 14C] acetate to an Aspergillus ochraceus D2306 PDB fermentation. A 100 ml scale PDB culture was inoculated with spores and incubated, shaken, at 28 °C. [1-' 4C] Acetate (5 i.tCi) was added to the flask 51, 75 and 99 hours post inoculation. The culture was harvested 69 hours after the last label addition. The broth was extracted and the diaporthin purified 155 11 10 H3 CH3 O,1CH 2i-. Sa HO2C OH XLIII Figure 7.1: The structure of citrinin (XLIII). Figure 7.2: The accumulation of extracellular citrinin in a P. citrinurn lEa potato dextrose broth fermentation. 200 180 160 140 120 E 100 80 60 40 20 0 0 10 20 30 40 50 60 70 80 Time (hours) 156 (see 3.2.6). The quantity and purity of the diaporthin was assessed using DAD-HPLC, whilst its radioactivity was measured using scintillation counting. The dynamics of citrinin production by lEa was investigated so as to be able to feed the A. ochraceus metabolites at an opportune time. Thus, two 100 ml scale lEa PDB cultures were inoculated using a spore suspension (spores of a mature lEa PDA slope were suspended in 4 ml sterile water containing a drop of Tween 80). The initial spore concentration of the lEa PDB cultures was approximately 2 x i0 5 spores/mi. The flasks were then incubated, shaken, at 28 °C and at appropriate time intervals, 10 ml samples were removed from alternate flasks for citnnin quantification using DAD- HPLC (see 2.2.5). Both the bicarbonate soluble fractions of the extracts (which contained the citrinin) and the bicarbonate insoluble fractions were analysed by DAD- HPLC using identical chromatographic conditions. For the addition of[14C] diaporthin, a 100 ml scale lEa PDB culture was initiated and incubated, shaken, at 28 °C. After 24 hours, 20 ml of the culture was removed to a sterile 500 ml conical flask containing 2.5 ml of a 5 ml sterile [ 14C] diaporthin solution (238 .tg of[ 14C] diaporthin was initially dissolved in 200 j.tl ethanol before being made up to 5 ml with sterile water) and allowed to continue to incubate. The remaining [ 14C] diaporthin was added 8 hours after the first feed, and the flask was incubated for a fhrther 16 hours before being extracted. An independent lEa PDB control flask in which 20 ml of the culture was removed to a sterile 500 ml flask 24 hours post inoculation and allowed to incubate for a further 24 hours before being extracted, was also analysed. All bicarbonate soluble and insoluble fractions were analysed by DAD-I{PLC, scintillation counting and TLC-autoradiography. For the experiment in which the effect of unlabelled diaporthin or orthosporin was studied, a 100 ml scale lEa PDB culture was initiated and incubated as above. At 24 hours post inoculation, three 30 ml aliquots were removed to three sterile 500 ml conical flasks. To two flasks was added 2.5 ml of a solution of either diaporthin or orthosporin (360 j.tg was dissolved in 200 ethanol which was then made up to 5 ml with sterile water). To the third (control) flask was added 2.5 ml sterile water 157 containing 100 .i.l ethanol. The cultures were incubated for a further 8 hours (32 hours post inoculation) prior to re-dosing exactly as before. At 48 hours post inoculation, half of all of the fed flasks' contents were harvested, with the remaining half being extracted 8 hours later (56 hours post inoculation). All of the samples were analysed by DAD-HPLC. 7.3: Results and discussion. The feeding of[1- 14C] acetate at three stages to D2306 PDB cultures was designed to ensure some success, since there was insufficient knowledge of the precise dynamics of diaporthin production. Nevertheless, in practise the label was added during the diaporthin production phase (figure 2.8). The [ 14C} diaporthin (238 p.g) recovered from the fed flask (an estimated extraction yield of 76%) had a radioactivity of 39222 dpm. The specffic radioactivity was 165 dpm/p.g, which bettered previous values obtained when D2306 was fed [1-' 4C] acetate (see 4.3). Perhaps an even higher specific radioactivity could have been achieved by feeding the labelled precursor at a much earlier stage of diaporthin biosynthesis (around 24 hours post inoculation) and then harvesting soon after (perhaps after only 1 hour). This technique worked very well when producing [ 14C} mellein of high specific radioactivity (see 5.2.4). The dynamics of citrinin production by lEa in PDB is shown in figure 7.2. Extracellular citrinin concentrations reached fairly high levels (approaching 200 mg/L) which compared favourably with previously published figures of 133 mgfL (Montani eta!., 1988) and 410 mg/L (Bhattacharya and Majumdar, 1984). The high yields of citnnin produced by P. citrinum in a simple medium presumably aided its early discovery and characterisation by Hetherington and Raistrick (1931). In addition to citrinin, there were two other prominent compounds in the bicarbonate soluble lEa DAD-HPLC metabolic profile: acidic 'A' and B', and another (neutral 'A') which did not partition into bicarbonate but yet had a DAD-HPLC retention time and 158 a UV absorption spectrum almost identical to acidic 'A'. Figure 7.3 shows the resolution of acidic 'A' and 'B' and neutral 'A' in the DAD-HPLC system, whilst figure 7.4 shows their UV absorbance spectra. The dynamics of acidic 'A' and 'B' and neutral 'A' production by lEa in PDB is shown in figure 7.5. Acidic 'A' was often not produced in lEa PDB fermentations or, when it was, in comparatively low amounts (up to 2.26 x 106 DAD peak area, mAU*sec, per litre at X 327 nm) which resulted in all attempts at its purification and characterisation being unsuccessfiul. The production of neutral 'A' mirrored that of acidic 'A', although yields of the former were consistently higher. Perhaps acidic and neutral 'A' are one and the same compound, and its occurrence in both bicarbonate soluble and insoluble fractions of the lEa PDB extracts is due to its poor solubility in bicarbonate. Notably, acid 'A' only occurred concurrently with neutral 'A' in the lEa PDB fermentations. Despite being produced in higher quantities than acidic 'A', neutral 'A' was produced erratically and therefore time was not devoted to its isolation and characterisation. Acidic 'B', however, was produced much more consistently, and considerable progress was made toward its isolation and purification. A 40 hours post inoculation 100 ml scale lEa PDB culture was extracted and the ensuing bicarbonate soluble fraction was subjected to preparative HPLC (acetonitrile-acetic acid-water: 39.5:1:59.5 flowing at 8.4 mI/mm, X 297 nm) and the eluate corresponding to the unknown acidic compound (retention time 28 minutes) was collected and dried. Compound purity was determined by DAD-HPLC and the compound was analysed by El and fast atom bombardment (FAB) mass spectrometry. FAB-MS was performed using Cs at 30-3 5 KeY as the primary ion beam and 3-mtrobenzyl alcohol as the matrix. Unfortunately, neither FAB nor El mass spectrometry were successflul in producing a meaningflul mass spectrum of acidic 'B' which could aid its identification. The reason for the poor mass spectrometric analyses may be due to the lack of material, but also to the compound not being volatile enough. Derivatisation of acidic 'B should be possible by forming methyl or trimethylsilyl esters to increase volatility, since there should be at least one carboxylic acid group. However, care must be taken 159 Figure 7.3: DAD-FIPLC chromatograms showing the resolution of acidic 'A' and 'B' and neutral 'A'. 160 Si Acidic 'A' #3 Acidic 'B' Figure 7.4: UV absorption spectra of acidic 'A' and 'B' and neutral 'A'. 161 Figure 7.5: The accumulation of acidic 'A' and 'B' and neutral 'A' in a P. citrinuin potato dextrose broth - fennentation. 25 ;-'0 II 10 0 0 10 20 30 40 50 60 70 80 Time (hours) [Acidic 'A'] ° [Acidic '13'] ' fNeutraI 'A'] 162 when interpreting any forthcoming MS data and the assignment of the molecular and fragmentation ions. According to figure 7.2, at the times of[ 14CJ diaporthin addition (24 and 32 hours post inoculation), citrinin should just be starting to accumulate in the broth, presumably indicating that the start of citrinin biosynthesis was not long before. At the time of harvesting (48 hours post inoculation), citrinin biosynthesis with its methylation step at C-7 should be firmly established, with an extracellular citrinin concentration of approximately 40 mgfL. However, DAD-HPLC of bicarbonate soluble extracts of the lEa PDB cultures fed [14C} diaporthin showed there to be no citrinin, although acidic 'B' was present in usual concentrations (approximately 27 x 106 mAU*secfL). Acidic and neutral 'A' were also detected, both at enhanced levels (72 and 69 x 106 mAU*sec/L, respectively). There was no sign of residual [14C} diaporthin. This apparent inhibition of citrinin biosynthesis and concurrent increase in acidic and neutral 'A' (by diaporthin) may be an indication of the biosynthetic connectivity of these compounds. Perhaps diaporthin may be used as an inhibitor to investigate other similar biosynthetic pathways. Scintillation counting of the lEa PDB extracts fed [14C] diaporthin showed 2540 and 13578 dpm radioactivity, corresponding to 6.5 and 34.6% of the added radiolabel, in the bicarbonate insoluble and soluble fractions, respectively. Thus, there was a shift of radioactivity from bicarbonate insoluble (i.e. [ 14C] diaporthin) to soluble as the [14C} diaporthin was metabolised. An autoradiograph of a developed TLC plate of the bicarbonate soluble and insoluble fractions (using toluene-ethyl acetate-formic acid, 50:40:10, as the mobile phase) is shown in figure 7.6. The intensity of the bands on the X-ray film reflects the distribution of the label into bicarbonate soluble and insoluble fractions suggested by the scintillation counting results. It is striking that [14C] diaporthin was biotransformed into essentially 3 compounds discernible by the extraction and TLC procedures. This is incompatible with [14C] diaporthin being catabolised to [ 14C] acetate before being distributed throughout the organism, especially as there was no radioactivity associated with the relatively non-polar, 163 • •••. 1 4 - c (-3 14 -. . • * -BiC +BiC Figure 7.6: An autoradiograph of a TLC plate of bicarbonate insoluble (-BiC) and soluble (+BiC) fractions of extracts of a lEa PDB culture fed [ 14C] diaporthin with the corresponding TLC information including the appearence of the compounds of interest in daylight and under UV light (at 254 and 350 nm). 164 acetate derived sterols in the bicarbonate insoluble fractions which in TLC run near the solvent front. The TLC information of the 3 biotransformation products is provided in figure 7.6. The silica corresponding to compound 3 was scraped off and extracted in propan-2-ol but unfortunately, there was too little for further investigation, perhaps suggesting that it was a biotransformation product of [ 14C] diaporthin that was not a native metabolite of P. citrinum. The independent control flask showed that removing 20 ml of the culture to a sterile 500 ml conical flask did not appear to affect the production of citrinin to a great extent: 30.83 and 37.04 mgfL citrinin were detected in the daughter and mother flask at 48 hours post inoculation, respectively. The DAD-HPLC analysis of the non-radioactive diaporthin and orthosporin feeding experiment showed that there was essentially no difference between the experimental and the control citrinin, acidic 'A' and 'B' and neutral 'A' concentration values at both harvest times. There was no residual diaporthin detected in the bicarbonate insoluble fractions of extracts of the culture fed diaporthin, although orthosporin was detected in the bicarbonate insoluble fractions of extracts of the culture fed orthosporin (40 and 20 tg detected at 48 and 56 hours post inoculation harvest times, respectively). There was an additional compound in the equivalent bicarbonate soluble fractions which had a retention time and UV absorbance spectrum identical to those of orthosponn. This apparently acidic orthosporin was detected in very small amounts (a total of approximately 16 and 13 g were estimated at 48 and 56 hours post inoculation harvest times, respectively, which corresponds to an 8% conversion of the added orthosporin). Thus, of the 360 .tg of orthosporin originally added, only 25% can be accounted for in the extracts. The DAD-HPLC chromatogram of the acidic orthosporin, along with citrinin, is shown in figure 7.7, whilst figure 7.8 shows its UV absorbance spectrum compared with orthosporin. Unfortunately, the second feeding experiment did not reproduce the inhibitory effect of diaporthin on citrinin biosynthesis, despite being performed in a seemingly similar 165 Figure 7.7: DAD-HPLC chromatogram of acidic orthosporin and citrinin. Figure 7.8: Comparing the UV absorbance spectrum of acidic orthosporin (-) with orthosponn (-). 166 manner, although both experiments have hinted at mechanisms which are able to transform neutral dihydroisocoumarin-like compounds into acidic ones. It is considered that these speculative experiments probably deserve further attention to determine whether exploitable biotransforming potential is present. 167 8: Isolation and characterisation of fungi from unroasted coffee beans. 8.1: Introduction Ochratoxin A as a contaminant of coffee products has been the subject of many investigations. The natural occurrence of ochratoxin A in green coffee beans has been regarded as infrequent and at low levels. Examples of contamination rates for commercial green coffee beans are 22/335 in the range 20-360 pg/kg (Levi eta!., 1974), 2/201 samples in the range 24 and 96 pg/kg (Levi, 1980), 9/40 samples in the range 0.5-23 pg/kg (Cantafora et aL, 1983), 4/22 samples in the range 10-46 pg/kg (Tsubouchi eta!., 1984) and 17/29 samples (0.2-15 pg/kg) by Micco eta!. (1989). The highest frequency of ochratoxin A contamination may be due to the high degree of sensitivity of the assay employed in that study, and the highest level of ochratoxin A contamination was from beans that were heavily moulded. Mycobiotal evaluations of green coffee beans have been carried out by Levi et aL (1974), Mislivec eta!. (1983) and Tsubouchi et a!. (1984) and the general consensus was that Aspergillus species dominated, A. g!aucus, A. niger, A. tamarii, A. flavus and A. ochraceus being the five most common species in decreasing order of prevalence. Penici!!ium species were also found regularly. Some strains of A. ochraceus isolated from green coffee beans were strongly ochratoxinogenic on substrates such as rice (producing between 50 and 2088 pg/g substrate) yet less so on coffee beans (0.45 to 130 j.tg/g substrate) (Stack et aL, 1983; Tsubouchi et aL, 1984, 1985). Coffee beans have been noted as a poor substrate for ochratoxin A production by ochratoxinogenic A. ochraceus strains that had not been isolated from coffee beans (Levi eta!., 1974; Tsubouchi eta!., 1987). It was originally thought that caffeine inhibited both the growth of A. ochraceus and its production of ochratoxin A. Indeed it was shown that A. ochraceus isolate NRRL 3174 (not isolated from coffee beans) grown as a stationary culture on 3% yeast extract-6% sucrose (YES) medium at 28 °C grew nearly as well when the medium 168 was supplemented with caffeine (0.3%), but ochratoxin A production was up to 98% inhibited (Buchanan et aL, 1981). It has been estimated that green coffee beans have a caffeine concentration of approximately 8-9 mg/g (Tsubouchi eta!., 1985). Tsubouchi eta!. (1985) grew an ochratoxinogenic A. ochraceus strain (isolated from rice) as a stationary culture on YES medium (2% yeast extract-15% sucrose) supplemented with caffeine (up to 1%) and showed that the growth and ochratoxin A production were significantly inhibited even at the lowest caffeine concentration (0.1%) and completely inhibited at the highest caffeine concentration (1%). However, when the experiment was repeated using an A. ochraceus strain isolated from coffee beans, caffeine had no effect on growth but had a stimulatory effect on ochratoxin A production. It was also shown that the caffeine in the medium of the coffee bean A. ochraceus fermentations was depleted very rapidly. There is discrepancy in the literature concerning the resistance of ochratoxin A to roasting. Levi et aL (1974) spiked green coffee beans with ochratoxin A and mimicked industrial coffee bean roasting. It was found that 87% of the ochratoxin A was lost within 5 minutes at 200 °C. Tsubouchi eta!. (1987) inoculated green coffee beans with an ochratoxinogenic A. ochraceus strain and then analysed the dried infected beans for ochratoxin A before and after roasting. It was found that there was a reduction in ochratoxin A of only 1-12% after 20 minutes at 200 °C if the beans were roasted whole or a reduction of only 6-20% if the beans were roasted after being ground. It was argued that ochratoxin A produced within the bean by A. ochraceus was more heat resistant because it became bound to components of the bean. In the same study it was shown that ochratoxin A was stable in the decoction of contaminated roasted coffee beans. However, Micco eta!. (1989) showed that naturally contaminated green coffee beans lost 90-100% of ochratoxin A after roasting for 5-6 minutes, yet beans artificially contaminated showed only a reduction of ochratoxin A of 48-87% after roasting. It was suggested that the artificially contaminated beans contained more moisture which suppressed the effect of roasting on ochratoxin A (a phenomenon observed in 169 ochratoxin A destruction by heat in cereals; Scott, 1984). The study also showed that decaffeination (by steaming and then extracting with dichioromethane) reduced ochratoxin A by 60% in naturally contaminated green coffee beans and that coffee beverages produced by a standard Italian coffee making machine using artificially contaminated roasted coffee contained no traces of ochratoxin A. The only survey of commercial roasted coffee products was by Tsubouchi et aL (1988) who found that 5/68 samples analysed were contaminated with ochratoxin A (3.2-17.0 jig/kg). Despite this evidence, two recent reviews of carcinogenic mycotoxins in coffee products stated that mycotoxins are seldom found in green coffee beans and probably not found in roasted coffee (Maier, 1991 a, 1991 b). editorial Prompted by a recent article in New Scientist (8 February 1995, page 8) concerning evidence that ochratoxin A is a contaminant of roasted coffee products (at levels of approximately 5 .tgfkg) and that it is found in the ensuing decoction, a survey involving the isolation and charactensation of the mycobiota of green coffee beans destined for the British coffee market was undertaken. 8.2: Materials and methods. 8.2.1: Mycological analysis. Twenty green coffee beans from thirteen sources around the world (Arabica, Colombian Decaffeinated, Colombian Medellin, Colombian Supremo, Costa Rica SHB, Guatemala SHE, Honduras SHG, Kenya A/A, Kenya 'C', Kenya Gethumbwini, Mocha, Mysore and Uganda SC 18 Bold Bean Robusta) were surface sterilized by immersion into a 10% (v/v) chioros solution, containing a drop of Tween 80, for 10 minutes. The beans were washed five times in sterile water before 15/20 of them were plated onto DG-18 medium (5 beans per 9 cm petri dish containing 25 ml of medium) to aid the isolation of Penicillia and Aspergilli by suppressing zygomycetes. DG-18 medium was prepared by steaming 700 ml distilled water containing 15 g agar, 10 g glucose, 5 g peptone, I g KH2PO4 and 5 g MgSO4.7H20 for half an hour before 170 adding 220 g of glycerol and 1 ml of dichioran (0.2%, w/v, in ethanol). The mixture was made up to 1 L with distilled water prior to autoclaving (Hocking and Pitt, 1980). The beans were incubated at 25 °C and were checked regularly for emerging Penicillium and yellow Aspergillus ochraceus-like fungi. In addition, the plates were checked for fluorescence under UV light (350 and 254 nm), the beans were rated for % infection and a preliminaiy identification of the predominant contaminating fungal types was attempted. Representatives of sporing Peniciiium and Aspergillus fungi were subcultured onto PDA slopes, using sterile drawn glass Pasteur pipettes, and incubated at 25 °C. The PDA slopes were checked regularly for fluorescence under UV light (350 and 254 nm) and contamination, subculturing if necessaly. Penicillium species were identified according to Pitt (1979, 1988). The persistent yellow A. ochraceus isolates (Colombian Supremo Y-1, Honduras Y-1, Mocha Y-3, and Uganda Y-4, 5 and 6) and the P. citrinum isolate (Uganda P-7), all maintained on PDA slopes, were used to inoculate shredded wheat cultures (20 g SW in 500 ml conical flasks was autoclaved and moistened with sterile water to 4O% prior to inoculation with spores). A sample (1 g) of culture was removed from each flask 13 and 34 days post inoculation for extraction and to separate bicarbonate soluble and insoluble fractions for DAD-HPLC and TLC analysis. A. niger-like isolates were plated onto Czapek yeast agar (CYA) medium and grown at either 25 or 37 °C (9 cm diameter petri dishes containing 25 ml CYA medium were inoculated with spores from the relevant PDA slopes). CYA medium was comprised of 30 g sucrose, 15 g agar, 10 ml Czapek concentrate, 5 g yeast extract and 1 g K2HPO4 in 1 L distilled water. Czapek concentrate was comprised of 30 g NaNO3, 5 g KC1, 5 g MgSO4.7H20, 0.1 g FeSO4.7H20, 0.1 g ZnSO4.7H20 and 0.05 g CuSO4.5H20 in 100 ml distilled water (Pitt, 1979). The A. niger-like cultures were observed after 5 days. The two Penicillium isolates (Colombian Supremo P-i and 2) were provisionally identified as either P. crustosum or P. roquefortii and were used to inoculate stationary Czapek-Dox yeast extract (CDYE) broth cultures (100 ml CDYE broth 171 medium in 500 ml conical flasks was inoculated by scattering spores from the relevant * PDA slope culture on the surface of the medium). CDYE broth medium was comprised of 30 g sucrose, 5 g yeast extract, 3 g NaNO 3, I g KH2PO4, 0.5 g KC1, 0.5 g MgSO4.7H20 and 0.01 g FeSO4.7H20 in 1 L distilled water (Laws and Mantle, 1989). The flasks were incubated at 28 °C for 3 weeks prior to extraction for penitrems and/or roquefortine. The Penicillium metabolites were extracted by homogenising each of the ensuing mycelial mats in 100 ml acetone using a Sorvall Omni-Mixer. The homogenate was allowed to soak overnight before being filtered through cotton wool. The filtrate was rotary evaporated to dryness prior to TLC analysis. TLC was performed on 20 x 10 cm Polygram SIL G/UV 254 TLC plates (0.25 mm silica gel with fluorescent indicator UV254 pre-coated onto plastic sheets) in chloroform-acetone (19:1). Penitrems A, B, C, D, E and F and roquefortine can be visualised either by their quenching of the fluorescent indicator or with the use of a FeC13 spray reagent (3% w/v in butan-1-ol), followed by gentle heating, which renders the penitrems characteristic shades of green and roquefortine bluish grey. 8.2.2: Ochratoxins A and B analysis. Twenty-five g of green Uganda SC 18 Bold Bean Robusta coffee beans were milled twice using a Glen Creston coffee mill and then homogenised with 330 ml of ethyl acetate-0.01 M phosphoric acid (10:1) using a Sorvall Omni-Mixer. The mix was partitioned for ochratoxin A in the manner employed for SW cultures. The subsequent bicarbonate soluble fraction was initially analysed for ochratoxin A using DAD-HPLC and TLC at a limit of detection of 0.01 pg/g substrate. To improve the limit of detection and to eliminate spurious fluorescences, the bicarbonate soluble fraction was subjected to a clean up step based on Terada et al. (1986). The bicarbonate soluble fraction was dissolved in 12.5 ml water-0.2 M phosphate buffer (J)H 5.0)-0.05 M cetyltrimethylammonium bromide (10:2:0.5) and then introduced to a CiS Sep-Pak Classic cartridge (Millipore, Watford, UK) preconditioned first with 20 ml methanol, then with 20 ml water and lastly with 2 ml 0.005 M cetyltrimethylammonium bromide. 172 The cartridge was washed with 15 ml 40% aqueous acetone followed by 5 ml water prior to elution of ochratoxin A with 15 ml 40% aqueous acetonitrile. The collected aqueous acetonitrile fraction was made up to 50 ml with slightly acidic water and extracted with an equal volume of ethyl acetate. The post Sep-Pak fraction was subjected to DAD-HPLC and TLC analysis for ochratoxrn A with an improved detection limit of 1.4 ng/g substrate. To assess the efficiency of the C18 Sep-Pak clean up operation, 1.3 and 0.15 mg of ochratoxins A and B, respectively, were dried in a 100 ml round bottomed flask then dissolved in 12.5 ml water-0.2 M phosphate buffer (pH 5.0}-O.05 M cetyltrimethylammonium bromide (10:2:0.5) before being subjected to the C18 Sep- Pak clean up procedure as described above. In addition to the 40% aqueous acetonitrile, the eluate corresponding to the loading solution (12.5 ml water-0.2 M phosphate buffer (pH 5.0)-0.05 M cetyltrimethylammonium bromide, 10:2:0.5) and the combined 40% aqueous acetone and water washes were also collected. All of the eluates were made up to 50 ml with acidic water before being extracted with an equal volume of ethyl acetate. The undissolved ochratoxins in the round bottomed flask were collected in a methanol and methanol was also used to wash off any ochratoxins retained on the Sep-Pak. All four additional fractions were analysed quantitatively, using DAD-HPLC, to ascertain the fate of ochratoxins A and B in the clean up procedure. In addition, 25 g of green Kenya Gethumbwini coffee beans were milled twice using a Glen Creston coffee mill. The ground beans were spiked with ochratoxins A and B (1.5 and 0.18 mg in methanol, respectively) and then homogenised with 330 ml of ethyl acetate-0.01 M phosphoric acid (10:1) using a Sorvall Omni-Mixer. The ethyl acetate-phosphoric acid mix was extracted for ochratoxin A in the manner employed for SW cultures and the ensuing bicarbonate soluble fraction was cleaned up using the Cl 8 Sep-Pak protocol described above. 173 8.3: ResuLts and discussion. 8.3.1: Mycological analysis. Details of the fungi isolated from the coffee beans are shown in table 8.1. The beans were vely likely to have come from the 1994 crop. They were generally of high quality destined for the ground coffee market and could have been used for blends or for coffee extracts, except the Arabica beans which originated from Papua New Guinea or Uganda. Some of the coffees were mycologically sterile, including the Colombian Decaffeinated beans (which had been treated by steaming then extraction with dichloromethane), whereas, all beans of some others were infected by fungi. None of the cultures on DG-18 plates showed any signs of fluorescence under UV light (350 and 254 nm). The four most common contaminating fungi, in the order of decreasing occurrence, were preliminarily identified as A. niger, A. glaucus, A. tamarii and A. flavus. This corresponds well with literature data (Tsubouchi ci aL, 1984; Mislivec eta!., 1983; Levi et al., 1974). A common mistake was isolating from young yellow A. fiavus or A. tamarii sporophores, in the belief that they were A. ochraceus, and finding that on PDA medium they produced cultures that later became green or brown sporing, respectively. However, some persistent yellow A. ochraceus cultures that also produced typical pink-purple scierotia with age were isolated for further investigation (Colombian Supremo Y-1, Honduras Y-1, Mocha Y-3, and Uganda Y-4, 5 and 6). The overall % infection by A. ochraceus was low, with the exception of the beans from Uganda, of which 3/15 were infected. This relatively high incidence of A. ochraceus infection prompted analysis of the green Ugandan beans for ochratoxins A and B. All the A. ochraceus isolates, grown on SW, failed to accumulate ochratoxins A or B according to DAD-HPLC and TLC analysis using an overall detection limit of approximately 0.3 .Lg/g substrate. However, the bicarbonate soluble DAD-HPLC metabolic profiles did show in common an unfamiliar metabolite, which had a UV absorbance spectrum similar to mellein, which it was not and is yet to be identified. 174 Bean source % Infected Predominant contain- % A. ochraceus mating fungi infection Colombian 0 Decaffeinated Kenya Get. 0 Costa Rica SI-LB 13 A.flavus 0 Kenya A/A 13 A. niger 0 Mysore 13 A. glaucus 0 Kenya 'C' 20 A. niger 0 Guatemala SHB 35 A. glaucus 0 Colombian 73 A. glaucus 0 Medellin A. lamar/i Colombian 93 A. lamarii 7 t' Supremo P. roquefortii t2 Arabica 100 A. niger t 0 A. glaucus A.flavus Honduras SHG 100 A. niger t 7 t A. lamaril A.flavus Mocha 100 A. niger t6 7f A. glaucus A.flavus UgandaSCl8 100 A. niger t8 20 A. ochraceus t A. tamarii P. citrinum t'° Table 8.1: Details of fungi isolated from green coffee beans. Notes: In order of decreasing occurrence. t' One example was isolated which was persistently yellow on PDA: "Colombian Supremo Y-3". 175 Notes continued: t2 Two examples were isolated: "Colombian Supremo P-I and 2". t One example was isolated "Arabica N-i". t One example was isolated: "Honduras N-i ". t One example was isolated which was persistently yellow on PDA: "Honduras Y-i ". t6 Two examples were isolated: "Mocha N-i and 2". t One example was isolated which was persistently yellow on PDA: "Mocha Y-3". t Four examples were isolated: "Uganda N-2, 3 long sporophore, 3 short sporophore and 4". t Three examples were isolated which were persistently yellow on PDA: "Uganda Y- 4, 5and6". tb One example was isolated: "Uganda P-7". 176 The DAD-FIPLC chromatogram of this acidic mellein is shown in figure 8.1, whilst figure 8.2 shows its UV absorbance spectrum compared with mellein. Bicarbonate insoluble DAD-HPLC metabolic profiles consistently showed two metabolites, one being positively identified as mellein, and the other, whilst not being diaporthin, having a UV absorbance spectrum similar to it (see chapter 9). Uganda P-7 was identified as P. citrinum; the PDA slope cultures fluoresced bright yellow under UV light (350 nm) and citrinin was detected in SW cultures (confirmed by DAD-HPLC and TLC analysis using a citrinin standard). A. niger var. niger has recently been implicated in ochratoxin A production (Abarca et a!., 1994). However, Frisvad (1995) believes that ochratoxinogenic A. niger strains belong to the species A. carbonarius which can be distinguished from A. niger by its poor growth at 37 °C on CYA. None of the A. niger isolates (Arabica N-i, Honduras N-i, Mocha N-i and 2 and Uganda N-2, 3 long sporophore, 3 short sporophore and 4) showed no signs of fluorescence under UV light (350 and 254 nm) and all grew equally well at 25 or 37 °C on CYA. Thus, the black Aspergilli isolated were just the common A. niger. The metabolic profile of Colombian Supremo P-i and 2 on TLC plates showed the absence of any penitrems using a detection limit of approximately 50 j.tg/g mycelial mat. However, roquefortine was provisionally identified in the extracts of both isolates, suggesting they are P. roquefortii. 8.3.2: Ochratoxins A and B analysis. DAD-HPLC analysis of the Ugandan bean extracts prior to a Sep-Pak clean up step showed the absence of ochratoxin A, although there was a compound which shared its retention time but had a dissimilar UV absorbance spectrum. Ochratoxin B could not be identified since the region of the chromatogram where it eluted was obscured by other compounds. The DAD-HPLC analysis of the post Sep-Pak sample showed the absence of both ochratoxins A and B consequent on removal of the interferences in the regions of the chromatograni corresponding to both ochratoxins A and B. 177 Figure 8.1: DAD-HPLC chromatogram of acidic mellein. fl;Ai; I-i2Th mAt) 1000 800 600 400 200 0 Figure 8.2: Comparing the UV absorbance spectrum of acidic mellein (-) with mellein (--) 178 Of the 1.3 mg of ochratoxin A used to assess the efficiency of the C18 Sep-pak clean up operation, 0.327 mg (25%) did not dissolve in the loading solution, 0.031 mg (2.4%) was retained on the Sep-Pak with only 0.644 mg (49.6%) being eluted in the 40% aqueous acetonitrile. The other 0.298 mg (23%) of ochratoxin A was unaccounted for. Of the 0.15 mg of ochratoxin B used, 23 .tg (15%) did not dissolve in the loading solution, 12 .Lg (8%) was eluted with 40% aqueous acetone with only 15 j.tg (10%) being eluted in the 40% aqueous acetonitrile. The majority of ochratoxin B (100 pig or 67%) was unaccounted for. The recovely of ochratoxins A and B from the spiked Kenya Gethumbwini beans was much worse, with only approximately 4% of each being recovered. Thus, the Sep-Pak clean up procedure was found to be very inefficient. Terada later published an improved clean up system based on a NH2 Sep- Pak (Tsubouchi eta!., 1988), but this was not attempted. Despite having the highest incidence of A. ochraceus, the coffee beans from Uganda did not contain ochratoxin A using the present analytical methodology. Ochratoxin B was essentially undetectable. In conclusion, this pilot study on the mycobiota of green coffee beans was in accordance with previous reports suggesting that the incidence of A. ochraceus is low and the contamination by ochratoxin A infrequent. 179 9: Isolation and characterisation of metabolite "429" of an Aspergillus ochraceus isolate from green coffee beans. 9.1: Introduction. The search for A. ochraceus in green coffee beans resulted in the discoveiy of six isolates (Colombian Supremo Y-1, Honduras Y-1, Mocha Y-3 and Uganda Y-4, 5 and 6) that were used to inoculate shredded wheat medium. DAD-HPLC analysis of extracts of these SW cultures revealed a common bicarbonate insoluble compound which was not diaporthin, yet had a UV absorbance spectrum somewhat similar to it (see 8.3.1). Figure 9.1 shows the DAD-HPLC chromatogram of this compound (provisionally named "429") and mellein, and figure 9.2 shows its UV absorbance spectrum compared with that of diaporthin. Quantitative analysis by DAD-HPLC showed that metabolite 429 was best produced by the Mocha Y-3 A. ochraceus isolate, that was the organism of choice for subsequent investigations. 9.2: Materials and methods. Shredded wheat medium (20 g SW in 500 ml conical flasks moistened to 40%) was inoculated with spores of Mocha Y-3 and incubated at 25 °C. At intervals, samples (1 g) were removed and extracted in the same manner as used for the ochratoxins. The bicarbonate insoluble fractions of the extracts were analysed by DAD-HPLC for 429 and mellein. Following subsequent purification, a calibration graph for quantitative 429 analysis was constructed using the appropriate ChemStation programme (see appendix Viii). To see whether 429 could be produced on a liquid medium by Mocha Y-3, spores from a SW culture were used to inoculate six flasks of potato dextrose broth (PDB) and three flasks of yeast extract-sucrose (YES) media. All liquid cultures were as 100 ml medium in 500 ml conical flasks and YES medium was comprised of 2% yeast 180 Figure 9.1: DAD-HPLC chromatogram of "429" and mellein. Figure 9.2: Comparing the UV absorbance spectrum of "429" (-) with diaporthin (--) 181 extract and 4% sucrose (wlv). The cultures were incubated, shaken, at 28 °C. A PDB flask was harvested 1, 2, 3, 5, 7 and 9 days post inoculation and one YES culture was harvested 2, 5 and 9 days post inoculation. The harvested flasks were filtered and the broth was extracted in the usual manner. The ensuing bicarbonate soluble and insoluble fractions were analysed by DAD-HPLC. The purification of 429 for identification and characterisation involved extracting 8 day old Mocha Y-3 SW cultures. The ensuing bicarbonate insoluble fractions were subjected to preparative HPLC using an isocratic mobile phase of water-acetic acid- acetomtrile (59.5:1:39.5) flowing at 8.4 mI/mm through a 300 x 25 mm Dynamax Macro C18 column. Detection was by UV absorbance at 239 nm. The eluate corresponding to 429 was collected (retention time approximately 13 minutes), dried and the HPLC clean up repeated. Subsequent purification involved preparative TLC (PLC) using 20 x 20 cm Camlab SIIJUV 254 TLC plates (1 mm silica gel with fluorescent indicator UV254 pre-coated on glass plates) developed in toluene-ethyl acetate-formic acid (50:40:10). The unknown compound had an Rf value of 0.4 in this solvent system and was visualised by its yellow appearance in daylight and its quenching of the UV fluorescent indicator under UV light at 254 nm. However, 429 was undetectable under UV light at 350 nm. The silica corresponding to the 429 was scraped ofl extracted in propan-2-ol, and then purified further by preparative HPLC. The purity of 429 was assessed using DAD-HPLC. The pure 429 was analysed by El (nominal and accurate mass measurement), ammonia CI and FAB mass spectrometry. FAB-MS was performed on a ZAB-2SE mass spectrometer fitted with a Cs gun operated at 20-25 KeY using 1-monothiolglycerol as the matrix. In addition, linked scan (B/E and B2IE) El-MS was performed on the major fragmentation ions (m/z 429, 414 and 359) to rationalise the fragmentation pattern of 429 in El-MS. The unknown compound was also analysed by 1H NMR spectroscopy, 1H NMR spectroscopy was performed on a Bruker NMR spectrometer at 500 MHz using CD3OD as the solvent. Aside from standard 1H NMR spectroscopy, 1H-1H COSY experiments were performed on 429 to try and ascertain the connectivity of the 1H 182 NMR signals. 1H observed 1H- 13C one bond correlation experiments were also performed on 429, which enabled the assignment of the 1H NMR signals more accurately. Both the 1H-1H COSY and the 1H observed 1H-13C one bond correlation experiments were performed at 400 MHz. 9.3: Results and discussion. The production of 429 by Mocha Y-3 in SW is shown in figure 9.3. The unknown compound proved very difficult to purilj despite being produced in comparatively high yields (approaching 7 mg/g substrate). It was noted that 429 on PLC plates became progressively more yellow with time and that a second preparative HPLC peak (retention time 19 minutes) persistently appeared in apparently "pure" 429 samples. Great care was needed to prevent unnecessary exposure of 429 to air and, as a precaution, all samples of 429 were kept refrigerated under nitrogen gas. Only mellein was produced in both PDB and YES media, which may be indicative of the media used or that 429 is not detected extracellularly. Alternatively, the increased temperature, from 25 °C to 28 °C, may adversely affect the production of 429. The accurate mass El spectrum of 429 is shown in figure 9.4. The signal at mlz 429 (M) corresponded to a molecular formula of C 3 1H27N0 and there was a related fragment at m/z 414 which corresponded to C30H24N0 (i.e. a loss of CH3). The ammonia CI mass spectrum is shown in figure 9.5, but neither of the expected ions mlz 430 (M + HJ nor 447 (M + NH4) were present. However, m/z 432 (414 + NH4 ) was evident in the CI mass spectrum, although 415 (414 + H) was not. The FAB mass spectrum is shown in figure 9.6. There appeared to be two quasimolecular ions (M + H) in the FAB mass spectrum: m/z 430 and 448. The ion at m/z 430 probably corresponded to the protonated 429 ion (i.e. M + H), whereas mlz 448 could have corresponded to the quasimolecular ion of 429 plus H 20 (i.e. M + H20 + H). Linked scan El-MS showed that m/z 429 fragmented to 414 and 359 and, since it had no parent ion, mlz 429 was proved to be the molecular ion. The ion at m/z 414 183 Figure 9.3: The accumulation of "429" in an A. ocbraceus Mocha Y-3 shredded wheat fermentation. 7 6 U 0 0 2 4 6 8 10 12 14 16 Time (days) 184 mlz Formula 429 C31H27N0 414 C30H24N0 359 C28H23 261 C18H15N0 Figure 94: Accurate mass electron impact spectrum of "429". 185 Figure 9.5: The ammonia chemical ionisation mass spectrum of "429". ri1•xMl11.E Id.n1_3 17,2O3O •21 Cs1.14J7M..35k2 7. 0E4 4. 44 3.5E4 , 715., au Figure 9.6: The fast atom bombardment mass spectrum of "429". 186 had 429 as its parent ion and fragmented to 399, 329 and 261. The ion corresponding to m/z 359 had 429 and, much weaker, 375 as its parents ions and fragmented to 343 and, to a lesser extent, 329. The fragmentation pattern of 429 in El-MS is summarised in figure 9.7. The lack of strong El-MS fragmentation ions suggests that 429 has a rather stable structure, despite being shown to be reactive. The 1H NMR spectrum of 429 is shown in figure 9.8. The solvent signals are clearly visible at 6 3.30 and 4.90, and there are two other sets of signals at 6 1.25 and 4.10 which may be due to a contaminant since their integration values are incompatible with the other signals. Integration of the signals showed that, in the low field strength region of the 1H NMR spectrum, there were 5 signals that were equivalent to single protons (6 7.38, 6.86, 6.55, 5.66 and 5.28). The signals at 6 4.60, 3.45, 2.80, 2.75 and 2.20 integrated to 2 protons and there were 3 proton integration values in the 6 2.00, 1.90, 1.40 and 1.10 regions of the high field strength 1H NMIR spectrum. The 1H-1H COSY spectra of the low and high field strength 'H NMR signals are shown in figures 9.9 and 9.10, respectively. There was clear coupling between the protons at 6 7.38 and 6.55 and also between the protons 6 6.86 and 5.66 in the low field strength region of the 1H NMR spectrum, although the proton at 6 5.28 seemed to have no proton neighbours. In addition, there was weak coupling between the hydroxyl proton of the solvent (64.90) and protons at 64.60 and 4.00 (which corresponded to an area of very weak signals). In the high field strength region of the 'H NMR spectrum, the contaminant protons at 64.10 and 1.25 seem to be strongly coupled and there was coupling between the protons at 6 3.45 with the methyl protons of the solvent (6 3.30). The only other clear coupling occurred between the protons at 62.80 and 2.20 and between the protons at 62.75 and 2.00. The 1H observed 1H-13C one bond correlation spectrum is shown in figure 9.11, with an expansion of the signals in high field strength region shown in figure 9.12. Single proton signals at 6 7.38, 6.86, 6.55, 5.66 and 5.28 in the 1H NMR spectrum are linked to carbon atoms, presumably as CH groups. The hydroxyl proton of the solvent (at 6 4.90) is, not surprisingly, missing from the spectrum as is the 2H signal at 64.60, 187 429 (C 1HNO) N 4 C3H4NO\ -16 414 359 (CH24NO) (CH23) / ..16 329 399 261 343 (C i8HiNO) Figure 9.7: Fragmentation pattern of "429" in electron impact mass spectrometry. 188 a a a if 0 C4- ______- - x 0. a. '.4 - 0 z I- z 0 t 0 0 U, U, N U. 0 0 m0 0 z 'U -I a- U, U. 0 x • 0 U, z a 0 189 JOHN HARRIS. COSY IN CO300. 0 45 _ ___ 3: _ 5.0 0 5.5 6.0 5.5 a S 7.0 S 7.5 8.0 • 7.5 7.0 6.5 • 6.0 5.5 5.0 1.5 PPM Figure 9.9: Low field strength region of the 1H-1H COSY spectrum of"429" in CD3OD. 190 JOHN HARRIS. COSY IN CD300. 10 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 • PPM 6.0 -- PPM Figure 9.10: High field strength region of the 1H-1H COSY spectrum of "429" in CD3OD. 191 Joirn Harris sa.ple in d4-.ethano]: Proton-carbon I bond correlation in an attempt to locate carbon signals. I. S S 50 , S S r pp. pp. 7 6 5 4 3 2 1 Figure 9.11: The 1H observed 1H-13C one bond correlation spectrum of "429" in CD3OD. 192 John Harris saaple in dl-.ethanol: Proton-carbon I bond correlation irran atte.pt to locate carbon signals. I I I I . I . I 10 • I I • I . I. I s' I • .1 I, 'L - 'ct.' . ,• I _1ff4• . II II• • I • •, ,. I jL• I • I ______I'• '_!.?I I . •I ' I i I - • • , Ii I • , PP. PP. 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Figure 9.12: The 1H observed 1H-13C one bond correlation spectrum of "429" in CD3OD (high field strength expanded). 193 which may correspond to a N}12 group. The strongest signal is from the CH3 of the solvent (at 6 3.30), but there are also strong signals from the CH2 and CH3 of the contaminating compound (at 6 4.10 and 1.25, respectively). Expansion of the spectrum in the high field strength region of the 1H observed 1H-13C one bond correlation spectrum shows signals at 62.80 and 2.20 to be clearly linked to carbon atoms (as CH2) and signals at 6 3.45 and 2.75 to be weakly linked to carbon atoms (again as CH2). The occurrence of a CH2 signal in such a low field strength region of the 1H NMR spectrum may indicate that the protons at 6 3.45 may be situated near an electronegative atom (such as oxygen). Protons corresponding to signals at 6 2.00, 1.90, 1.40 and 1.10 in the 1H NMR spectrum are clearly linked to carbon atoms, presumably as CH3 groups. The 1H NMR spectroscopy results are summarised in table 9.1. All 27 protons have been accounted for in the 1H NMR spectrum along with 13 of the 31 carbons. The remaining 18 quarternary carbons are presumably involved in a complex unsaturated system containing 19 double bond equivalents (calculated from the molecular formula). Since 429 seemed only to be produced by Mocha Y-3 on a stationaty solid substrate, feeding experiments to determine the biosynthetic origin of this hydrophilic neutral aromatic alkaloid would be difficult to execute. However, [1- 14C] acetate, [methyl- 14C] methionine and [U-'4C] phenylalanine (since the most likely origin of the lone N atom was an amino acid) were all added (as a 200 il aqueous solution) to a 2 g aliquot of SW culture which was then incubated overnight prior to extraction. DAD- HPLC of the ensuing bicarbonate insoluble fractions was used to collect the eluate Only corresponding to 429, which was then scintillation counted. weak incorporation of [1-'4C] acetate into [14C] 429 was detected, although the peak purity analysis of the DAD-HPLC peak corresponding to 429 showed it to be impure. El-MS of 429 showed no evidence of an isoprene in the structure (no mlz 69 fragment ion which is equal to C5H9), although a structure based on acetate could be consistent with a terpenoid origin. Limited time precluded further investigation into the characterisation of 429. 194 5 J (Hz) Assignmen t Linkage 1.10 (s)3H- CH3 1.40 (s)3H- CH3 1.90 (s)3H- CH3 2.00 (s)3H- CH3 52.75 2.20 (m)2H - CH2 52.80 2.75 (m)2H- CH2 52.00 2.80 (m)2H - CH2 52.20 3.45 (m)2H - CH2(O?) 4.60 (s)2H- 2 (?) 5.28 (s) H- CH 5.66 (d) H 9.74 CH 56.86 6.55 (d) H 8.30 CH 5 7.38 6.86 (d) H 9.78 CH 5 5.66 7.38 (d) H 8.35 CH 5 6.55 Table 9.1: 1H NMR data of '429" in CD3OD. 195 10: Summary. This study has attempted to elucidate further the biosynthetic events of the important mycotoxin ochratoxrn A using two different approaches. First, the occurrence and biosynthetic dynamics of ochratoxin A and other structurally related polyketides including ochratoxins B, a and 13, mellein, diaporthin and orthosporin produced by the saprophytic fungus Aspergillus ochraceus, strain D2306, was explored. Secondly, isotopic labelling experiments using putative advanced intermediates of ochratoxin A biosynthesis were performed. There have been veiy few isolates of A. ochraceus that have been used in ochratoxin A biosynthetic studies, CSIR 804 and NRRL 3174 being the two most popular, which reflects the low frequency of ochratoxinogenic strains in nature. A. ochraceus is, however, widely distributed in tropical and sub-tropical latitudes and has been isolated from a variety of foodstuffs including, as described in chapter 8, green coffee beans. Two important related questions raised are why expression of ochratoxin A biosynthesis in the laboratory is apparently such a rare event and is this a fair reflection of A. ochraceus in nature? These, unfortunately, are not easily answered, although future experiments designed to elucidate regulation of expression of this biosynthetic pathway fully will certainly aid in resolving these issues. Even the A. ochraceus strain D2306 was able to produce large amounts of ochratoxin A only under special conditions i.e. shaken solid substrate with carefully controlled moisture content. Ochratoxin A was produced by D2306 erratically in potato dextrose broth but not at all in yeast extract-sucrose medium, the latter facilitating the production of aspergillic acid and deoxyaspergillic acid instead. This indicates the presence of a control mechanism attuned to the environment of the fungus as well as emphasising the difficulty of these types of investigations. In contrast, other structurally related polyketides, such as mellein, are produced more consistently by a much larger number of A. ochraceus strains. When ochratoxin A was produced, it was found to be closely linked with the production of ochratoxin B and was 196 concurrent with growth. Thus, it is unwise to regard all secondary metabolites as biosynthesized only within the "idiophase" of a fungal fermentation. Presumably, ochratoxin A biosynthesis is not essential for fimgal growth as non-ochratoxinogenic strains will attest. However, this has serious implications in food spoilage since moderate, undetectable infection of a foodstuff by an ochratoxinogenic A. ochraceus strain could result in significant ochratoxin A contamination. This could account for the small amounts of ochratoxin A detected in some coffees, but it is by no means certain that A. ochraceus has to be the only source of ochratoxin A in tropical commodities. It has also been noted in the present studies that the most prolific ochratoxin A production occurred prior to the fungus sporulating. This would indicate that ochratoxin A probably may not exist in the spores and that perhaps sporulation may inhibit ochratoxin A biosynthesis. Ochratoxin B yields were consistently lower than ochratoxin A. Experiments using [ 14C] labelled precursors of ochratoxin A biosynthesis fed to D2306 submerged cultures have shown that there was a temporal separation of biosynthetic events. The early phase of ochratoxin A biosynthesis involved acetate, presumably in the construction of the dihydroisocoumarin moiety, and occurred approximately 50 hours prior to the time of maximum ochratoxin A yield in the broth. Experiments involving administering the methylation inhibitor ethionine to D2306 submerged cultures showed that methylation was, like acetate, linked to early biosynthetic events. This was also seen when [14C] labelled precursors of ochratoxin A biosynthesis were fed to shaken solid substrate D2306 fermentations, since there was an approximate 4 day lag between the time of maximum incorporation of[1- 14C] acetate and [methyl-14C] methionine into ['4C] ochratoxins A and B and the time of maximum ochratoxin A yield in the fermentation. However, there was also a lag (of 2 days) between the time of maximum incorporation of [U- 14C] phenylalanine into [ 14C] ochratoxins A and B and the time of maximum ochratoxin A yield in the fermentation. The production of the ochratoxins has been shown to be a very dynamic affair with regard to their apparently simultaneous biosynthesis and catabolism. They appear to 197 be a part of a substrate cycle (rather like citrinin is thought to be, see appendix IX) which may even signify a possible role in primary metabolism. The dynamics of the structurally related polyketide biosynthesis in potato dextrose broth (figure 2.8) shows that there was a distinct sequence involving three groups: ochratoxins A and B, followed by diaporthin and orthosporin, followed by mellein. It may not be coincidence that this sequence corresponds to a decrease in complexity of their chemical structure as well as a decrease in the variety of primary metabolite precursors required for their construction. The ochratoxins require acetate, methionine and phenylalanine as well as a chlorine source. Diaporthin and orthosporin require acetate and methionine, whereas mellein requires acetate alone, which may be the reason why it is produced more consistently by a larger array of organisms (including animals). It may also be true that ochratoxins a and f3 also feature in this sequence, closely linked with ochratoxins A and B, although the identification of the two former compounds was not definitive. This polyketide biosynthesis sequence may be an indication of the changing state of nutrient resources within the substrate. Therefore, an interesting experiment would be to add precursors suspected of being exhausted to see whether biosynthesis could be reinstated. The temporal sequence of polyketide biosyntheses was less apparent when the fungus was grown in solid substrate: diaporthin was produced concurrently with ochratoxins A and B in greatly reduced yields and orthosponn and mellein were undetected. There seems to be a metabolic hierarchy involving the three polyketides groups and their constituent primary metabolites which is dependent on the environment, although there is little evidence to suggest that they are linked any closer i.e. part of each othe?s metabolic pathways. This is surprising with respect to mellein and the dihydroisocoumarin moiety of the ochratoxins as they are very similar. Diaporthin and orthosporin had never been previously found as A. ochraceus metabolites, and [ 14C] labelled precursor feeding studies showed that the metabolic origin of diaporthin was acetate and methionine. Diaporthin and orthosporin were found to be produced concurrently and it was reasoned that diaporthin was probably 198 derived by methylation of orthosporm. However, ethionine was found to reduce yields of both diaporthin and orthosporin suggesting that the orthosporin in the culture broth may have been derived from the de-methylation of diaporthin. This could be further evidence of metabolic turn-over of secondary metabolites. Diaporthin was shown to have an inhibitory effect on citrinin production by a P. citrinum isolate (although not reproducibly) and by using orthosporin and [14C] diaporthin feeding experiments, it was demonstrated that there was a P. citrinum mechanism that was able to transform these neutral dihydroisocoumarin-like compounds into, as yet undefined, acidic ones. In the second approach to understanding the ochratoxin A biosynthetic pathway, the isotopic labelling experiments showed that there was a very low incorporation of [14C] mellein into [ 14C] ochratoxins A and B when fed to either liquid or solid substrate D2306 fermentations. Any radiolabel incorporation that was detected may have be due to the incorporation of[ 14C] acetate derived from [ 14C] mellein. [14C] Ochratoxin a, however, was shown to be incorporated into [14C] ochratoxin A almost ten times more readily than [ 14C] mellein, and approximately 33% of the [14C] ochratoxin 13 fed to D2306 solid substrate fermentations was incorporated into [14C] ochratoxins A and B. This evidence suggests that one possible ochratoxin A biosynthetic pathway involves ochratoxin 13 being converted to ochratoxin a which is subsequently converted into ochratoxin A. A separate pathway presumably converts ochratoxin 13 into ochratoxin B, and mellein is not involved at all. Another possible pathway involves ochratoxin (3 being incorporated into ochratoxin A via the chlorination of ochratoxin B. Using dual labelled [1O-' 4C, phenylalanyl-3H1 ochratoxin A feeding experiments, it was shown that there was a mechanism that can convert ochratoxin A into ochratoxin B, although non-specifically and at a low rate. It is not clear whether this is a step in the degradation of ochratoxin A. A very low percentage incorporation of dual labelled ochratoxin B into dual labelled ochratoxin A was recorded. Since the dual labelled feeding experiments did not achieve the high percentage incorporation of the [1O- 14C] ochratoxins a and 13 feeding experiments, it could be argued that the interconversion of ochratoxins A and B, although shown to 199 exist, is not as important in the ochratoxin A biosynthetic pathway as the reactions involving ochratoxins a and 13 . It is also difficult to envisage the origin of ochratoxin a Wit is not from ochratoxin 13. However, it would be advisable to repeat the dual labelled ochratoxin B feeding experiment to avoid any ambiguity due to possible experimental error. A desirable additional experiment would be to try to incorporate [1O-14C] 7- methylmellein into ochratoxins A and B. This would have been attempted except that an organism that produced 7-methylmellein was unavailable and its chemical synthesis was impractical. It would also be desirable to complete the chemical degradation experimentation in order to demonstrate that any incorporation of fed radiolabels was specific. The discovery of 5-methyl-, 5-formyl- and 5-carboxymellein in a culture broth of Nummulariella marginata (Whalley and Edwards, 1985) provides a precedent for a suitable oxidation system of a substituted methyl group, yet if such compounds are stable enough to be isolated from a fungal culture broth why have 7-methyl and 7- formylmellein not, so far, been discovered? If oxidation of 7-methylmellein to ochratoxin 13 is part of the ochratoxin A pathway, the enzymes involved are particularly efficient. New physico-chemical data was obtained for diaporthin including a fully assigned 13C NMR spectrum, 13C NMR DEPT analysis and improved assignment of 1H NMR signals. Linked scanned and accurately measured El mass spectra were obtained for ochratoxins a and 13, diaporthin, mellein and a compound reasoned to be 4- hydroxymellein. Similarly, spectroscopic data was obtained for a partially characterised alkaloid named "429" which was extracted from cultures of an A. ochraceus strain isolated from green coffee beans, and was a consistent metabolite of all A. ochraceus isolates from coffee. Accurate mass measurement El spectra were also obtained for ochratoxins A and B, 0-methyl, methylochratoxin A, 0- methylochratoxin A, 0-methyl, methylochratoxin a, orthosporin, cycloechinulin, aspergilhic acid and a compound reasoned to be deoxyaspergillic acid. In addition to 200 the mass spectral data, 1H NMR, 1H-1H COSY and 'H observed 1H-13C 1 bond correlation spectra were obtained for alkaloid "429" that indicated that this is a novel alkaloid and provided some of the data for eventual full characterisation. 201 Appendix I: DAD-HPLC calibration details of ochratoxin A. C: \HPCHEM\l\METHODS\OA.M Calibration Curve Ochratoxjn A at exp. RT: 8.067 DAD1 A, Sig332,4 Ref=450,80 Correlation: 1.00000 Residual Std. Dev.: 58.13652 Formula: y - mx a: 9.13495 x: Amount y: Area 202 ppendix II: DAD-HPLC calibration details of ochratoxin B. C: \HPCHEM\l\METHoDS\OA.M Calibration Curve Ochratoxin B at exp. RT: 4.500 DAD1 A, Sig=332,4 Ref45O,80 Correlation: 1.00000 Residual Std. Dev.: 1.16790 Forunila: V ax 8.22717 x: Amount y: Area 203 kppendix ifi: DAD-HPLC calibration details of orthosporin. C: \HPCHEM\1\METHODS\DIAPORT .14 calibration Curve - = -- -=-- Orthosporin at exp. RT: 1.900 DAD1 A, Sig=329,4 Ref=450,80 Correlation: 0.99997 Residual Std. Dev.: 108.54487 Formula: v = ax a: 24.02029 x: Amount y: Area 204 ppendix W: DAD-HPLC calibration details of diaporthin. C: \HPCHEM\l\HETHODs\DIAPORT . M Calibration Curve Diaporthin at exp. RT: 3.970 DAD1 A, Sig=329,4 Ref=450,80 Correlation: 0.99996 Residual Std. Dev.: 117.21866 Foriu1a: y = 24.01941 x: Aiiount y: Area ______ 205 Appendix V: DAD-HPLC calibration details of mellein. C: \HPCHEM\1\METHODS\DIAPORT .14 Calibration Curve Mellein at exp. RT: 4.990 DAD1 A, Sig=329,4 Ref=450,80 Correlation: 1.00000 Residual std. Dev.: 7.21900 Formula: v mx 8.22235 x: Amount y: Area 206 Appendix VI: DAD-HPLC calibration details of ergosterol. C: \HPCHEM\1\METHODS\ERGO . H Calibration Curve Ergosterol at exp. RT: 8.132 DAD1 A, Sig=282,4 Ref=450,80 Correlation: 1.00000 Residual Std. Dev.: 5.16613 Formula: y = mx a: 11.27668 x: Amount y: Area 207 Appendix VII: DAD-HPLC calibration details of citrinin. C: \HPCIIEM\1\METHODS\CIT . M Calibration Curve Citrinin at exp. RT: 5.000 DAD1 A, Sig=327,4 Ref=45080 Correlation: 0.99986 - Residual Std. Dev.: 1315.28826 Formula: y = mx 13.09606 x: Amount y: Area 208 Appendix Yffi: DAD-HPLC calibration details of "429". C: \HPCREfl\l\METflODS\429 .H Calibration Curve 429 at exp. RT: 3.740 DAD1 A, Sig=329,4 Ref=450,80 Correlation 1.00000 Residual Std. Dev. 0.15393 Foraula: y ax a: 4.06472e-1 x: Amount - y: Area 209 Appendix IX: The biosynthesis of citrinin. Citrinin (figure 7.1, XLIII) is a pentaketide secondary metabolite of several Penicillium and Aspergillus species. Citrinin was first isolated from Peniciiium citrinum grown on a glucose based medium (Hetherington and Raistrick, 1931) and was later found to possess antibacterial activity (Raistrick and Smith, 1941). Citrinin is both nephrotoxic (Arai and Hibmo, 1983) and carcinogenic (Ueno and Kubota, 1976) so its toxicity precludes its use as a therapeutic drug. Citrinin may be produced exclusively, or concurrently with other mycotoxins such as ochratoxin A. Evidence for citrinin and ochratoxin A toxicity acting synergistically has been published (Creppy et a!. 1980). Citrinin is regarded as an important mycotoxin that may be a contaminant of animal or human food (Wilson, 1982). Citrinin has been classified as toxic to moderately toxic (Frank, 1992) but alarming recent research has shown that heating citrinin to moderate temperatures (100-140 °C) in the presence of water resulted in the formation of a citrinin complex (citnnin Hi) which showed ten-fold more cytotoxicity to HeLa cells than citrinin (Trivedi eta!, 1993). Citrinin chemistry and toxicology were reviewed by Saito et a!. (1971) and citrinin production, isolation, separation and purification were reviewed by Betina (1984), who wrote a more comprehensive review article concerning citrinin five years later (Betina,, 1989). Citrinin production, natural occurrence, toxicology and analysis were reviewed by Frank (1992). Birch et a!. (1958), using [1- 14C] acetate and [14C] formate fed to cultures of P. citrinum and partial chemical degradation studies, showed that citrinin biosynthesis originated from five acetate units and three C 1 units. Also using 14C labelling studies and partial chemical degradation, Schwenk et a!. (1958) showed that C-12 of citrinin was derived from methionine whereas C-I was derived from acetate. One third of the total radioactivity of citrinin isolated from a P. ci/rinum culture fed with [methyl- 14C] methionine was located in C-12 and one fifth of the total radioactivity of citrinin isolated from a P. citrinum culture fed with [1- 210 14C] acetate was located in C-i. Thus, it was postulated that citrinin was constructed via the polyacetate pathway i.e. head-to-tail additions of acetate, to give the main skeleton of the molecule (C atoms 1, 3-9) with the extraskeletal methyl groups (C atoms 10-12) originating, in preference to formate, from methionine. Rodig eta!. (1966) devised a complete chemical degradation method for citrinin to allow the isolation of each constituent carbon atom. [1-' 4C] and [6-'4C] glucose were fed to cultures of P. citrinum and the resulting [14C] citrinin was degraded to determine the pattern of 14C labelling. From both labelled compounds, there was an alternation of radioactive carbon atoms within the skeleton of citrinin (only C atoms: 9, 4, 5, 7 and 8a were labelled) and there was also activity in the citrinin methyl carbons (C atoms 10-12). Glucose is catabolised to acetate via the Embden-Meyerhof pathway and labelling glucose at either C-i or C-6 would result in the production of [2-14C] acetate. The C-i and C-6 atoms of glucose also contribute to the C 1 pool. Rodig et a!. also reported that the extraskeletal methyl groups differed in radioactivity with C-i2 > C-Il > C-b. This led to the theoiy that the methyl groups were added sequentially in the order C-i2, C-li and then C-10, as decreasing radioactivity reflected the dilution of the 14C1 pool. Curtis eta!. (1968) isolated scierotinin A and dihydrocitrinone (figure A.!, XL1V and XLV, respectively) from mutant P. citrinum strains and proposed a citrinin biosynthetic pathway with these two compounds as intermediates. Carter et a!. (1979) synthesized two potential advanced intermediates: an isocoumarin and a trunethyl isocoumarin (figure A. 1, XLVI and XLVII, respectively). These compounds were based on isocoumarin intermediates of sclerotinin A biosynthesis and both were radiolabelled with 14C at position C-9. The radiolabelled compounds were fed to surface cultures ofF. citrinum and the ensuing citrinin was harvested and chemically degraded. Radiolabel from the isocoumarin was incorporated into citrinm, although not specifically. The isocoumarin was thought to be incorporated into citrinin only after being catabolised into C 2 units (namely acetate). The trimethyl isocoumarin gave significant, specific incorporation into citrinin. It was concluded that the failure of the 211 CH3 CH3 OH 0 OH 0 XLW XLV I-K CH OH 0 OH 0 XLVI XLVII Figure A. 1: The structures of scierotinin A (XLIV), dihydrocitrinone (XLV), the isocoumarin (XLV!) and the trimethyl isocoumarin (XLVII). 212 isocoumarin to be specifically incorporated into citnnin was due to the necessity of at least one, or possibly all three, methylations of the polyketide chain prior to aromatisation. Barber and Staunton (1979) investigated the origin of the C-H bonds in citrinin using [1,2-J C2] acetate fed to cultures of P. citrinum grown on a medium containing 2H20. This allows protium to be the tracer in a deutenated environment. The 13C NMR spectrum of citrinin produced under these circumstances can be compared with the control citrinin 13C NMR spectrum i.e. of citrinin derived from [1,2- 13C2] acetate produced in a medium containing H20. The control 13C NMR spectrum of citrinin confinned that the polyketide nucleus is formed by head-to-tail linkage of five intact C2 units. The 13C NMIR spectrum of citrinin produced in 21120 showed changes in C- H signals as protons were exchanged with deuterons, this being most apparent in the methyl groups (C atoms 10-12) which are ultimately derived from glucose. Significantly, the 13C NMR spectrum of citrinin produced in 21120 showed the presence of protons at C-4, which would suggest that the C-H bond at C-4 originated from acetate. Consequently, a trimethyl isocoumarin (such as the one proposed by Carter et aL, 1979) was thought to be an unlikely intermediate of citrinin biosynthesis since the necessary reduction of the C-4 - C-3 double bond would result in an increase of deuterium at the C-H bond at C-4. The trimethyl isocoumarin was presumed to access the citrinin biosynthetic pathway indirectly. Furthermore, the incorporation of protium at C-i and C-3 was found to be different. It was assumed that whilst on the polyketide synthase (PKS) enzyme, reductions are via a common pool of nicotinamide co-enzyme resulting in a constant protium: deuterium incorporation ratio. Only when released from the PKS can this ratio become variable. Therefore, the authors reasoned that the reductions at positions C-i and C-3 were carried out at different times and as enzyme free intermediates. Perception of the first enzyme free intermediate of citrinin biosynthesis was narrowed to the lactone (figure A.2, XLVIII) in which a reduction had occurred at C-3, but not C-i or the keto-aldehyde (figure A.2, XLIX) in which C-i but not C-3 had been reduced. The aldehydo-alcohol (figure A.2, L) could not 213 11 II CH3 CH3 CH3 CH3 HO\)J,CH I II F1 10 CH3 CHO OH 0 OH XLVffl XUX CH3 CH3 CH3 CH3 HO\))CH HO\J CH 3J I Ii 'OH CHO JLII3 CH3 CH( T CO2H OH OH L LI OH LII Figure A.2: The structures of the lactone (XLVIII), the keto-aldehyde (XLIX), the aldehydo-alcohol (L), the keto-acid (LI) and the quinone methide (LII). 214 have been the first enzyme free intermediate of citrinin biosynthesis since it has been reduced at both C-i and C-3. The keto-acid (figure A.2, Li) was not considered. Barber and Staunton (1980 a) prepared both of the potential advanced intermediates labelled with a single deuteron in the methyl group C-i 1. After separately administering the compounds to cultures of P. citrinum , the resultant citrinin was subjected to 2H NMR spectroscopy. The citrinin derived from the lactone showed no deutenum enrichment at C-i 1, whereas that derived from the keto-aldehyde did (along with marginal enrichment at C-9/C-10 and C-4). In conclusion, it was proposed that the keto-aldehyde was the first enzyme free intermediate of citrinin biosynthesis and that there is a competitive enzyme degradation process resulting in the formation of CH22HCO2H which is incorporated back into citrinin. Barber and Staunton (1980 b) prevented citrinin and any aromatic intermediates of citrinin biosynthesis from being produced by a P. citrinum culture with the use of the methylation inhibitor ethionine. This was consistent with the current theory that the methylation of the polyketide chain precedes the formation of the first aromatic intermediate. When the keto-aldehyde which had been labelled with deuterium at C-i was added with ethionine to a culture of P. citrinum, citrinin was produced. 1H NMR spectroscopy showed that the incorporation of the keto-aldehyde into citrinin was higher that earlier experiments, and that there was a low level of dilution by exogenous material resulting in higher enrichment of deuterium. Barber et aL (1981) shed some more light on the sequence of the methylations in citrinin biosynthesis by studying the incorporation of [methyl- 13 C] methionine into the three methyl groups of citrinin. After feeding [methyl- 13C] methionine to cultures of P. citrinum, it was found that the ratios of signal intensity in the 13C NMR spectrum for C-b, C-li and C-i2 of citrinin were the same for the natural abundance and 13C enriched samples. It was speculated that the three methylation steps in citrinin biosynthesis are carried out in rapid succession on the PKS by molecules from a single pool of methionine. 215 Colombo et a!. (1981) chemically synthesized various putative advanced precursors of citrinin biosynthesis including: scierotinin A, the tnmethyl isocoumarin, the lactone, the keto-aldehyde and a quinone methide (figure A.2, LII). Each of the compounds was 14C labelled at the methyl positions (C atoms 10-12). Each labelled compound was added to surface cultures ofF. citrinum and the ensuing citrinin harvested. Subsequent chemical degradation studies showed a good incorporation of the keto- aldehyde but also significant incorporation of the quinone methide into citrinin. These two compounds were included in a proposed citrinin biosynthetic pathway (figure A.3). The second cyclisation does not involve the lactone, thereby leaving the cyclic hemiacetal (Lifi), derived from the aldehydo-alcohol, as the only alternative. Some scrambling of the label occurred, which was attributed to the catabolism of the intermediates into acetate prior to their incorporation. Sankawa et a!. (1983) used multiple labelled acetate molecules, namely: [2- 13C, 2- 2H3] and [1 3C, 1802] acetate, to incorporate 2H and 180 into citrinin produced by Aspergillus terreus. The labels were detected by 13C NMR spectroscopy through 2H- 13C coupling and isotope shift induced by 2H and 180. Another multiple labelled acetate, [1- 13C, 170] acetate, was also tested for its potentiality as a precursor to trace the fate of acetate oxygen. 13C-170 coupling in 13C NMR spectroscopy was too small to be detected, although the incorporation of 170 was directly measured with 170 NMR spectroscopy. The origin of the hydrogen, carbon and oxygen atoms of citrinin is shown in figure A.4. The results do not contradict the pathway depicted in figure A.3, suggesting the pathway is common to both Peniciiium and Aspergillus species. In addition, X-ray analysis was prefonned on citrinin which provided bond length data and evidence of the presence of a resonance form (figure A.5). Barber eta!. (1987 a) investigated the structure of citrinin in vivo using 1H and '3C NMR spectroscopy. It was shown that in an aqueous culture medium environment, citrinin does not exist in the quinone methide fonn, but as a diastereoisomeric mixture of hydrates (figure A6). This refutes theories that citrinin acts as a simple Michael acceptor in vivo which can intercalate with DNA. A mechanism by which citrinin may 216 3XC1 CH3 CH3 4XCH2(CO2H)2 PKS EnZS...&1,.L1.,L.,CH3 IX CH3CO2H 0 0 0 0 0 ,/"Fokling HO 0 -' CH CH o1 cyclatii CH3 HO Reductive 3 Enz cleavage Reduction Keto-akiehyde (XLIX) JTIO\ H CII Cyclic hemtceta1 (Lifi) Aldehydo-alcohol (L) Dehydration CH3 CH3 Cl!3 CH3 O J IICH3 Oxidation ci-i( H TOH OH Quinone inethide (LIT) Citrinin (XLffl) Figure A.3: The biosynthesis of citrinin. 217 .. CHJ U. * CH3DQ 0.çXCH3 0 00 U HO2C DH H *0 0 Figure A.4: The origin of the citrinin atoms. * C-i of acetate, D = C-2 of acetate, U = methionine and o = other. 0 H( - HO2C HO2 OH Figure A. 5: The resonance formulae of citrinin. çH3 CH HO CH3 0 HO2C OHH OH Figure A.6: The structures of the citnnin hydrates. Acetate Citrinin R1 Figure Al: A generalised substrate cycle between citrinin and acetate. 218 pass through cell membranes was also put forward: citrinin hydrate is produced inside the cell and, if the hydration in under enzymic control, may be stereochemically pure. At the cytosol-membrane interface, dehydration to give the lipid soluble citrinin will occur and, after transport through the membrane, rehydration will give the observed mixture of stereoisomers. Barber eta!. (1987 b) used [1,2-' 3C2, 1-2H3] acetate feeding experiments successfully to trace the origins of hydrogen atoms in citrinin produced by P. citrinum. This novel labelled precursor had the advantage of allowing deuterium monitoring by analysis of a and 3 shifts in the 13C NMR spectrum with 13C-13C coupling which enabled the acetate derived carbon atoms to be distinguished from those arising from natural abundance 13C from other carbon sources. The results were in accordance with figure A.4. Barber eta!. (1988) investigated further the degradation of citnnin by P. citrinum. In a series of experiments it was shown that: i) P. citrinum scrambled specifically labelled [1 1-2H] keto-aldehyde in a carbon unlimited environment. ii) Ethionine inhibited degradation of citrinin by P. citrinum. iii) Citnnin biosynthesis and degradation by P. citrinum occurred concurrently and iv) citrinin was degraded to acetate, and ultimately to CO2. This led to the theory that the citrinin - acetate interconversion is by two separate pathways in a substrate (or "futile") cycle (figure A.7). The relative rates of citrinin biosynthesis (R2) and citrmin degradation (R 1) may alter in a controlled manner signifying a possible role of citrinin in primary metabolism. The degradation of citrinin to acetate may have a bearing on the results of Patterson and Damoglou (1987) where a both citrininogenic and ochratoxinogenic Penicillium viridicatum culture was fed [U-14C] citrinin. The main radiolabelled breakdown product was identified as dihydrocitrinone but ochratoxin A was also found to be radioactive. A resurrection of the Curtis eta!. (1968) citrinin biosynthetic pathway was used to explain how citrinin was directly converted to ochratoxin A via dihydrocitrinone. It now seems that perhaps [14C] ochratoxin A was biosynthesized from [ 14C] acetate derived from the catabolism of[U- 14C] citrinin, since no specific 219 incorporation of citrinin into ochratoxin A was shown. The role of dihydrocitrinone in citrinin biosynthesis is still unknown although it has been proposed as an intermediate of citrinin degradation (Barber eta!., 1988). Citrinin biosynthesis has been reviewed by Saito et aL (1971), Vleggaar and Steyn (1980) and Betina (1989). 220 References. Abarca, M.L.; Bragulat, M.R.; Caste!lá, G. and Cabafles, F.J. (1994). App!. Environ. Microbiol. 60, (7): 2650-2652. Abell, C.; Garson, M.J.; Leeper, F.J. and Staunton, J. (1982). J. Chem. Soc. Chem. 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