Fate of the methyl group during the conversion of sterigmatocystin into O-methylsterigmatocystin and aflatoxin B1 by cell-free preparations of Aspergillus parasiticus

Cell-free extracts of fungal mycelia of two aflatoxin non-producing isolates of Aspergillus parasiticus (SRRC 163 and SRRC 2043) were utilized for the study of enzyme activities involved in the latter stages of aflatoxin biosynthesis. The post-microsomal fractions (105,000 x g supernatant) of both SRRC 163 and SRRC 2043 were able to convert sterigmatocystin (ST) into O-methylsterigmatocystin (OMST); whereas the microsomal (105,000 x g pellet) preparation of only SRRC 163 was able to convert OMST into aflatoxin B1 (AFB1). S-Adenosylmethionine (SAM) was the primary substrate for the ST to OMST (methyltransferase) enzymatic conversion; [3H]OMST of specific activity 0.93 Ci/mmol was obtained in a reaction containing the [3H]SAM substrate (specific activity 1 Ci/mmol). After the terminal enzymatic conversion of OMST into AFB1, none of the radiolabel of the methyl group from OMST was found in AFB1. It is postulated that the methylation of ST may be required for subsequent enzymatic oxidation of OMST to aflatoxin B1.     

Enzymological evidence for separate pathways for aflatoxin B1 and B2 biosynthesis

Aflatoxins B1 (AFB1) and B2 (AFB2) are biologically active secondary metabolites of Aspergillus flavus and Aspergillus parasiticus. These toxins are synthesized by the fungi from pathway precursors: sterigmatocystin (ST)----O-methylsterigmatocystin (OMST)----AFB1; dihydrosterigmatocystin (DHST)----dihydro-O-methylsterigmatocystin (DHOMST)----AFB2. The late stages of AFB1 synthesis are carried out by two enzyme activities, a methyltransferase (MT) (ST----OMST), and an oxidoreductase (OR) (OMST----AFB1). Properties of the purified MT have been identified in a previous investigation [Bhatnagar et al. (1988) Prep. Biochem. 18, 321]. In the current study, the OR was partially purified (150-fold of specific activity) from fungal cell-free extracts and characterized with extended investigation of the late stages of AFB1 and AFB2 synthesis. Whole cells of an isolate of A. flavus (SRRC 141), which produce only AFB2, were able to produce AFB1 in ST and OMST feeding studies; the results suggested that the enzymes involved in AFB2 biosynthesis also carry out AFB1 synthesis. Substrate competition experiments carried out with the OR showed that an increasing concentration of either OMST or DHOMST in the presence of a fixed, nonsaturating concentration of either DHOMST or OMST, respectively, resulted in a decline in production of one aflatoxin (B1 or B2) with a corresponding increase in the synthesis of the other toxin (B2 or B1). OMST was a preferred substrate (Km, 1.2 microM) for the oxidoreductase as compared to DHOMST (Km, 13.4 microM). Similar, substrate competition experiments showed that ST (Km, 2.0 microM) was a preferred substrate over DHST (Km, 22.5 microM) for a homogeneous MT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus.

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.     

Two distinct O-methyltransferases in aflatoxin biosynthesis

The substances belonging to the sterigmatocystin group bear a close structural relationship to aflatoxins. When demethylsterigmatocystin (DMST) was fed to Aspergillus parasiticus NIAH-26, which endogenously produces neither aflatoxins nor precursors in YES medium, aflatoxins B1 and G1 were produced. When dihydrodemethylsterigmatocystin (DHDMST) was fed to this mutant, aflatoxins B2 and G2 were produced. Results of the cell-free experiment with S-adenosyl-[methyl-3H]methionine showed that first the C-6-OH groups of DMST and DHDMST are methylated to produce sterigmatocystin and dihydrosterigmatocystin (O-methyltransferase I) and then the C-7-OH groups are methylated to produce O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST) (O-methyltransferase II). However, no methyltransferase activity was observed when either OMST, DHOMST, 5,6-dimethoxysterigmatocystin, 5-methoxysterigmatocystin, or sterigmatin was incubated with the cell extract. Treatment of the cell extract with N-ethylmaleimide inhibited O-methyltransferase I activity but not that of O-methyltransferase II. Furthermore, these O-methyltransferases were different in their protein molecules and were involved in both the reactions from DMST to OMST and DHDMST to DHOMST. The reactions described in this paper were not observed when the same mold had been cultured in YEP medium.     

Two distinct O-methyltransferases in aflatoxin biosynthesis.

The substances belonging to the sterigmatocystin group bear a close structural relationship to aflatoxins. When demethylsterigmatocystin (DMST) was fed to Aspergillus parasiticus NIAH-26, which endogenously produces neither aflatoxins nor precursors in YES medium, aflatoxins B1 and G1 were produced. When dihydrodemethylsterigmatocystin (DHDMST) was fed to this mutant, aflatoxins B2 and G2 were produced. Results of the cell-free experiment with S-adenosyl-[methyl-3H]methionine showed that first the C-6-OH groups of DMST and DHDMST are methylated to produce sterigmatocystin and dihydrosterigmatocystin (O-methyltransferase I) and then the C-7-OH groups are methylated to produce O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST) (O-methyltransferase II). However, no methyltransferase activity was observed when either OMST, DHOMST, 5,6-dimethoxysterigmatocystin, 5-methoxysterigmatocystin, or sterigmatin was incubated with the cell extract. Treatment of the cell extract with N-ethylmaleimide inhibited O-methyltransferase I activity but not that of O-methyltransferase II. Furthermore, these O-methyltransferases were different in their protein molecules and were involved in both the reactions from DMST to OMST and DHDMST to DHOMST. The reactions described in this paper were not observed when the same mold had been cultured in YEP medium.     

Biosynthetic relationship among aflatoxins B1, B2, G1, and G2

Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4 (NRRL 2999), produces neither aflatoxins nor precursors. When sterigmatocystin (ST) or O-methylsterigmatocystin was fed to this mutant in YES medium, aflatoxins B1 (AFB1) and G1 (AFG1) were produced. When dihydrosterigmatocystin (DHST) or dihydro-O-methylsterigmatocystin was fed to this mold, aflatoxins B2 (AFB2) and G2 (AFG2) were produced. The reactions from ST to AFB1 and DHST to AFB2 were also observed in the cell-free system and were catalyzed stepwise by the methyltransferase and oxidoreductase enzymes. In the feeding experiments of strain NIAH-26, the convertibility from ST to AFB1-AFG1 was found to be remarkably suppressed by the coexistence of DHST in the medium, and the convertibility from DHST to AFB2-AFG2 was also suppressed by the presence of ST. When some other mutants which endogenously produce a small amount of aflatoxins (mainly AFB1 and AFG1) were cultured with DHST, the amounts of AFB1 and AFG1 produced were significantly decreased, whereas AFB2 and AFG2 were newly produced. In similar feeding experiments in which 27 kinds of mutants including these mutants were used, most of the mutants which were able to convert exogenous ST to AFB1-AFG1 were also found to convert exogenous DHST to AFB2-AFG2. These results suggest that the same enzymes may be involved in the both biosynthetic pathways from ST to AFB1-AFG1 and DHST to AFB2-AFG2. The reactions described herein were not observed when the molds had been cultured in the YEP medium.     

Biosynthetic relationship among aflatoxins B1, B2, G1, and G2.

Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4 (NRRL 2999), produces neither aflatoxins nor precursors. When sterigmatocystin (ST) or O-methylsterigmatocystin was fed to this mutant in YES medium, aflatoxins B1 (AFB1) and G1 (AFG1) were produced. When dihydrosterigmatocystin (DHST) or dihydro-O-methylsterigmatocystin was fed to this mold, aflatoxins B2 (AFB2) and G2 (AFG2) were produced. The reactions from ST to AFB1 and DHST to AFB2 were also observed in the cell-free system and were catalyzed stepwise by the methyltransferase and oxidoreductase enzymes. In the feeding experiments of strain NIAH-26, the convertibility from ST to AFB1-AFG1 was found to be remarkably suppressed by the coexistence of DHST in the medium, and the convertibility from DHST to AFB2-AFG2 was also suppressed by the presence of ST. When some other mutants which endogenously produce a small amount of aflatoxins (mainly AFB1 and AFG1) were cultured with DHST, the amounts of AFB1 and AFG1 produced were significantly decreased, whereas AFB2 and AFG2 were newly produced. In similar feeding experiments in which 27 kinds of mutants including these mutants were used, most of the mutants which were able to convert exogenous ST to AFB1-AFG1 were also found to convert exogenous DHST to AFB2-AFG2. These results suggest that the same enzymes may be involved in the both biosynthetic pathways from ST to AFB1-AFG1 and DHST to AFB2-AFG2. The reactions described herein were not observed when the molds had been cultured in the YEP medium.     

Aflatoxin production via cross-feeding of pathway intermediates during cofermentation of aflatoxin pathway-blocked Aspergillus parasiticus mutants.

Cofermentation of Aspergillus parasiticus strains (SRRC 163 and SRRC 2043) blocked at different steps in the aflatoxin B1 (AFB1) biosynthetic pathway in a synthetic liquid medium or on seeds (cottonseed, corn kernels, and peanuts) resulted in production of AFB1. Strain SRRC 2043 accumulated O-methylsterigmatocystin (OMST), a late precursor in AFB1 biosynthesis, whereas SRRC 163 accumulated averantin, an early precursor in the pathway. Strain SRRC 2043 secreted large amounts of OMST in culture relative to the amounts of several other pathway intermediates secreted into media (by other AFB1 pathway-blocked strains). AFB1 production occurred even when colonies of SRRC 163 and SRRC 2043 strains (producing no detectable AFB1) were grown together on an agar medium while physically separated from each other by a filter membrane (0.22-micron pore size). In addition, when mycelia of strain SRRC 163 were added to culture filtrates (containing no mycelia but containing secreted OMST) of strain SRRC 2043, AFB1 production occurred. The results suggested a chemical (rather than genetic) mechanism of complementation for AFB1 production between AFB1 pathway-blocked strains, since no mycelial contact was required between these strains for AFB1 production. The mechanism for chemical complementation involves secretion of OMST by SRRC 2043 and subsequent absorption and conversion of OMST to AFB1 by mycelia of strain SRRC 163.     

Aflatoxin production via cross-feeding of pathway intermediates during cofermentation of aflatoxin pathway-blocked Aspergillus parasiticus mutants.

Cofermentation of Aspergillus parasiticus strains (SRRC 163 and SRRC 2043) blocked at different steps in the aflatoxin B1 (AFB1) biosynthetic pathway in a synthetic liquid medium or on seeds (cottonseed, corn kernels, and peanuts) resulted in production of AFB1. Strain SRRC 2043 accumulated O-methylsterigmatocystin (OMST), a late precursor in AFB1 biosynthesis, whereas SRRC 163 accumulated averantin, an early precursor in the pathway. Strain SRRC 2043 secreted large amounts of OMST in culture relative to the amounts of several other pathway intermediates secreted into media (by other AFB1 pathway-blocked strains). AFB1 production occurred even when colonies of SRRC 163 and SRRC 2043 strains (producing no detectable AFB1) were grown together on an agar medium while physically separated from each other by a filter membrane (0.22-micron pore size). In addition, when mycelia of strain SRRC 163 were added to culture filtrates (containing no mycelia but containing secreted OMST) of strain SRRC 2043, AFB1 production occurred. The results suggested a chemical (rather than genetic) mechanism of complementation for AFB1 production between AFB1 pathway-blocked strains, since no mycelial contact was required between these strains for AFB1 production. The mechanism for chemical complementation involves secretion of OMST by SRRC 2043 and subsequent absorption and conversion of OMST to AFB1 by mycelia of strain SRRC 163.     

Conversion of 11-hydroxy-O-methylsterigmatocystin to aflatoxin G1 in Aspergillus parasiticus

In aflatoxin biosynthesis, aflatoxins G(1) (AFG(1)) and B(1) (AFB(1)) are independently produced from a common precursor, O-methylsterigmatocystin (OMST). Recently, 11-hydroxy-O-methylsterigmatocystin (HOMST) was suggested to be a later precursor involved in the conversion of OMST to AFB(1), and conversion of HOMST to AFB(1) was catalyzed by OrdA enzyme. However, the involvement of HOMST in AFG(1) formation has not been determined. In this work, HOMST was prepared by incubating OrdA-expressing yeast with OMST. Feeding Aspergillus parasiticus with HOMST allowed production of AFG(1) as well as AFB(1). In cell-free systems, HOMST was converted to AFG(1) when the microsomal fraction, the cytosolic fraction from A. parasiticus, and yeast expressing A. parasiticus OrdA were added. These results demonstrated (1) HOMST is produced from OMST by OrdA, (2) HOMST is a precursor of AFG(1) as well as AFB(1), and (3) three enzymes, OrdA, CypA, and NadA, and possibly other unknown enzymes are involved in conversion of HOMST to AFG(1).     

Precursor recognition by kinetic pulse-labeling in a toxigenic aflatoxin B1-producing strain of Aspergillus.

Kinetic pulse-labeling of aflatoxin pathway compounds was carried out in Aspergillus parasiticus, beginning with radioactive acetate. Norsolorinic acid, averufin, versicolorin A, and sterigmatocystin (all known as compounds which can be incorporated into the aflatoxin molecule) were radiotraced to follow their order of appearance. Aflatoxin species B1, B2, G1, and G2 were included. Norsolorinic acid and averufin appeared as early transient intermediates followed in order by versicolorin A, aflatoxins, and sterigmatocystin. To date, a mutually confirming array of results has been obtained with established precursors in wild-type strains of A. parasiticus and A. versicolor (as well as with an aflatoxin pathway mutant of A. parasiticus), which together establish a practical methodology for recognition of new pathway intermediates. The kinetic of pulse-labeling for sterigmatocystin in relation to aflatoxins suggests that duel branchlets may exist to flatoxins; i.e., sterigmatocystin may not be an obligatory aflatoxin precursor.     

Individual reaction requirements of two enzyme activities, isolated from Aspergillus parasiticus, which together catalyze conversion of sterigmatocystin to aflatoxin B1

Individual reaction requirements were determined for each of two enzyme activities present in Aspergillus parasiticus mycelia which together catalyze conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1). A postmicrosomal activity (PMA) catalyzed conversion of ST to O-methylsterigmatocystin (OMST) and a microsomal activity (MA) catalyzed conversion of OMST to AFB1. PMA was stimulated two- to three-fold in the presence of S-adenosylmethionine. Addition of NADPH promoted the maximum MA; this activity was not detected when FAD, FMN, NAD, or NADH were utilized individually as cofactors in reaction mixtures. A substantial amount (62%) of MA was lost during isolation of the microsomal fraction, but the activity was completely restored by reconstitution with a heat-treated (100 degrees C) postmicrosomal fraction. The reaction catalyzed by MA was optimum at pH 7.0 and at 17-23 degrees C, whereas the PMA reaction was optimum at pH 8.0-8.5 and at 35-40 degrees C. Apparent Km values of approximately 2.6 X 10(-6) M (for ST) and 6.6 X 10(-7) M (for OMST) were determined for PMA and MA, respectively.     

Enzymes in atlatoxin B1 biosynthesis: Strategies for identifying pertinent genes

Recent work on the aflatoxin biosynthetic pathway is reviewed, with special emphasis on the enzymes of the late stages of the pathway involving conversion of sterigmatocystin (ST) to aflatoxin B₁ (AFB₁) through an O-methylsterigmatocystin intermediate. Two enzyme activities were discovered in subcellular fractions of cell-free extracts of a mutant strain ofAspergillus parasiticus (SRRC 163): 1)A post-microsomal methyltransferase (MT) catalyzed conversion of ST to OMST, and 2) a microsomal-associated activity (oxido-reductase) converted OMST to AFB₁. The 168 KDa, anionic MT was purified to homogeneity and characterized (two subunits, 110 KDa and 58 KDa). Preliminary evidence indicated the presence of a cationic isozyme of the MT in mycelial extracts. The oxido-reductase has been partially purified and characterized. Polyclonal antibodies were prepared to the anionic MT and the enzyme's amino acid composition determined. A cDNA library has been constructed from mRNA isolated fromAspergillus parasiticus mycelia during the onset of AFB₁ biosynthesis for the purpose of identifying the genes responsible for aflatoxin biosynthesis.     

adhA in Aspergillus parasiticus is involved in conversion of 5'-hydroxyaverantin to averufin

Two routes for the conversion of 5'-hydroxyaverantin (HAVN) to averufin (AVF) in the synthesis of aflatoxin have been proposed. One involves the dehydration of HAVN to the lactone averufanin (AVNN), which is then oxidized to AVF. Another requires dehydrogenation of HAVN to 5'-ketoaverantin, the open-chain form of AVF, which then cyclizes spontaneously to AVF. We isolated a gene, adhA, from the aflatoxin gene cluster of Aspergillus parasiticus SU-1. The deduced ADHA amino acid sequence contained two conserved motifs found in short-chain alcohol dehydrogenases-a glycine-rich loop (GXXXGXG) that is necessary for interaction with NAD(+)-NADP(+), and the motif YXXXK, which is found at the active site. A. parasiticus SU-1, which produces aflatoxins, has two copies of adhA (adhA1), whereas A. parasiticus SRRC 2043, a strain that accumulates O-methylsterigmatocystin (OMST), has only one copy. Disruption of adhA in SRRC 2043 resulted in a strain that accumulates predominantly HAVN. This result suggests that ADHA is involved in the dehydrogenation of HAVN to AVF. Those adhA disruptants that still made small amounts of OMST also accumulated other metabolites, including AVNN, after prolonged culture.     

Purification and characterization of a methyltransferase from Aspergillus parasiticus SRRC 163 involved in aflatoxin biosynthetic pathway

A five step scheme has been developed for the purification of a methyltransferase (MT) from mycelia of 3-day old Aspergillus parasiticus (SRRC 163), which catalyzes one step in the aflatoxin biosynthetic pathway. The S-adenosylmethionine (SAM) requiring MT activity is essential for the conversion of sterigmatocystin (ST) to O-methylsterigmatocystin (OMST) prior to being converted to aflatoxin B1. The purification of the MT was carried out from cell-free extracts by CDR (Cell Debris Remover, a cellulosic weak anion exchanger, Whatman) treatment, QMA ACELL, Hydroxylapatite-Ultrogel, PBE 94 chromatofocusing and FractoGel TSK HW-50F filtration chromatography. The purified enzyme was only about 0.1% of the total extractable proteins. The pI of the protein was about 5.0 as judged by chromatofocusing. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 160-KDa. SDS-polyacrylamide gel electrophoresis revealed two protein subunit bands of molecular masses approximately 110-KDa and 58-KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.87 X 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5). The reaction catalyzed by the MT was optimum at pH 7.5 and between 25-35 degrees C. The Km of the enzyme for ST and SAM was determined to be 1.8 microM and 42 microM, respectively with an estimated turnover number of the enzyme for ST of 2.2 X 10(-2) per sec.     

Production of M-/GM-group aflatoxins catalyzed by the OrdA enzyme in aflatoxin biosynthesis

Aspergillus parasiticus produces the minor aflatoxins M(1) (AFM(1)), M(2) (AFM(2)), GM(1) (AFGM(1)), and GM(2) (AFGM(2)), as well as the major aflatoxins B(1) (AFB(1)), B(2) (AFB(2)), G(1) (AFG(1)), and G(2) (AFG(2)). Feeding of A. parasiticus with aspertoxin (12c-hydroxyOMST) caused AFM(1) and AFGM(1), and cell-free experiments using the microsomal fraction of A. parasiticus and aspertoxin caused production of AFM(1), indicating that aspertoxin is a precursor of AFM(1) and AFGM(1). Feeding of the same fungus with O-methylsterigmatocystin (OMST) caused AFM(1) and AFGM(1) together with AFB(1) and AFG(1); feeding with dihydroOMST (DHOMST) caused AFM(2) and AFGM(2) together with AFB(2) and AFG(2). Incubation of either the microsomal fraction or OrdA enzyme-expressing yeast with OMST caused production of aspertoxin together with AFM(1) and AFB(1). These results demonstrated that the OrdA enzyme catalyzes both 12c-hydroxylation reaction from OMST to aspertoxin and the successive reaction from aspertoxin to AFM(1). In contrast, feeding of the fungus with AFB(1) did not produce any AFM(1), demonstrating that M-/GM-aflatoxins are not produced from B-/G-aflatoxins. Furthermore, AFM(1) together with AFB(1) and AFG(1) was also produced from 11-hydroxyOMST (HOMST) in feeding experiment of A. parasiticus, whereas no aflatoxins were produced when used the ordA deletion mutant. These results demonstrated that OrdA enzyme can also catalyze 12c-hydroxylation of HOMST to produce 11-hydroxyaspertoxin, which serves as a precursor for the production of AFM(1) and AFGM(1). The same pathway may work for the production of AFM(2) and AFGM(2) from DHOMST and dihydroHOMST through the formation of dihydroaspertoxin and dihydro-11-hydroxyaspertoxin, respectively.     

Aflatoxin production by entomopathogenic isolates of Aspergillus parasiticus and Aspergillus flavus

Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.     

Synthesis of sterigmatocystin derivatives and their biotransformation to aflatoxins by a blocked mutant of Aspergillus parasiticus

Seven alkyl and aryl homologues of O-methylsterigmatocystin (OMST) were synthesised and fed in separate experiments to a mutant of Aspergillus parasiticus capable of converting sterigmatocystin (ST) to aflatoxin B₁ (AFB₁). Their conversion to AFB₁ was followed over a time period and it was found that O-propylsterigmatocystin (OPRST) was converted to AFB₁ more rapidly than O-ethylsterigmatocystin (OEST) or OMST or ST itself. The aryl derivative O-benzoylsterigmatocystin (OBzST) was converted at the slowest rate. These results show that alkyl and aryl homologues of OMST may be converted to AFB₁, suggesting that the methylation of ST is not an absolute requirement for its conversion to AFB₁. It seems likely that whatever enzyme(s) are involved in this process exhibit relative specificity. As to whether alkylation of ST is an obligatory step in AFB₁ biosynthesis is neither supported nor disproved as the fungal cells used are presumably capable of methylating ST. The fact that the propyl derivative showed fastest conversion is not necessarily significant as this may be due to faster diffusion of the least polar of the derivatives through the cell membrane. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biosynthetic origin of aflatoxin G1: confirmation of sterigmatocystin and lack of confirmation of aflatoxin B1 as precursors

The origin of aflatoxin G1 was studied using mutant strains of Aspergillus parasiticus blocked early in the pathway and by tracing 14C-labelled aflatoxin B1 (AFB1) in wild-type A. flavus and A. parasiticus strains. Sterigmatocystin (ST) was a precursor of AFB1, AFG1 and AFG2 in the four mutants examined. The identity of AFG1 was confirmed by mass spectrometry. No evidence for conversion of AFB1 to AFG1 was found. A rigorously controlled study of conversions of radioactivity based on preparative thin-layer chromatography of aflatoxins demonstrated that low levels of aflatoxin interconversions previously reported in the literature might actually be artifacts.