Sterigmatocystin Production Research Articles

Emericella venezuelensis, a New Species With Stellate Ascospores Producing Sterigmatocystin and Aflatoxin B 1

Emericella venezuelensis is a new species, differing from two other species with stellate ascospores, E. variecolor and E. pluriseminata, by triangular flaps on the convex sides of the ascospores, and further from E. variecolor by producing an Aspergillus anamorph only on unconventional growth media. The three species also differ in their profiles of extrolites (secondary metabolites). Emericella venezuelensis produces aflatoxin B1, sterigmatocystin, and terrein and compounds with chromophores of the shamixanthone, emerin and desertorin type of compounds. E. variecolor produces asteltoxin, shamixanthone, asperthecin, and terrein, in addition to metabolites unequivocally recorded in the literature or tentatively identified here as astellolide A & B, andibenin A, B, C, andilesin A, B, C, anditomin, astellatol, stellatic acid, stellatin, tajixanthone, radixanthone, najamxanthone, ajamxanthone, variecoxanthone A, B, C, isoemericellin, kojic acid, varitriol, varioxiran, dihydroterrein, 7-hydroxyemodin, avariquinone and stromemycin. E. pluriseminata produces several unknown specific extrolites. E. venezuelensis is the first organism of marine origin reported to produce aflatoxin. Aflatoxin production by E. venezuelensis makes this species an attractive model organism for the study of the regulation of this important type of carcinogenic mycotoxins in combination with the knowledge on sterigmatocystin production by E. nidulans, soon to be whole genome sequenced. The isolates were also analyzed cladistically using partial sequences of the beta-tubulin gene. Since three species of Emericella have stellate ascospores, and the type material of E. variecolor is equivocal, this species is epitypified with CBS 598.65. Emericella species normally do not appear to cause problems for food safety, as they are most often found in litter and soil.

Increased conidiation associated with progression along the sterigmatocystin biosynthetic pathway

The Aspergillus nidulans sterigmatocystin (ST) gene cluster contains both regulatory (aflR) and biosynthetic genes (stc genes) required for ST production. A total of 26 genes are in the cluster, 13 of which have been assigned a known function in the biosynthetic pathway. This complex secondary pathway represents a physiological cost to the fungus. We tested the amount of asexual spore production using a series of isogenic lines of A. nidulans, differing only in a mutation in aflR (resulting in a strain containing no ST intermediates) or a mutation in three stc genes that produced either no ST intermediates (ΔstcJ), an early ST intermediate, norsoloroinic acid (ΔstcE) or a late ST intermediate, versicolorin A (ΔstcU). In two independently replicated experiments we compared the numbers of conidia produced by each of these mutant strains and a wild type ST producer in a neutral (growth media) and a host (corn seed) environment. A stepwise increase in asexual spore production was observed with each progressive step in the ST pathway. Thus, the data suggest that recruitment or loss of these secondary metabolite pathway genes has a selective advantage apart from the physiological activity of the metabolite.

Connection of Propionyl-CoA Metabolism to Polyketide Biosynthesis in Aspergillus nidulans

Propionyl-CoA is an intermediate metabolite produced through a variety of pathways including thioesterification of propionate and catabolism of odd chain fatty acids and select amino acids. Previously, we found that disruption of the methylcitrate synthase gene, mcsA, which blocks propionyl-CoA utilization, as well as growth on propionate impaired production of several polyketides-molecules typically derived from acetyl-CoA and malonyl-CoA-including sterigmatocystin (ST), a potent carcinogen, and the conidiospore pigment. Here we describe three lines of evidence that demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis. First, inactivation of a putative propionyl-CoA synthase, PcsA, which converts propionate to propionyl-CoA, restored polyketide production and reduced cellular propionyl-CoA content in a DeltamcsA background. Second, inactivation of the acetyl-CoA synthase, FacA, which is also involved in propionate utilization, restored polyketide production in the DeltamcsA background. Third, fungal growth on several compounds (e.g., heptadecanoic acid, isoleucine, and methionine) whose catabolism includes the formation of propionyl-CoA, were found to inhibit ST and conidiospore pigment production. These results demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis.

Emericella astellata, a new producer of aflatoxin B, B and sterigmatocystin.

To report on aflatoxin B(1) and B(2) production from a species of Emericella. Aflatoxins and sterigmatocystin were determined by high-pressure liquid chromatography (HPLC) with diode array detection and confirmed by HPLC with mass spectrometry detection. Among 30 known species of Emericella only one species produced aflatoxin. Strains originating from the same geographical source material had different patterns of aflatoxin and sterigmatocystin production on different media, indicating that epigenetic factors may be involved in the regulation of aflatoxin production. However, two cultures from the same original genet were very similar. Emericella astellata can produce small amounts of sterigmatocystin and aflatoxin B(1) and B(2). Emericella has been used extensively in genetic studies and therefore the isolates producing aflatoxin can be used to elucidate the genetic, evolutionary and maybe ecological role of aflatoxins using molecular genetic methods.

Blockage of methylcitrate cycle inhibits polyketide production in Aspergillus nidulans.

Aspergillus nidulans produces the polyketide toxin sterigmatocystin (ST) of which the biosynthetic and pathway specific regulatory genes compose a stc gene cluster. A previous mutagenesis screen identified 23 mutants defective in production of ST. Five mutants constitute a single locus. Genetic complementation and sequencing analysis revealed the mutant locus to be mcsA encoding methylcitrate synthase that converts propionyl-CoA to methylcitrate. Feeding downstream products of methylcitrate synthase, methylcitrate and pyruvate, did not restore ST production in mcsA mutants, indicating that loss of methylcitrate cycle products is not the cause of the ST defect. However, propionate, a precursor for propionyl-CoA, inhibited ST production and induced transcription of mcsA in the wild type. Furthermore, propionate impaired formation of two polyketide spore pigments whereas overexpression of mcsA relieved inhibition of ST production by propionate. Transcription analyses revealed that disruption of mcsA did not affect expression of the specialized fatty acid synthase genes (stcJ and stcK) or polyketide synthase gene (stcA) required for formation of norsolorinic acid (NOR), the first stable intermediate in the ST biosynthetic pathway. Feeding studies showed that NOR but not hexanoic acid (the fatty acid produced by StcJ/StcK and primer unit of StcA) or malonate (source of the extender unit of StcA) restored ST production in the mcsA mutant. We hypothesize that excess buildup of propionyl-CoA in mcsA mutants interferes with polyketide synthase activity.

Suppressor mutations bypass the requirement of fluG for asexual sporulation and sterigmatocystin production in Aspergillus nidulans.

Asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans requires the early developmental activator fluG. Loss of fluG results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin (ST). To investigate molecular mechanisms of fluG-dependent developmental activation, 40 suppressors of fluG (SFGs) that conidiate without fluG have been isolated and characterized. Genetic analyses showed that an individual suppression is caused by a single second-site mutation, and that all sfg mutations but one are recessive. Pairwise meiotic crosses grouped mutations to four loci, 31 of them to sfgA, 6 of them to sfgB, and 1 each to sfgC and sfgD, respectively. The only dominant mutation, sfgA38, also mapped to the sfgA locus, suggesting a dominant negative mutation. Thirteen sfgA and 1 sfgC mutants elaborated conidiophores in liquid submerged culture, indicating that loss of either of these gene functions not only bypasses fluG function but also results in hyperactive conidiation. While sfg mutants show varying levels of restored conidiation, all recovered the ability to produce ST at near wild-type levels. The fact that at least four loci are defined by recessive sfg mutations indicates that multiple genes negatively regulate conidiation downstream of fluG and that the activity of fluG is required to remove such repressive effects.

Storage and drying of grain in Canada: low cost approaches

Most Canadian grain (>70% of harvests) is stored on the farm. High moisture content of grain at harvest rapidly leads to spoilage and occasionally the production of the mycotoxins sterigmatocystin, ochratoxin A, or citrinin. Near ambient drying systems that consist of an electrical fan at the base of a granary blowing air into a plenum beneath a perforated floor under stored grain is relatively economical. Heat produced by the fan can dry grain by 2% moisture content in 2 months at a cost for electricity of Can. $0.87/tonne. Hot air dryers are used on wet grain at harvest to rapidly lower 21% moisture content maize to 15% moisture content at a cost of Can. $7.80/tonne for propane plus aeration costs to cool the grain. Procedures for safe storage of grain are outlined and the capabilities and planned use of a new grain storage research facility at the University of Manitoba are discussed.

Requirement of spermidine for developmental transitions in Aspergillus nidulans

Deletion of the spermidine synthase gene in the fungus Aspergillus nidulans results in a strain, deltaspdA, which requires spermidine for growth and accumulates putrescine as the sole polyamine. Vegetative growth but not sporulation or sterigmatocystin production is observed when deltaspdA is grown on media supplemented with 0.05-0.10 mM exogenous spermidine. Supplementation of deltaspdA with >/= 0.10 mM spermidine restores sterigmatocystin production and >/= 0.50 mM spermidine produces a phenotype with denser asexual spore production and decreased radial hyphal growth compared with the wild type. DeltaspdA spores germinate in unsupplemented media but germ tube growth ceases after 8 h upon which time the spores swell to approximately three times their normal diameter. Hyphal growth is resumed upon addition of 1.0 mM spermidine. Suppression of a G protein signalling pathway could not force asexual sporulation and sterigmatocystin production in deltaspdA strains grown in media lacking spermidine but could force both processes in deltaspdA strains supplemented with 0.05 mM spermidine. These results show that increasing levels of spermidine are required for the transitions from (i) germ tube to hyphal growth and (ii) hyphal growth to tissue differentiation and secondary metabolism. Suppression of G protein signalling can over-ride the spermidine requirement for the latter but not the former transition.

Relationship between secondary metabolism and fungal development.

Filamentous fungi are unique organisms-rivaled only by actinomycetes and plants-in producing a wide range of natural products called secondary metabolites. These compounds are very diverse in structure and perform functions that are not always known. However, most secondary metabolites are produced after the fungus has completed its initial growth phase and is beginning a stage of development represented by the formation of spores. In this review, we describe secondary metabolites produced by fungi that act as sporogenic factors to influence fungal development, are required for spore viability, or are produced at a time in the life cycle that coincides with development. We describe environmental and genetic factors that can influence the production of secondary metabolites. In the case of the filamentous fungus Aspergillus nidulans, we review the only described work that genetically links the sporulation of this fungus to the production of the mycotoxin sterigmatocystin through a shared G-protein signaling pathway.

Identification of Aspergillus nidulans Genes Essential for the Accumulation of Sterigmatocystin

The fungus Aspergillus nidulans (Emericella nidulans) was used as a genetic model for the identification of genes required for efficient accumulation of sterigmatocystin (ST). The required gene for sterigmatocystin expression was stc, which is an intermediate penultimate product in the aflatoxin biosynthetic pathway. Genetic analysis included studies of the sexual and parasexual cycles. The allelic segregation rates and recombination frequencies between linked and nonlinked genetic markers were determined by the crossing of the strains UT448 (stc) to UT196 (stc+) and UT448 (stc) to UT184 (stc). Low ST accumulation (4.0 ppm) in the UT196 strain and in 7.4% of the meiotic segregants allowed us to map the stc locus at chromosome I, 3.4% distant from riboA1. The diploid UT448 (stc)//UT184 (stc), prepared from nonproducing strains, was analyzed based on the parasexual cycle, and 28% of the haploid segregants accumulated the ST toxin. The results suggest that UT448 carries the stc mutant (or an inactivated) allele and that UT184, although carrying the stc+ allele, is reactivated only by the R2+ factor, which is located at chromosome VIII of UT448. In such a configuration, the diploid accumulates large amounts of sterigmatocystin (40 ppm). Another regulator factor (R1), located at the meth-w (II) chromosomic interval, was identified in the UT448 strain. At DNA level in chromosome I, the R1 product acts and blocks the stcZ+ gene transcription. In a different genotypic configuration, the R1 product interacts with the R2 product (of chromosome VIII), allowing the stcZ+ gene expression. Furthermore, the diploid UT448 (stc)//UT196 (stc+) accumulated the ST toxin at high level (40 ppm), indicating similar interaction between mentioned factors and the stc gene. Obtained data suggest that R1 (II) regulates the stcZ+ transcription, by interacting with chromosome I (at the DNA level) and that R2 (VIII) controls R1 activity at the cytoplasm level. Based on these results, we propose a regulation model for the sterigmatocystin production.

RcoA has pleiotropic effects on Aspergillus nidulans cellular development.

Aspergillus nidulans rcoA encodes a member of the WD repeat family of proteins. The RcoA protein shares sequence similarity with other members of this protein family, including the Saccharomyces cerevisiae Tup1p and Neurospora crassa RCO1. Tup1p is involved in negative regulation of an array of functions including carbon catabolite repression. RCO1 functions in regulating pleiotropic developmental processes, but not carbon catabolite repression. In A. nidulans, deletion of rcoA (DeltarcoA), a recessive mutation, resulted in gross defects in vegetative growth, asexual spore production and sterigmatocystin (ST) biosynthesis. Expression of the asexual and ST pathway-specific regulatory genes, brlA and aflR, respectively, but not the signal transduction genes (i.e. flbA, fluG or fadA) regulating brlA and aflR expression was delayed (brlA) or eliminated (aflR) in a DeltarcoA strain. Overexpression of aflR in a DeltarcoA strain could not rescue normal expression of downstream targets of AflR. CreA-dependent carbon catabolite repression of starch and ethanol utilization was only weakly affected in a DeltarcoA strain. The strong role of RcoA in development, vegetative growth and ST production, compared with a relatively weak role in carbon catabolite repression, is similar to the role of RCO1 in N. crassa.

Genetic Involvement of a cAMP-Dependent Protein Kinase in a G Protein Signaling Pathway Regulating Morphological and Chemical Transitions in Aspergillus nidulans

In the filamentous fungus Aspergillus nidulans, a heterotrimeric G protein alpha-subunit and an RGS domain protein, encoded by fadA and flbA, respectively, regulate production of the carcinogenic metabolite sterigmatocystin (ST) and asexual spores (i.e., conidia). We investigated the genetic involvement of the cAMP-dependent protein kinase catalytic subunit (PkaA), a potential downstream target of FadA activity, in ST production and conidiation. Relative to wild type, sporulation was decreased in the pkaA overexpression strain but was not totally absent, as occurs in DeltaflbA or fadA(G42R) (fadA-dominant active) strains. Deletion of pkaA resulted in a hyper-conidiating strain with limited radial growth. This phenotype was epistatic to mutation in flbA or fadA; the double mutants DeltapkaA; DeltaflbA and DeltapkaA; fadA(G42R) recovered sporulation and their radial growth was severely restricted. PkaA overexpression also negatively regulated AflR, the ST biosynthesis-specific transcription factor, both transcriptionally and post-transcriptionally. Deletion of pkaA restored ST production in the DeltaflbA background but not in the fadA(G42R) background. These data provide genetic evidence that the FlbA/FadA signaling pathway regulating ST production and morphological development is partially mediated through PkaA.

Production of mycotoxins on artificially inoculated building materials

Isolates of Stachybotrys chartarum, Aspergillus versicolor and Trichoderma spp. from water damaged Danish buildings were grown on old and new gypsum boards, wood pieces, chipboard, gypsum board with wallpaper and acoustic ceiling tiles. Production of sterigmatocystin and 5-methoxysterigmatocystin by each of 5 isolates of A. versicolor growing on pine wood, wallpaper, gypsum board and chipboard, were detected using HPLC with diode array detection and TLC with AlCl 3 staining. After derivatization to the heptafluorobutyrlated ester and using gas chromatography ion trap mass spectrometry, negative ion chemical ionisation, for detection, trichothecenes of the verrucarol type were found in 4 of 5 isolates of S. chartarum growing on old and new gypsum boards. None of 8 Trichoderma isolates produced the trichothecenes T-2 toxin, HT-2 toxin, diacetoxyscirpenol, fusarenon-X, deoxynivalenol, nivalenol or trichothecenes of the verrucarol or trichodermol type on any of the above mentioned materials.

Aspergillus sporulation and mycotoxin production both require inactivation of the FadA G alpha protein-dependent signaling pathway.

The filamentous fungus Aspergillus nidulans contains a cluster of 25 genes that encode enzymes required to synthesize a toxic and carcinogenic secondary metabolite called sterigmatocystin (ST), a precursor of the better known fungal toxin aflatoxin (AF). One ST Cluster (stc) gene, aflR, functions as a pathway-specific transcriptional regulator for activation of other genes in the ST pathway. However, the mechanisms controlling activation of aflR and synthesis of ST and AF are not understood. Here we show that one important level for control of stc gene expression requires genes that were first identified as early acting regulators of asexual sporulation. Specifically, we found that loss-of-function mutations in flbA, which encodes a RGS domain protein, or dominant activating mutations in fadA, which encodes the alpha subunit of a heterotrimeric G protein, block both ST production and asexual sporulation. Moreover, overexpression of flbA or dominant interfering fadA mutations cause precocious stc gene expression and ST accumulation, as well as unscheduled sporulation. The requirement for flbA in sporulation and ST production could be suppressed by loss-of-function fadA mutations. The ability of flbA to activate stc gene expression was dependent upon another early acting developmental regulator, fluG, and AflR, the stc gene-specific transcription factor. These results are consistent with a model in which both asexual sporulation and ST production require inactivation of proliferative growth through inhibition of FadA-dependent signaling. This regulatory mechanism is conserved in AF-producing fungi and could therefore provide a means of controlling AF contamination.

Seed Lipoxygenase Products Modulate Aspergillus Mycotoxin Biosynthesis

Oilseed crops are frequently subject to contamination by mycotoxins produced by Aspergillus spp., particularly aflatoxin (AF) and to a lesser extent sterigmatocystin (ST). Several studies have suggested that metabolites generated from the plant lipoxygenase (LOX) pathway may either decrease or increase mycotoxin production by Aspergillus spp. We tested the possibility that the occurrence of seed LOX isozymes that produce distinct hydroperoxy fatty acids may account for these different effects on AF biosynthesis. For example, soybean LOX1 catalyzes the addition of O2 to the C13 position of linoleic and linolenic acids while maize embryo LOX catalyzes the addition of O2 to the C9 position. In vitro experiments showed that 13S-hydroperoxy fatty acids at concentrations of 10 and 100 μM repressed AF and ST pathway gene expression and significantly (P = 0.05) reduced AF and ST production in both A. parasiticus (AF producer) and A. nidulans (ST producer). Treatment with 1 μM 13S-hydroperoxy linoleic acid also significantly decreased AF production when introduced into growth media at continuous 24-h intervals. In contrast, the same concentrations of 9S-hydroperoxy linoleic acid did not reduce AF or ST production but extended the length of time AF and ST transcripts were detectable. These results show that 13S-hydroperoxy fatty acids directly or indirectly repress AF and ST biosynthesis and provide in vitro evidence that specific seed lipoxygenase activity could provide resistance to mycotoxin contamination by Aspergillus spp.

A Convenient Method for Assessing Mycotoxin Production in Cultures of Aspergilli and Penicillia

Production of aflatoxins, sterigmatocystin, citrinin, and ochratoxin A by Aspergillus and Penicillium species on agar in 50-ml flasks after 21 days was detected and confirmed by thin-layer chromatography. Toxins were extracted with 2.5 ml of acidified methylene chloride for 20 to 60 min. Mycotoxin concentrations were estimated by liquid chromatography. Addition of formic acid to the methylene chloride extraction solvent effected an increase in recovery of ochratoxin A from Czapek-yeast (CY) agar from 20% to 91%. Recoveries of added mycotoxins were generally higher from CY agar than from yeast extract-sucrose (YES) agar and ranged from 11% (citrinin from YES agar) to 91% (ochratoxin A from CY agar). The method described is convenient, rapid, and reproducible, since it samples the entire culture surface. Human exposure to volatile solvents is also minimized.

Incidence of toxigenic molds in farm-level cheesemaking units from Arzua (La Coruña, Spain)

Four hundred ten strains of Penicillium and 120 strains of Aspergillus isolated from air, surfaces, and cheese, in 10 farm-level cheesemaking farm units from Arzúa (La Coruña, Spain) were classified. The mycotoxin production using the APA medium and thin-layer chromatography was proved. With respect to the production of aflatoxins, 19% from Aspergillus strains resulted positive in agar APA and 18.3% by thin-layer chromatography. With regard to citrinin, 22.2% from Penicillium strains and 5% from the Aspergillus strains were producers of this mycotoxin; 16.1% from Penicillium and 1.67% from Aspergillus produced ochratoxin A. With regard to patulin, 5.4% from the fungi belonging to the genus Penicillium studied were producers of this mycotoxin as well as 3.3% from the Aspergillus. Just 6.6% from the Aspergillus strains resulted in the production of sterigmatocystin.

Antibacterial, antidermatophytic and antitoxigenic activities of onion ( Allium cepa L.) oil

The inhibitory effect of onion oil against the growth of various isolates of bacteria representing Gram-positive (4 isolates) and Gram-negative (4 isolates) species were studied. Results show that onion oil was highly active against all Gram-positive bacteria tested and only one isolate (Klebsiella pneumoniae) of Gram-negative bacteria. The inhibitory effect of onion oil against nine different species of dermatophytic fungi were also studied. Onion oil (200 ppm) completely inhibited the growth of Microsporum canis, M. gypseum and Trichophyton simii while the growth of both, Chrysosporium queenslandicum and Trichophyton mentagrophytes was completely inhibited by 500 ppm of onion oil. The growth of four other species of dermatophytic fungi was gradually reduced by increasing the concentrations of onion oil. The inhibitory effect of onion oil was also tested against four toxigenic isolates of fungi. Onion oil at different concentrations (100, 200 and 500 ppm) tested gradually reduced fungal growth and aflatoxin production by Aspergillus flavus IMI 89 717 and A.parasiticus var. globosus IMI 120920. Fungal growth and production of sterigmatocystin and rubratoxin A by A. versicolor IMI 16139 and Penicillium rubrum IMI 136127 were completely inhibited by the addition of 200 ppm onion oil.

Genetic modification of an echinocandin B-producing strain ofAspergillus nidulans to produce mutants blocked in sterigmatocystin biosynthesis

The production of echinocandin B (ECB), a lipopolypeptide used for chemical manufacture of the anti-Candida agent Cilofungin, was accomplished by fermentation using a strain of Aspergillus nidulans. In addition to ECB, this fermentation also produces a significant amount of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Mutants blocked in the ST biosynthetic pathway were created by genetic modification of the polyploid production strain C747. The following steps were involved: (i) reduction of the genotype to haploid by treatment with the spindle fiber poison methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate (MBC), using colony morphology, conidia size, and the ability to obtain 5-fluoro-orotic acid (5-FOA)-resistant mutants as criteria for ploidy; (ii) mutagenesis of a haploid isolate using UV irradiation; and (iii) screening of mutants for inability to produce ST by thin layer chromatography. Six mutants blocked in ST production were isolated. All six remained capable of producing ECB equivalent in quantity to the haploid strain C747-GR14. One of the mutants was shown to be the result of a chromosomal translocation.

Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin

Aspergillus nidulans produces the carcinogenic mycotoxin sterigmatocystin (ST), the next-to-last precursor in the aflatoxin (AF) biosynthetic pathway found in the closely related fungi Aspergillus flavus and Aspergillus parasiticus. We identified and characterized an A. nidulans gene, verA, that is required for converting the AF precursor versicolorin A to ST. verA is closely related to several polyketide biosynthetic genes involved in polyketide production in Streptomyces spp. and exhibits extended sequence similarity to A. parasiticus ver-1, a gene proposed to encode an enzyme involved in converting versicolorin A to ST. By performing a sequence analysis of the region 3' to verA, we identified two additional open reading frames, designated ORF1 and ORF2. ORF2 is closely related to a number of cytochrome P-450 monooxygenases, while ORF1 shares identity with the gamma subunit of translation elongation factor 1. Given that several steps in the ST-AF pathway may require monooxygenase activity and that AF biosynthetic genes are clustered in A. flavus and A. parasiticus, we suggest that verA may be part of a cluster of genes required for ST biosynthesis. We disrupted the verA coding region by inserting the A. nidulans argB gene into the center of the coding region and transformed an A. nidulans argB2 mutant to arginine prototrophy. Seven transformants that produced DNA patterns indicative of a verA disruption event were grown under ST-inducing conditions, and all of the transformants produced versicolorin A but negligible amounts of ST (200-fold to almost 1,000-fold less than the wild type), confirming the hypothesis that verA encodes an enzyme necessary for converting versicolorin A to ST.