Next Article in Journal
Quantification of Signaling Lipids by Nano-Electrospray Ionization Tandem Mass Spectrometry (Nano-ESI MS/MS)
Next Article in Special Issue
Genetics of Polyketide Metabolism in Aspergillus nidulans
Previous Article in Journal
Mass Spectrometry Based Lipidomics: An Overview of Technological Platforms
Previous Article in Special Issue
Role of Cereal Secondary Metabolites Involved in Mediating the Outcome of Plant-Pathogen Interactions
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Metabolites
Volume 2
Issue 1
10.3390/metabo2010039
metabolites-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparative Chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357)

by
Christian Rank
1,†,
Marie Louise Klejnstrup
1,†,
Lene Maj Petersen
1,
Sara Kildgaard
1,
Jens Christian Frisvad
1,
Charlotte Held Gotfredsen
2 and
Thomas Ostenfeld Larsen
1,*
1
Department of Systems Biology, Center for Microbial Biotechnology, Technical University of Denmark, Søltofts Plads B221, DK-2800 Kgs. Lyngby, Denmark
2
Department of Chemistry, Technical University of Denmark, Kemitorvet B201, DK-2800 Kgs. Lyngby, Denmark
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Metabolites 2012, 2(1), 39-56; https://doi.org/10.3390/metabo2010039
Submission received: 18 November 2011 / Revised: 14 December 2011 / Accepted: 22 December 2011 / Published: 5 January 2012
(This article belongs to the Special Issue Secondary Metabolites and Metabolism)
Browse Figures
Versions Notes

Abstract

:
Aspergillus oryzae and A. flavus are important species in industrial biotechnology and food safety and have been some of the first aspergilli to be fully genome sequenced. Bioinformatic analysis has revealed 99.5% gene homology between the two species pointing towards a large coherence in the secondary metabolite production. In this study we report on the first comparison of secondary metabolite production between the full genome sequenced strains of A. oryzae (RIB40) and A. flavus (NRRL 3357). Surprisingly, the overall chemical profiles of the two strains were mostly very different across 15 growth conditions. Contrary to previous studies we found the aflatrem precursor 13-desoxypaxilline to be a major metabolite from A. oryzae under certain growth conditions. For the first time, we additionally report A. oryzae to produce parasiticolide A and two new analogues hereof, along with four new alkaloids related to the A. flavus metabolites ditryptophenalines and miyakamides. Generally the secondary metabolite capability of A. oryzae presents several novel end products likely to result from the domestication process from A. flavus.

1. Introduction

Aspergillus oryzae is one of industry’s most used “workhorses” and has been used for centuries in food fermentation for the production of e.g., sake, soy sauce and other traditional Asian foods [1]. A. oryzae is also a widely used organism for production of amylase, lipases and proteases and more recently also for heterologous expression of secondary metabolite genes and non-fungal proteins [2,3,4]. For many years, A. oryzae has been suspected to be a domesticated form of A. flavus, a plant and mammalian pathogenic saprophyte, capable of producing some of the most carcinogenic compounds known: the aflatoxins. Genetic work and subsequent genome sequencing of strains of both species have verified the tight link between the species [1,5,6,7,8].
The relationship of the two species has resulted in extensive screening of the toxic potential of A. oryzae, but no genuine evidence of aflatoxin production in validated A. oryzae isolates has ever been shown. Other important toxins, known from A. flavus, have on the other hand been shown in A. oryzae: 3-Nitropropionic acid [9] and cyclopiazonic acid (CPA) [10] along with kojic acid [11,12] (Figure 1). Additional metabolites previously reported from A. oryzae are asperfuran [13], sporogen AO1 [14,15], maltoryzine [16], and aspergillomarasmine A [17,18]. Aspirochlorine has been found in A. flavus, A. oryzae and A. tamarii [19,20,21,22,23] (Figure 1). For reviews on the safety and taxonomy of A. oryzae, see [7,24,25,26].
Metabolites 02 00039 g001 1024
Figure 1. Known compounds from Aspergillus flavus or A. oryzae.
Figure 1. Known compounds from Aspergillus flavus or A. oryzae.
Metabolites 02 00039 g001
The few predicted differences between the genomes of A. oryzae and A. flavus (ca. 99.5% genome homology and 98% at the protein level for RIB40/ATCC 42149 and NRRL 3357 [27]), could lead one to expect A. oryzae to produce most of the metabolites found in A. flavus [1,28,29,30,31], but published metabolic data indicates a very low chemical correlation [32]. It is with reference to the established genetic heritage of A. oryzae from A. flavus remarkable that maltoryzine, sporogen AO1, asperfuran and aspergillomarasmine A have never been unambigously identified in A. flavus. Though research on A. flavus chemistry has focused primarily on toxic compounds, one would expect that these metabolites should be part of its chemical potential as they are for the domesticated A. oryzae. The preliminary bioinformatic studies in conjunction with the genome sequencing shows roughly the same number of predicted genes: 32 Polyketide synthases (PKSs) and 28 non-ribosomal synthases (NRPSs) for A. flavus and 32 PKSs and 27 NRPSs for A. oryzae with 2 NRPSs apparently unique for each strain [33]. The exclusiveness of these genes in terms of end product has yet to be verified chemically.
Most of the predicted genes for secondary metabolites of A. oryzae (or A. flavus) have not been mapped to specific metabolic products, despite the genome sequencing of RIB40 in 2005 [27]. Only genes of the most important toxins: Aflatoxin [31,34,35], CPA [36,37] and aflatrem [38] have been fully annotated in both species, which leaves much to be explored. The full chemical potential of either species is unknown and epigenetic modifiers [39,40,41,42,43] may be necessary, alongside with the use of different growth conditions to aid triggering the full potential of secondary metabolite expression in these two closely related species.
The aim of the current work has been to perform an initial comparative investigation of the chemistry from the two genome sequenced strains of A. oryzae (RIB40) and A. flavus (NRRL 3357), in order to get further insights into possible homologies and differences in secondary metabolite production for these two important species.

2. Results and Discussion

2.1. De-Replication of A. oryzae RIB40

For the analysis of A. oryzae RIB40 chemistry, we investigated a series of solid media (YES, YESBEE, DRYES, CYA, CYAS, CY20, CY40, DUL, GAK, GMMS, MEA, OAT, PDA, TGY, WATM (see Methods and Materials for explanation) cultivations with micro-scale extractions [44,45] and subsequently analyzed with HPLC-DAD-MS for selection of optimal conditions. The different media were tested on a collection of A. oryzae (RIB40, IBT 28103) and A. flavus (NRRL 3357, IBT 23106, IBT 3642) and these strains were cultivated at 25 °C in the dark for 7 and 14 days. The media screening for A. oryzae and A. flavus indicated the greatest chemodiversity and metabolite production from CYA, YES and WATM agar for our purpose.
The comparison of the secondary metabolite profiles of the two strains, NRRL 3357 and RIB40, exposed a surprisingly high degree of chemical difference on all media as illustrated in Figure 2 and Table 1 for the WATM medium. The major metabolite repetitions between the two genome sequenced strains were merely kojic acid and ergosterol, like a number of minor metabolites (not analyzed here) seemed to be shared between the two strains. Altogether, this is in sharp contrast to the high gene homology, particularly for the secondary metabolite genes.
Metabolites 02 00039 g002 1024
Figure 2. ESI+ BPC chromatogram of 7 day micro scale extract from WATM, bottom: A. oryzae RIB40, top: A. flavus NRRL 3357. Besides kojic acid (shown in box) and analogues in the beginning of chromatogram and ergosterol in the end (not shown), there are very few identical compounds between the genetically almost identical strains. Note that aspirochlorine is only detectable in negative ionization, and therefore not visible in this chromatogram.
Figure 2. ESI+ BPC chromatogram of 7 day micro scale extract from WATM, bottom: A. oryzae RIB40, top: A. flavus NRRL 3357. Besides kojic acid (shown in box) and analogues in the beginning of chromatogram and ergosterol in the end (not shown), there are very few identical compounds between the genetically almost identical strains. Note that aspirochlorine is only detectable in negative ionization, and therefore not visible in this chromatogram.
Metabolites 02 00039 g002
Table
Table 1. LC-MS de-replication of some of the important secondary metabolite pathways from the two full genome sequenced siblings, A. flavus (NRRL 3357) and A. oryzae (RIB40). Based on 7 day fermentation on solid WATM agar in the dark. (+) indicates the presence of these types of compounds in A. flavus based on UV spectroscopic analysis. * New compounds reported here for the first time.
Table 1. LC-MS de-replication of some of the important secondary metabolite pathways from the two full genome sequenced siblings, A. flavus (NRRL 3357) and A. oryzae (RIB40). Based on 7 day fermentation on solid WATM agar in the dark. (+) indicates the presence of these types of compounds in A. flavus based on UV spectroscopic analysis. * New compounds reported here for the first time.
MetaboliteA. flavus NRRL 3357A. oryzae RIB40
Kojic acid++
Aflatoxin+-
Aflavinines(+)+
Aflatrem+13-desoxypaxilline
Miyakamides(+)Oryzamides *
Aspirochlorine-+
Cyclopiazonic acid+-
Ditryptophenaline+Ditryptoleucine *
Parasiticolide A-14-deacetyl parasiticolide A *
Known metabolites to A. oryzae were de-replicated and we found that the RIB40 strain did not produce detectable levels (LC-MS) of CPA (as also noted by Tokuoka et al. [36]), asperfuran, sporogen AO1, maltoryzine or aspergillomarasmine under these growth conditions. It did, however, produce kojic acid and aspirochlorine and a series of potentially new metabolites of which some were isolated, structurally characterized and reported here.

2.2. New Metabolites to A. oryzae RIB40

During fermentation of the chemically potent RIB40 strain, we have been interested in the tremorgenic compounds, allegedly coupled to fungal sclerotia [46,47,48,49,50,51,52,53,54] and whether these could be found in A. oryzae as they have been in A. flavus. The RIB40 strain produces large and abundant sclerotia, especially on WATM agar, a fact not widely announced in literature although sclerotia have been observed in A. oryzae sporadically [55,56,57]. No sclerotia were observable after 14 days on YES agar, but although these metabolites are often characterized as sclerotial metabolites, there is not a strict correlation between the biosynthesis of these metabolites and the formation of sclerotia, as also noted by Wilson [58], and this extract was used for the described isolations.
Here, we report the discovery of the aflatrem precursor 13-desoxypaxilline (13-dehydroxypaxilline) in A. oryzae RIB40, originally isolated from Penicillium paxilli [59,60,61,62,63]. Aflatrem is known from A. flavus and was discovered by Wilson and Wilson in 1964 [64] and structure elucidated by Gallagher et al. in 1978 and 1980 [65,66]. 13-desoxypaxilline was present in YES, CYA, OAT and WATM agar 7 day old micro-scale extracts. From the 14 days old YES 200 plate extract used for isolation, 13-desoxypaxilline was recovered as an intermediate metabolite. LC-MS, LC-MS/MS and NMR data analysis (Supplementary Material) confirmed the structure. Naturally the prospect of finding aflatrem itself was investigated, though no apparent peak was visible in HPLC-DAD data files. The use of a LC-MS/MS method further confirmed 13-desoxypaxilline as an end-product of A. oryzae RIB40 for the above cultivation conditions, since none of the proposed intermediate steps towards aflatrem could be detected (LC-MS/MS) and only one sample (WATM, 7d) showed traces of paspaline, a precursor for 13-desoxypaxilline (Figure 3).
Metabolites 02 00039 g003 1024
Figure 3. The final steps in the proposed biosynthesis of aflatrem (in A. flavus). A. oryzae RIB40 biosynthesis stops at 13-desoxypaxilline [38].
Figure 3. The final steps in the proposed biosynthesis of aflatrem (in A. flavus). A. oryzae RIB40 biosynthesis stops at 13-desoxypaxilline [38].
Metabolites 02 00039 g003
A second extract was made from 100 plates of a 14-day old A. oryzae RIB40 culture grown on WATM agar with abundant sclerotia formation to validate the findings from the YES extract. The analysis of the WATM extract showed 13-desoxypaxilline as a major metabolite alongside other sclerotium-related metabolites, such as aflavinines (based on UV-data not shown). The discovery of 13-desoxypaxilline as the apparent end-product of A. oryzae RIB40 is in agreement with the analysis of Nicholson et al. [38], who showed that a frameshift mutation in the atmQ gene presumably accounts for 13-desoxypaxilline not being converted into paspalicine and paspalinine. This mutation is therefore likely responsible for terminating the aflatrem biosynthesis in RIB40 prematurely, short of the acetal ring closure, C-13 hydroxylation and isoprene attachment. Nicholson et al. [38] demonstrated that AtmQ is a multifunctional cytochrome P450 monooxygenase likely to catalyze the several oxidative steps needed for biosynthesis of the acetal ring present in the structures of paspalicine, paspalinine and aflatrem, altogether pointing towards a more complex biosynthesis than shown here. Contrary to our discovery, Nicholson et al. did not find the aflatrem gene cluster of RIB40 to be transcribed during their fermentations [38].
The isolated 13-desoxypaxilline is a member of the paspalitrem tremorgens, a widely distributed group of metabolites that have been isolated from several genera: Penicillium, Eupenicillium, Claviceps, Emericella, Aspergillus and Phomopsis [67,68,69]. Besides the tremorgenic activity in animals, these metabolites have been shown to be insecticides [68], which is believed to be their ecological function together with aflatoxin and CPA for protection of the sclerotia against fungivorous insects [46,47].
In addition to 13-desoxypaxilline, two new analogues of parasiticolide A were also isolated and are here reported for the first time. The metabolites showed to be dide- and 14-deacetoxy analogues, and are most likely precursors to the sesquiterpene parasiticolide A (= astellolide A) (see Figure 4). The metabolites were present in CYA, YES and WATM extracts and isolated from the same 14 day old YES extract as 13-desoxypaxilline, and the dide- and 14-deacetyl analogues were also found in the sclerotia enriched WATM extract. Again the metabolites were analyzed using LC-MS and NMR. Several different extraction procedures were tested to verify the correctness of the compounds as genuine metabolites and not as in vitro degraded parasiticolide A products, but all samples showed only dide- and 14-deacetyl parasiticolide A and no traceable (LC-MS) levels of parasiticolide A itself, even with different non-acidic extractions. Parasiticolide A have been isolated from A. flavus var. columnaris (FKI-0739) once [71] and was originally isolated and characterized from A. parasiticus (IFO 4082) [72,73] and later also from a mutant of Emericella variecolor (= A. stellatus Curzi) [74]. Recently parasiticolides have been detected in the newly described species A. arachidicola (CBS 117610) and A. minisclerotigenes (CBS 117635) [75]. There have to our knowledge not been published any toxic studies on the parasiticolides, but the related peniopholides from the fungus Peniophora polygonia have been reported to have antifungal properties [76].
Metabolites 02 00039 g004 1024
Figure 4. Structures of dide- and 14-deacetyl parasiticolide A and parasiticolide A.
Figure 4. Structures of dide- and 14-deacetyl parasiticolide A and parasiticolide A.
Metabolites 02 00039 g004
In our observations parasiticolides are more often detectable metabolites of A. oryzae than of A. flavus under the same fermentation conditions, suggesting that the pathway is partly silenced for A. flavus and may need epigenetic modification to be expressed under otherwise normal growth conditions. It is interesting that parasiticolide A is scarcely observed in A. flavus, when it is an important product of A. oryzae and also of A. parasiticus. As for 13-desoxypaxilline, dide- and 14-deacetyl parasiticolide A are almost certainly products of a prematurely ended biosynthesis, here parasiticolide A. We also isolated and elucidated a third parasiticolide A analogue; a formyl variant of parasiticolide A, but it was not possible to exclude the possibility of in vitro chemistry due to the formic acid added during the ethyl acetate extraction, so the correctness of this metabolite remains tentative. (Hamasaki et al. used benzene to extract parasiticolide A [77]). All paraciticolide analogues were structure elucidated by NMR and shift values correlated with the published data for parasiticolide A, except for the missing signals of the acetate units and their minor influence on the chemical shifts values of adjacent protons and carbons (See Supplementary Material for NMR data).
To further verify these observations, a MS/MS method was used to analyze several different microscale extracts of RIB40 for parasiticolide A itself. Trace amounts of parasiticolide A was found under these conditions and compared to an isolate of A. parasiticus (IBT 4387) capable of producing parasiticolide A. In the A. parasiticus isolate no dide- or 14-deacetyl parasiticolide A could be measured, indicating a complete transformation into the end-product (Figure 4). The small amount of parasiticolide A in RIB40 (roughly 1:1.000 ratio compared to 14-acetyl parasiticolide A, presuming the same response factor) might be the result of spontaneous acetylation involving the first acetylating enzyme. When the gene cluster of this metabolite is mapped, it is likely that the gene responsible for the last (specific) acetylation will be found to be mutated. Except for the section Nidulantes member Emericella variecolor, all other producers of these metabolites have been members of section Flavi. No indication points to these metabolites being part of the previously mentioned sclerotium metabolites since they are not found in selective sclerotium extracts, but are found for example in A. arachidicola, which is not known to produce sclerotia.
Four new A. flavus related NRPS compounds were also isolated from A. oryzae RIB40 and characterized based on standard interpretation of 1H, COSY, HMQC and HMBC NMR data (see Supplementary Material). The compounds showed to be two isomeric metabolites named ditryptoleucine related to the ditryptophenalines [78,79,80,81] and the two indole-enamides named oryzamides A1-2 (cis- and trans-forms) structurally similar to the antibiotic miyakamide B1-2 [71] (Figure 5).
Metabolites 02 00039 g005 1024
Figure 5. Structure of the new A. oryzae metabolites: Ditryptoleucine and oryzamide A1-2.
Figure 5. Structure of the new A. oryzae metabolites: Ditryptoleucine and oryzamide A1-2.
Metabolites 02 00039 g005
It proved difficult to unambiguously isolate the cis- and trans-isomer of oryzamide A in a pure form due to isomerization around the double bond. Due to this the structure elucidation has been performed on mixtures of compounds. It is evident from the structure elucidation that oryzamides A1-2 only differ in the configuration around the double bond of the enamine as evident from the size of the coupling constant. Marfey analysis of the amino acid derived compounds only established the presence of L-tyrosine in the oryzamides. NMR structural data on the trans isomer (A1) is present in the supporting material.
Intriguingly, these A. oryzae metabolites have apparently exchanged phenylalanine with leucine compared to the similar A. flavus metabolites, indicating either a common trait in the domestication process or a coupling between the two pathways. The diketopiperazine ditryptoleucine was isolated in two variants with 1H-NMR shifts varying around the C-C dimeric bond, but with the same base structure. This unspecific dimerization is in line with previously isolated compounds. A hybrid between the ditryptophenaline and ditryptoleucine was isolated from an Aspergillus sp. as WIN 64745, with both a phenylalanine and leucine moiety, but with no N-methylation [79]. These compounds were tested and proved antagonistic against substance P at the NK1 receptor [82]. The oryzamides A1-2 are variants of the miyakamides B1-2 [71]. The exchanged phenylalanine has been substituted with leucine between a tryptophan and a tyrosine in the oryzamides. However this exchange of phenylalanine and leucine has often been found for compounds produced within the same species. Penicillium polonicum (= P. fructigenum) [83,84,85] produces both fructigenine A = puberuline = rugulosuvine A, containing phenylalanine and fructigenine B = verrucofortine, containing leucine. We could also detect two slightly later eluting compounds in the A. oryzae RIB40 extracts likely to be two further indole-enamides having phenylalanine incorporated instead of tyrosine as evident from LC-DAD-MS analysis (data not shown) as also seen for the miyakamides [71].

3. Experimental

General procedures and methods for analysis are described in [43,44,86].
Mass spectrometric analysis was performed using a Agilent HP 1100 liquid chromatograph with a DAD system (Waldbronn, Germany) on a LCT oaTOF mass spectrometer (Micromass, Manchester, UK) with a Z-spray ESI source and a LockSpray probe. For general procedures see [87]; method 1 for LC-DAD-TOF was used in this study. All solvents used were HPLC grade from Sigma-Aldrich (St. Louis, MO, USA).

3.1. Fungal Material and Fermentation

A. oryzae RIB40 (IBT28103); A. flavus NRRL3357 (IBT3696), (IBT15934), NRRL 13462; A. parvisclerotigenus IBT16807 and A. minisclerotigenes IBT13353 were obtained from the IBT Culture Collection at DTU Systems Biology, Technical University of Denmark. The RIB40 isolate used for isolation of 13-dehydroxypaxilline was cultured for 14 days at 25 °C in the dark on 200 Petri dishes with Yeast Extract Sucrose agar (YES). All strains were grown for 1 week at 25 °C on YES, Czapek Yeast Autolysate (CYA), Wickerhams Antibiotic Test Medium (WATM) agar [88], YESBEE [89] (YES+50 g Bee pollen Type III, granulate, Sigma, P-8753, pr. 1 L medium), DRYES (Dichloran rose Bengal chloramphenicol agar), AFPA (Aspergillus flavus, A. parasiticus agar), CYAS (CYA + 50 g NaCl pr. 1 L medium), CY20 (CYA + 170 g sucrose pr. 1 L), CY40 (CYA with added 370 g sucrose pr. 1 L medium), DUL (Dulaney’s medium for Penicillin), GAK (Potato-carrot agar), GMMS (Glucose minimal media (GMM) + 2% sorbitol), MEA (Malt extract agar), OAT (Oat meal agar), PDA (Potato-dextrose agar). For medium formulations see Samson et al. [90].

3.2. Extraction and Isolation of Pure Compounds

13-Desoxypaxilline. The plates were homogenized using a Stomacher and 100 mL EtOAc with 1% HCO2H pr. 10 plates. The extract was filtered and dried down on a freeze drier. The crude extract was separated on a KP-C18-HS 60 g SNAP column using a Biotage Isolera One (Biotage, Uppsala, Sweden), resulting in a 22 mg fraction. The fraction was segmented with a 10 g ISOL Diol column, using 12 steps of stepwise Heptane-dichloromethane-EtOAc-MeOH. 13-desoxypaxilline was predominant in a 100% EtOAc fraction (6 mg), and purified on a Waters HPLC W600/996PDA (Milford, MA, USA) and a RP column (Phenomenex Luna C18(2), 250 × 10 mm, 5 µm, Torrance, CA, USA) using a gradient of 80% MeCN (H2O–Milli-Q (Millipore, MA, USA)) to 90% over 10 min. with 50 ppm TFA added to the solvents. The collection was concentrated on a rotary evaporator (Büchi V-855/R-215, Flawil, Switzerland) and dried under N2 to yield 0.5 mg of white, amorphous 13-desoxypaxilline.
Dideacetyl-, 14-deacetyl-, and 18-formyl parasiticolide A. From the same fermentation described for 13-desoxypaxilline a more polar, 90 mg fraction was fractionated with a 10 g ISOL Diol column, using 12 steps of stepwise Heptane-dichloromethane-EtOAc-MeOH. The parasiticolide A-analogues were predominant in a 100% EtOAc fraction (10 mg), and purified on a Waters HPLC W600/996PDA (Milford, MA, USA) and a RP column (Phenomenex Luna C18(2), 250 × 10 mm, 5 µm, Torrance, CA, USA) using a gradient of 72% MeCN (H2O–Milli-Q (Millipore, MA, USA)) to 87% over 15 min. with 50 ppm TFA. The collection was concentrated on a rotary evaporator (Büchi V-855/R-215) and dried under N2 to yield 0.3, 1.0 and 0.8 mg of white, amorphous di-, 14-deactyl- and 18-formyl parasiticolide A, respectively. See Supplementary Material for NMR data.
Ditryptoleucine. 400 plates of A. oryzae RIB40 were cultured on YES medium. The plates were homogenized using a Stomacher and 100 mL EtOAc pr. 10 plates. The extract was filtered and dried down on a freeze drier. The crude extract were separated into three phases by dissolving it in 9:1 MeOH:H2O–Milli-Q and extracted into a heptan phase and afterwards a DCM phase. The DCM phase was separated on a KP-C18-HS SNAP column using a Biotage Isolera One (Biotage, Uppsala, Sweden) using a gradient of 10% MeOH (H2O–Milli-Q (Millipore, MA, USA)) to 100% over 15 CVs (column volumes) resulting in a 402 mg fraction. The fraction was further separated on another KP-C18-HS SNAP column using a Biotage Isolera One (Biotage, Uppsala, Sweden) using a gradient of 10% MeOH (H2O–Milli-Q (Millipore, Ma, USA)) to 25% over 2 CVs, 25% MeOH to 50% over 8 CVs and 50% to 100% over 4 CVs resulting in a 60.1 mg fraction. The fraction was fractionated on a LH-20 column using 100% MeOH. The ditryptoleucines were present in the earlier fractions and were purified on a Waters HPLC W600/996PDA (Milford, MA, USA) using a RP column (Phenomenex Luna C18(2), 250 × 10 mm, 5 µm, Torrance, CA, USA) using a gradient of 50% MeCN (H2O–Milli-Q (Millipore, MA, USA)) to 100% over 20 min. with 50 ppm TFA and a flow of 4 mL/min. The collections were concentrated on a rotary evaporator (Büchi V-855/R-215) and dried under N2 to yield 4.0 mg of the two isomers. See Supplementary Material for NMR data.
Oryzamides A1-2. The remaining broth from the fermentation described for ditryptoleucine was extracted with EtOAc + 1% HCO2H for 24 h. The extract was filtered and dried down on a freeze drier. The crude extract were separated into three phases by dissolving it in 9:1 MeOH:H2O–Milli-Q and extracted into a heptan phase and afterwards a DCM phase. The DCM phase was separated on a KP-C18-HS SNAP column using a Biotage Isolera One (Biotage, Uppsala, Sweden) using a gradient of 10% MeOH (H2O–Milli-Q (Millipore, MA, USA)) to 100% over 15 CVs (column volumes) resulting in a 654 mg fraction. The fraction was fractionated with a 10 g ISOL Diol column, using 13 steps of stepwise Hexane-dichloromethane-EtOAc-MeOH. The miyakamides were predominant in the 40:60 DCM:EtOAc and 20:80 DCM:EtOAc fractions (43.8 mg), and purified on a Waters HPLC W600/996PDA (Milford, MA, USA) and a RP column (Phenomenex Luna C18(2), 250 × 10 mm, 5µm, Torrance, CA, USA) using a gradient of 45% MeCN (H2O–Milli-Q (Millipore, MA, USA)) to 62% over 15 min. with 50 ppm TFA. The collection was concentrated on a rotarvap (Büchi V-855/R-215) and dried down under N2 to yield 4.0 and 2.0 mg of oryzamide A1 and A2, respectively. See Supplementary Material for NMR data.

3.3. Marfeys Method

100 µg of each peptide was hydrolysed with 200 µL 6 M HCl at 110 °C for 20 h. To the hydrolysis products (or 50 µL (2.5 µmol) solutions of standard D- and L-amino acids) was added 50 µL water, 20 µL 1 M NaHCO3 solution and 100 µL 1% FDAA in acetone, followed by reaction at 40 °C for 1 h. The reaction mixture was removed from the heat, neutralized with 10 µL 2 M HCl and the solution was diluted with 820 µL MeOH to a total volume of 1 mL. The FDAA derivatives were analysed by UHPLC-DAD on a Dionex Ultimate 3000 RS DAD equipped with a Kinetex C18 column (2.6 μm, 150 × 2.10mm, Phenomenex). The analyses were run with a gradient elution of MeCN/H2O–Mili-Q (Millipore, MA, USA) added 50 ppm TFA from 15 to 100% MeCN over 7 min (60 °C, 0.8 mL/min). The retention times of the FDAA derivatives were compared to retention times of the standard amino acid derivatives.

3.4. Selective Extraction of Sclerotium Metabolites

The selective extraction of sclerotia from IBT 15934, NRRL 13462, IBT 16807 and IBT 13353 was made from harvested sclerotia of a 7 day old cultivation on WATM and CYA agar (25 °C in dark). The sclerotia were washed several times with Milli-Q (Millipore, Millford, USA) 0.22 µm H2O and dried. The sclerotia were transferred to a 2 mL Eppendorf tube together with three stainless steel balls (2 × 1 mm and 1 × 5 mm) and frozen with liquid N2 before mechanical crushed. The pulverized sclerotia were suspended in 1mL methanol and transferred to a 2 mL vial with 1 mL of 1:2:3 methanol:dichloromethane:ethylacetate and left for evaporation overnight in a fume hood. The dried extract was resolved in 1 mL methanol in ultrasonicated for 10 min and then filtered with a 0.45 µm PTFE filter to a clean vial for analysis.

3.5. HPLC-DAD-TOF Method

Mass was measured using a Agilent HP 1100 liquid chromatograph with a DAD system (Waldbronn, Germany) on a LCT oaTOF mass spectrometer (Micromass, Manchester, UK) with a Z-spray ESI source and a LockSpray probe. For general procedures see [86], method 1.

3.6. NMR Instrumentation

NMR spectra were recorded on a Varian Unity Inova 500 MHz spectrometer equipped with a 5 mm probe using standard pulse sequences. The signals of the residual solvent protons and solvent carbons were used as internal references (δH 2.50 and δC 39.5 ppm for DMSO). In cases where higher field was needed the NMR spectra were recorded on a Bruker Avance 800-MHz spectrometer equipped with a 5-mm TCI Cryoprobe at the Danish Instrument Center for NMR Spectroscopy of Biological Macromolecules.

3.7. MS/MS Method Used for Aflatrem and Parasiticolide Screening

Liquid chromatography was performed on an Agilent (Torrence, CA, USA) 1100 HPLC system coupled to a triple-quadropole mass spectrometer (Waters-Micromass, Manchester, UK) with a Z-spray ESI operated in positive mode source using a flow of 700 L/h nitrogen desolvated at 350 °C. The hexapole was held at 50 V. The system was controlled by MassLynx v4.1 (Waters-Micromass). Nitrogen was used as collision gas, and the MS operated in MRM mode (dwell time = 100 ms) with the parameters listed in Table 2. Extracts of 2 µL were injected and separated on a Phenomenex Gemini C6-phenyl, 3 µm, 2 × 50 mm column with a flow of 0.3 µL/min. Water contained 20 mM ammonium formate. Oven temperature 40 °C. Two different methods were applied to the aflatrem- and parasiticolide screen: Aflatrem inlet method: Linearly gradient from 50 to 100% MeCN over 5 min, increased to 0.5 µL/min over 1.5 min. The column was washed additionally 1.5 min with 100% MeCN at 0.5 µL/min, followed by a return to 50% MeCN over 2.5 min and kept at this level for another 1 min with a linearly decrease in flow to 0.3 µL/min, prior to the next sample. Standards used for analysis of this pathway were from Sigma-Aldrich Aldrich (St. Louis, MO, USA). Parasiticolides: linearly gradient from 20 to 90% MeCN over 15 min, increased to 0.5µL/min from 90 to 100% MeCN in additional 1 min. The column was washed from 2 min with 100% MeCN at 0.5 µL/min, followed by a return to 20% MeCN over 1.5 min and kept at this level for another 3.5 min with a linearly decrease in flow to 0.3 µL/min, before the next sample. Standards were internal standards from other extracts of the known parasiticolide A producer A. parasiticus: IBT 4387 (= CBS 260.67) and IBT 11863 (= CBS 115.37).
Table
Table 2. MS/MS method including scan event, retention times, transition ions and the cone and collision energies used.
Table 2. MS/MS method including scan event, retention times, transition ions and the cone and collision energies used.
CompoundScan eventRT(min)Ion typeTransition ( m/z) aCone (V)Collision energy (eV)
Paxilline14.0Quantifier 436 → 13025253030
Qualifier436 → 182
Paspalinine24.3Quantifier 434 → 130 25252020
Qualifier434 → 376
13-desoxypaxilline34.8Quantifier 420 → 182 25253030
Qualifier420 → 130
Aflatrem45.2Quantifier 502 → 19825252020
Qualifier502 → 445
Paspaline55.5Quantifier 422 → 13025252020
Qualifier422 → 275
Dideacetyl-parasiticolide A17.0Quantifier 387 → 21730304040
Qualifier387 → 189
14-deacetyl parasiticolide A28.7Quantifier 429 → 21730304040
Qualifier429 → 189
Parasiticolide A310.4Quantifier 488 → 229 30303030
Qualifier488 → 247
a All transitions were made from [M+H]+, except for paraciticolide A: [M+NH4]+.

4. Conclusions

The tremorgenic 13-desoxypaxilline has been isolated from A. oryzae RIB40 and verified under several different growth conditions contrary to previous studies. We believe that 13-desoxypaxilline is the end-product of the aflatrem biosynthesis for the RIB40 strain since no aflatrem could be detected in any fermentation using LC-MS/MS as detection.
The new metabolites dide- and 14-deacetyl parasiticolide A were also found as genuine products from the RIB40 strain and the compounds were present in multiple fermentations, however parasiticolide A was only detected in trace amounts using a LC-MS/MS method. This indicates a defective acetylation of the 14-deacetyl parasiticolide A and the small amount of parasiticolide A in RIB40 could be the result of spontaneous acetylation in the cell cytosol. The mono-deacetylated analogue detected in both A. flavus and A. oryzae had same retention times, suggesting a selective acetylation.
The new NRPS compounds ditryptoleucine and oryzamides A1-2 appear to be natural variants of known A. flavus metabolites. They share the exchange of a phenylalanine for a leucine, although they are believed to originate from two unrelated pathways.
Altogether, our findings contribute to understanding why the overall chemical profiles of A. oryzae (RIB40) and A. flavus (NRRL 3357) appear quite different since some of the end-products usually seen in A. flavus are apparently not reached in A. oryzae. Whether the different chemical profiles are merely the result of different regulation that can be overcome by the use of epigenetic modifiers or are a result of genuine mutations remains to be settled. A. oryzae RIB40 is clearly a chemically potent strain, and as more of its chemistry is unfolded we believe that most of the biosynthetic pathways of A. flavus will be found to be more or less functional. Future research will reveal whether the many different A. oryzae strains that are used as industrial workhorses are as chemically potent as the RIB40 strain.

Supplementary Materials

Supplementary materials can be accessed at: https://www.mdpi.com/2218-1989/2/1/39/s1.

Acknowledgments

This work was funded by the Danish Research Agency for Technology and Production (Grant: 09-064967). We thank the Danish Instrument Center for NMR Spectroscopy of Biological Macromolecules at the Carlsberg Laboratory for 800 MHz NMR time.

Conflict of Interest

The authors declare no conflict of interest.

References and Notes

  1. Machida, M.; Yamada, O.; Gomi, K. Genomics of Aspergillus oryzae: Learning from the history of koji mold and exploration of its future. DNA Res. 2008, 15, 173–183. [Google Scholar] [CrossRef]
  2. Punt, P.J.; Biezen, N.V.; Conesa, A.; Albers, A.; Mangnus, J.; van den Hondel, C. Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol. 2002, 20, 200–206. [Google Scholar] [CrossRef]
  3. Meyer, V. Genetic engineering of filamentous fungi—Progress, obstacles and future trends. Biotechnol. Adv. 2008, 26, 177–185. [Google Scholar] [CrossRef]
  4. Fisch, K.M.; Bakeer, W.; Yakasai, A.A.; Song, Z.; Pedrick, J.; Wasil, Z.; Bailey, A.M.; Lazarus, C.M.; Simpson, T.J.; Cox, R.J. Rational domain swaps decipher programming in fungal highly reducing polyketide synthases and resurrect an extinct metabolite. J. Am. Chem. Soc. 2011, 133, 16335–16641. [Google Scholar]
  5. Geiser, D.M.; Pitt, J.I.; Taylor, J.W. Cryptic speciation and recombination in the aflatoxin-producing fungus Aspergillus flavus. Proc. Natl. Acad. Sci. USA 1998, 95, 388–393. [Google Scholar]
  6. Geiser, D.M.; Dorner, J.W.; Horn, B.W.; Taylor, J.W. The phylogenetics of mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae. Fungal Genet. Biol. 2000, 31, 169–179. [Google Scholar] [CrossRef]
  7. Abe, K.; Gomi, K.; Hasegawa, F.; Machida, M. Impact of Aspergillus oryzae genomics on industrial production of metabolites. Mycopathologia 2006, 162, 143–153. [Google Scholar] [CrossRef]
  8. Kobayashi, T.; Abe, K.; Asai, K.; Gomi, K.; Juvvadi, P.R.; Kato, M.; Kitamoto, K.; Takeuchi, M. Genomics of Aspergillus oryzae. Biosci. Biotechnol. Biochem. 2007, 71, 646–670. [Google Scholar] [CrossRef]
  9. Iwasaki, T.; Kosikowski, F.V. Production of beta-nitropropionic acid in foods. J. Food Sci. 1973, 38, 1162–1165. [Google Scholar] [CrossRef]
  10. Orth, R. Mycotoxins of Aspergillus oryzae strains for use in food-industry as starters and enzyme producing molds. Annales de la Nutrition et de l’Alimentation 1977, 31, 617–624. [Google Scholar]
  11. Manabe, M; Tanaka, K.; Goto, T.; Matsuura, S. Production capabilities of kojic acid and aflatixin by koji mold. In Toxigenic Fungi—Their Toxins and Health Hazards; Kurata, H., Ueno, Y., Eds.; Elsevier: Amsterdam, The Netherlands, 1984. [Google Scholar]
  12. Bentley, R. From miso, sake and shoyu to cosmetics: A century of science for kojic acid. Nat. Prod. Rep. 2006, 23, 1046–1062. [Google Scholar] [CrossRef]
  13. Pfefferle, W.; Anke, H.; Bross, M.; Steffan, B.; Vianden, R.; Steglich, W. Asperfuran, a novel antifungal metabolite from Aspergillus oryzae. J. Antibiot. 1990, 43, 648–654. [Google Scholar] [CrossRef]
  14. Tanaka, S.; Wada, K.; Katayama, M.; Marumo, S. Isolation of sporogen AO1, a sporogenic substance, from Aspergillus oryzae. Agric. Biol. Chem. 1984, 48, 3189–3191. [Google Scholar] [CrossRef]
  15. Tanaka, S.; Wada, K.; Marumo, S.; Hattori, H. Structure of sporogen AO1, a sporogenic substance of Aspergillus oryzae. Tetrahedron Lett. 1984, 25, 5907–5910. [Google Scholar] [CrossRef]
  16. Iizuka, H.; Iida, M. Maltoryzine, a new toxic metabolite produced by a strain of Aspergillus oryzae var. microsporus isolated from poisonous malt sprout. Nature 1962, 196, 681–682. [Google Scholar] [CrossRef]
  17. Barbier, M.; Vetter, W.; Bogdanov, D.; Lederer, E. Synthese und eigenschafgen eines analogen des lycomarasmins und der aspergillomarasmine. Annalen der Chemie-Justus Liebig 1963, 668, 132. [Google Scholar] [CrossRef]
  18. Robert, M.; Barbier, M.; Lederer, E.; Roux, L.; Bieman, K.; Vetter, W. Two new natural phytotoxins—Aspergillomarasmines A and B and their identity to lycomarasmine and its derivatives. Bulletin de la Societe Chimique de France 1962, 187–188. [Google Scholar]
  19. Monti, F.; Ripamonti, F.; Hawser, S.P.; Islam, K. Aspirochlorine: A highly selective and potent inhibitor of fungal protein synthesis. J. Antibiot. 1999, 52, 311–318. [Google Scholar] [CrossRef]
  20. Sakata, K.; Masago, H.; Sakurai, A.; Takahashi, N. Isolation of aspirochlorine (=antibiotic A30641) posessing a novel dithiodiketopiperazine structure from Aspergillus flavus. Tetrahedron Lett. 1982, 23, 2095–2098. [Google Scholar] [CrossRef]
  21. Sakata, K.; Kuwatsuka, T.; Sakurai, A.; Takahashi, N.; Tamura, G. Isolation of aspirochlorine (=antibiotic A30641) as a true anti-microbial constituent of the antibiotic, oryzachlorin, from Aspergillus oryzae. Agric. Biol. Chem. 1983, 47, 2673–2674. [Google Scholar] [CrossRef]
  22. Sakata, K.; Maruyama, M.; Uzawa, J.; Sakurai, A.; Lu, H.S.M.; Clardy, J. Structural revision of aspirochlorine (=antibiotic A30641), a novel epidithiopiperazine-2,5-dione produced by Aspergillus spp. Tetrahedron Lett. 1987, 28, 5607–5610. [Google Scholar]
  23. Klausmeyer, P.; McCloud, T.G.; Tucker, K.D.; Cardellina, J.H.; Shoemaker, R.H. Aspirochlorine class compounds from Aspergillus flavus inhibit azole-resistant Candida albicans. J. Nat. Prod. 2005, 68, 1300–1302. [Google Scholar] [CrossRef]
  24. Barbesgaard, P.; Heldt-Hansen, H.P.; Diderichsen, B. On the safety of Aspergillus oryzae: A review. Appl. Microbiol. Biotechnol. 1992, 36, 569–572. [Google Scholar]
  25. Tanaka, K.; Goto, T.; Manabe, M.; Matsuura, S. Traditional Japanese fermented foods free from mycotoxin contamination. Jpn. Agric. Res. Quart. 2002, 36, 45–50. [Google Scholar]
  26. Varga, J.; Frisvad, J.C.; Samson, R.A. Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Stud. Mycol. 2011, 69, 57–80. [Google Scholar] [CrossRef]
  27. Rokas, A.; Payne, G.; Fedorova, N.D.; Baker, S.E.; Machida, M.; Yu, J.; Georgianna, D.R.; Dean, R.A.; Bhatnagar, D.; Cleveland, T.E. What can comparative genomics tell us about species concepts in the genus Aspergillus? Mycol. 2007, 59, 11–17. [Google Scholar]
  28. Machida, M.; Asai, K.; Sano, M.; Tanaka, T.; Kumagai, T.; Terai., G.; Kusumoto, K.-I.; Arima, T.; Akita, O.; Kashiwagi, Y.; et al. Genome sequencing and analysis of Aspergillus oryzae. Nature 2005, 438, 1157–1161. [Google Scholar] [CrossRef]
  29. Machida, M.; Terabayashi, Y.; Sano, M.; Yamane, N.; Tamano, K.; Payne, G.A.; Yu, J.; Cleveland, T.E.; Nierman, W.C. Genomics of industrial aspergilli and comparison with toxigenic relatives. Food Addit. Contam. Part A 2008, 25, 1147–1151. [Google Scholar] [CrossRef]
  30. Payne, G.A.; Nierman, W.C.; Wortman, J.R.; Pritchard, B.L.; Brown, D.; Dean, R.A.; Bhatnagar, D.; Cleveland, T.E.; Machida, M.; Yu, J. Whole genome comparison of Aspergillus flavus and A. oryzae. Med. Mycol. 2006, 44, S9–S11. [Google Scholar] [CrossRef]
  31. Yu, J.; Payne, G.A.; Nierman, W.C.; Machida, M.; Bennett, J.W.; Campbell, B.C.; Robens, J.F.; Bhatnagar, D.; Dean, R.A.; Cleveland, T.E. Aspergillus flavus genomics as a tool for studying the mechanism of aflatoxin formation. Food Addit. Contam. Part A 2008, 25, 1152–1157. [Google Scholar] [CrossRef]
  32. Laatsch, H. AntiBase 2010. Available online: http://www.wiley-vch.de/stmdata/antibase2010.php (accessed on 27 December 2011).
  33. Cleveland, T.E.; Yu, J.; Fedorova, N.; Bhatnagar, D.; Payne, G.A.; Nierman, W.C.; Bennett, J.W. Potential of Aspergillus flavus genomics for applications in biotechnology. Trends Biotechnol. 2009, 27, 151–157. [Google Scholar] [CrossRef]
  34. Lee, Y.-H.; Tominaga, M.; Hayashi, R.; Sakamoto, K.; Yamada, O.; Akita, O. Aspergillus oryzae strains with a large deletion of the aflatoxin biosynthetic homologous gene cluster differentiated by chromosomal breakage. Appl. Microbiol. Biotechnol. 2006, 72, 339–345. [Google Scholar] [CrossRef]
  35. Tominaga, M.; Lee, Y.H.; Hayashi, R.; Suzuki, Y.; Yamada, O.; Sakamoto, K.; Gotoh, K.; Akita, O. Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains. Appl. Environ. Microbiol. 2006, 72, 484–490. [Google Scholar]
  36. Tokuoka, M.; Seshime, Y.; Fujii, I.; Kitamoto, K.; Takahashi, T.; Koyama, Y. Identification of a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene required for the biosynthesis of cyclopiazonic acid in Aspergillus oryzae. Fungal Genet. Biol. 2008, 45, 1608–1615. [Google Scholar] [CrossRef]
  37. Chang, P.-K.; Horn, B.W.; Dorner, J.W. Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus. Fungal Genet. Biol. 2009, 46, 176–182. [Google Scholar] [CrossRef]
  38. Nicholson, M.J.; Koulman, A.; Monahan, B.J.; Pritchard, B.L.; Payne, G.A.; Scott, B. Identification of two aflatrem biosynthetic gene loci in Aspergillus flavus and metabolic engineering in Penicillium paxilli to elucidate gene function. Appl. Environ. Microbiol. 2009, 75, 7469–7481. [Google Scholar]
  39. Shwab, E.K.; Bok, J.W.; Tribus, M.; Galehr, J.; Graessle, S.; Keller, N.P. Histone deacetylase activity regulates chemical diversity in Aspergillus. Eukaryot. Cell 2007, 6, 1656–1664. [Google Scholar] [CrossRef]
  40. Shwab, E.K.; Keller, N.P. Regulation of secondary metabolite production in filamentous ascomycetes. Mycol. Res. 2008, 112, 225–230. [Google Scholar] [CrossRef]
  41. Williams, R.B.; Henrikson, J.C.; Hoover, A.R.; Lee, A.E.; Cichewicz, R.H. Epigenetic remodeling of the fungal secondary metabolome. Org. Biomol. Chem. 2008, 6, 1895–1897. [Google Scholar] [CrossRef]
  42. Henrikson, J.C.; Hoover, A.R.; Joyner, P.M.; Cichewicz, R.H. A chemical epigenetics approach for engineering the in situ biosynthesis of a cryptic natural product from Aspergillus niger. Org. Biomol. Chem. 2009, 7, 435–438. [Google Scholar]
  43. Yakasai, A.A.; Davison, J.; Wasil, Z.; Halo, L.M.; Butts, C.P.; Lazarus, C.M.; Bailey, A.M.; Simpson, T.J.; Cox, R.J. Nongenetic reprogramming of a fungal highly reducing polyketide synthase. J. Am. Chem. Soc. 2011, 133, 10990–10998. [Google Scholar]
  44. Frisvad, J.C.; Thrane, U. Standardized high-performance liquid-chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone retention indexes and UV-Vis spectra (Diode-Array Detection). J. Chromatogr. A 1987, 404, 195–214. [Google Scholar] [CrossRef]
  45. Smedsgaard, J. Micro-scale extraction procedure for standardized screening of fungal metabolite production in cultures. J. Chromatogr. A 1997, 760, 264–270. [Google Scholar] [CrossRef]
  46. Wicklow, D.T.; Cole, R.J. Tremorgenic indole metabolites and aflatoxins in sclerotia of Aspergillus flavus—An evolutionary perspective. Can. J. Bot. 1982, 60, 525–528. [Google Scholar] [CrossRef]
  47. Gloer, J.B.; Tepaske, M.R.; Sima, J.S.; Wicklow, D.T.; Dowd, P.F. Antiinsectan aflavinine derivatives from the sclerotia of Aspergillus flavus. J. Org. Chem. 1988, 53, 5457–5460. [Google Scholar]
  48. Gloer, J.B.; Rinderknecht, B.L.; Wicklow, D.T.; Dowd, P.F. Nominine—A new insecticidal indole diterpene from the sclerotia of Aspergillus nomius. J. Org. Chem. 1989, 54, 2530–2532. [Google Scholar]
  49. Staub, G.M.; Gloer, J.B.; Wicklow, D.T.; Dowd, P.F. Aspernomine—A cytotoxic antiinsectan metabolite with a novel ring-system from the sclerotia of Aspergillus nomius. J. Am. Chem. Soc. 1992, 114, 1015–1017. [Google Scholar] [CrossRef]
  50. Staub, G.M.; Gloer, K.B.; Gloer, J.B.; Wicklow, D.T.; Dowd, P.F. New paspalinine derivatives with antiinsectan activity from the sclerotia of Aspergillus nomius. Tetrahedron Lett. 1993, 34, 2569–2572. [Google Scholar]
  51. Tepaske, M.R.; Gloer, J.B.; Wicklow, D.T.; Dowd, P.F. The structure of tubingensin B—A cytotoxic carbazole alkaloid from the sclerotia of Aspergillus tubingensis. Tetrahedron Lett. 1989, 30, 5965–5968. [Google Scholar]
  52. Tepaske, M.R.; Gloer, J.B.; Wicklow, D.T.; Dowd, P.F. 3 new aflavinines from the sclerotia of Aspergillus tubingensis. Tetrahedron 1989, 45, 4961–4968. [Google Scholar] [CrossRef]
  53. Tepaske, M.R.; Gloer, J.B.; Wicklow, D.T.; Dowd, P.F. Aflavazole—A new antiinsectan carbazole metabolite from the sclerotia of Aspergillus flavus. J. Org. Chem. 1990, 55, 5299–5301. [Google Scholar] [CrossRef]
  54. Tepaske, M.R.; Gloer, J.B.; Wicklow, D.T.; Dowd, P.F. Aflavarin and beta-aflatrem—New anti-insectan metabolites from the sclerotia of Aspergillus flavus. J. Nat. Prod. 1992, 55, 1080–1086. [Google Scholar] [CrossRef]
  55. Raper, K.B.; Fennell, D.I. The genus Aspergillus; Williams & Wilkins: Baltimore, MD, USA, 1965. [Google Scholar]
  56. Wicklow, D.T.; Mcalpin, C.E.; Yeoh, Q.L. Diversity of Aspergillus oryzae genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia. Mycoscience 2007, 48, 373–380. [Google Scholar] [CrossRef]
  57. Jin, F.J.; Takahashi, T.; Utsushikawa, M.; Furukido, T.; Nishida, M.; Ogawa, M.; Tokuoka, M. A trial of minimization of chromosome 7 in Aspergillus oryzae by multiple chromosomal deletions. Mol. Genet. Genomics 2010, 283, 1–12. [Google Scholar]
  58. Wilson, B.J. Toxins other than aflatoxins produced by Aspergillus flavus. Bacteriol. Rev. 1966, 30, 478–484. [Google Scholar]
  59. Springer, J.P.; Clardy, J.C.; Wells, J.M.; Cole, R.J.; Kirksey, J.W. Structure of paxilline, a tremorgenic metabolite of Penicillium paxilli Bainier. Tetrahedron Lett. 1975, 2531–2534. [Google Scholar]
  60. Longland, C.L.; Dyer, J.L.; Michelangeli, F. The mycotoxin paxilline inhibits the cerebellar inositol 1,4, 5-trisphosphate receptor. Eur. J. Pharmacol. 2000, 408, 219–225. [Google Scholar] [CrossRef]
  61. Bilmen, J.G.; Wootton, L.L.; Michelangeli, F. The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by paxilline. Arch. Biochem. Biophys. 2002, 406, 55–64. [Google Scholar] [CrossRef]
  62. Sabater-Vilar, M.; Nijmeijer, S.; Fink-Gremmels, J. Genotoxicity assessment of five tremorgenic mycotoxins (fumitremorgen B, paxilline, penitrem A, verruculogen, and verrucosidin) produced by molds isolated from fermented meats. J. Food Protec. 2003, 66, 2123–2129. [Google Scholar]
  63. Sheehan, J.J.; Benedetti, B.L.; Barth, A.L. Anticonvulsant effects of the BK-channel antagonist paxilline. Epilepsia 2009, 50, 711–720. [Google Scholar] [CrossRef]
  64. Wilson, B.J.; Wilson, C.H. Toxin from Aspergillus flavus—Production on food materials of substance causing tremors in mice. Science 1964, 144, 177–178. [Google Scholar]
  65. Gallagher, R.T.; Wilson, B.J. Aflatrem, the tremorgenic mycotoxin from Aspergillus flavus. Mycopathologia 1978, 66, 183–185. [Google Scholar] [CrossRef]
  66. Gallagher, R.T.; Clardy, J.C.; Wilson, B.J. Aflatrem, a tremorgenic toxin from Aspergillus flavus. Tetrahedron Lett. 1980, 21, 239–242. [Google Scholar] [CrossRef]
  67. Cole, R.J.; Dorner, J.W.; Springer, J.P.; Cox, R.H. Indole metabolites from a strain of Aspergillus flavus. J. Agric. Food Chem. 1981, 29, 293–295. [Google Scholar] [CrossRef]
  68. Steyn, P.S.; Vleggaar, R. Tremorgenic mycotoxins. Fortschritte der Chemie Organischer Naturstoffe 1985, 48, 1–80. [Google Scholar] [CrossRef]
  69. Bills, G.F.; Giacobbe, R.A.; Lee, S.H.; Peláez, F.; Tkacz, J.S. Tremorgenic mycotoxins, paspalitrem A and C, from a tropical Phomopsis. Mycol. Res. 1992, 96, 977–983. [Google Scholar] [CrossRef]
  70. Laakso, J.A.; Gloer, J.B.; Wicklow, D.T.; Dowd, P.F. A new penitrem analog with antiinsectan activity from the sclerotia of Aspergillus sulphureus. J. Agric. Food Chem. 1993, 41, 973–975. [Google Scholar]
  71. Shiomi, K.; Hatae, K.; Yamaguchi, Y.; Masuma, R.; Tomoda, H.; Kobayashi, S.; Omura, S. New antibiotics miyakamides produced by a fungus. J. Antibiot. 2002, 55, 952–961. [Google Scholar] [CrossRef]
  72. Fukuyama, K.; Kawai, H.; Tsukihara, T.; Tsukihara, K.; Katsube, Y.; Hamasaki, T.; Hatsuda, Y.; Kuwano, H. Structure-analysis of a bromo derivative of parasiticolide A by X-ray-diffraction method. Bull. Chem. Soc. Jpn. 1975, 48, 2949–2950. [Google Scholar] [CrossRef]
  73. Ishikawa, Y.; Morimoto, K.; Hamasaki, T. Flavoglaucin, a metabolite of Eurotium chevalieri, its antioxidation and synergism with tocopherol. J. Am. Oil Chem. Soc. 1984, 61, 1864–1868. [Google Scholar] [CrossRef]
  74. Gould, R.O.; Simpson, T.J.; Walkinshaw, M.D. Isolation and X-ray crystal structures of astellolides A and B, sesquiterpenoid metabolites of Aspergillus variecolor. Tetrahedron Lett. 1981, 22, 1047–1050. [Google Scholar] [CrossRef]
  75. Pildain, M.B.; Frisvad, J.C.; Vaamonde, G.; Cabral, D.; Varga, J.; Samson, R.A. Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts. Int. J. Syst. Evol. Microbiol. 2008, 58, 725–735. [Google Scholar]
  76. Ayer, W.A.; Trifonov, L.S. Drimane sesquiterpene lactones from Peniophora polygonia. J. Nat. Prod. 1992, 55, 1454–1461. [Google Scholar] [CrossRef]
  77. Hamasaki, T.; Kuwano, H.; Isono, K.; Hatsuda, Y.; Fukuyama, K.; Tsukihara, T.; Katsube, Y. New metabolite, parasiticolide A, from Aspergillus parasiticus. Agric. Biol. Chem. 1975, 39, 749–751. [Google Scholar] [CrossRef]
  78. Springer, J.P.; Buchi, G.; Kobbe, B.; Demain, A.L.; Clardy, J.C. The structure of ditryptophenaline—New metabolite of Aspergillus flavus. Tetrahedron Lett. 1997, 27, 2403–2406. [Google Scholar]
  79. Barrow, C.J.; Cai, P.; Snyder, J.K.; Sedlock, D.M.; Sun, H.H.; Cooper, R. WIN 64821, a new competitive antagonist to substance-P, isolated from an Aspergillus species—Structure determination and solution conformation. J. Org. Chem. 1993, 58, 6016–6021. [Google Scholar]
  80. Oleynek, J.J.; Sedlock, D.M.; Barrow, C.J.; Appell, K.C.; Casiano, F.; Haycock, D.; Ward, S.J.; Kaplita, P.; Gillum, A.M. WIN-64821, a novel neurokinin antagonist produced by an Aspergillus sp .2. Biological-activity. J. Antibiot. 1994, 47, 391–398. [Google Scholar]
  81. Barrow, C.J.; Sedlock, D.M. 1′-(2-Phenyl-ethylene)-ditryptophenaline, a new dimeric diketopiperazine from Aspergillus flavus. J. Nat. Prod. 1994, 57, 1239–1244. [Google Scholar] [CrossRef]
  82. Movassaghi, M.; Schmidt, M.; Ashenhurst, J. Concise total synthesis of (+)-WIN 64821 and (−)-ditryptophenaline. Angew. Chem. Int. Ed. Engl. 2008, 47, 1485–1487. [Google Scholar]
  83. Arai, K.; Kimura, K.; Mushiroda, T.; Yamamoto, Y. Structures of fructigenines A and B, new alkaloids isolated from Penicillium fructigenum Takeuchi. Chem. Pharm. Bull. 1989, 37, 2937–2939. [Google Scholar] [CrossRef]
  84. Frisvad, J.C.; Samson, R.A. Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of the food and air-borne terverticillate Penicillia and their mycotoxins. Stud. Mycol. 2004, 49, 1–173. [Google Scholar]
  85. Frisvad, J.C.; Smedsgaard, J.; Larsen, T.O.; Samson, R.A. Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Stud. Mycol. 2004, 49, 201–241. [Google Scholar]
  86. Frisvad, J.C.; Skouboe, P.; Samson, R.A. Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp nov. Syst. Appl. Microbiol. 2005, 28, 442–453. [Google Scholar] [CrossRef]
  87. Nielsen, K.F.; Mogensen, J.M.; Johansen, M.; Larsen, T.O.; Frisvad, J.C. Review of secondary metabolites and mycotoxins from the Aspergillus niger group. Anal. Bioanal. Chem. 2009, 395, 1225–1242. [Google Scholar] [CrossRef]
  88. Raper, K.B.; Thom, C. Manual of the Penicillia; Williams and Wilkins: Baltimore, MD, USA, 1949. [Google Scholar]
  89. Medina, A.; Gonzalez, G.; Saez, J.M. Bee pollen, a substrate that stimulates ochratoxin A production by Aspergillus ochraceus. Syst. Appl. Microbiol. 2004, 27, 261–267. [Google Scholar] [CrossRef]
  90. Samson, R.A.; Hoekstra, E.S.; Frisvad, J.C. Introduction to Food- and Airborne Fungi. 7; Centraalbureau voor Schimmelcultures: Utrecht, The Netherlands, 2004. [Google Scholar]

Supplementary Files

  • Supplementary File 1:

    DOCX-Document (DOCX, 110 KB)

    • Share and Cite

    MDPI and ACS Style

    Rank, C.; Klejnstrup, M.L.; Petersen, L.M.; Kildgaard, S.; Frisvad, J.C.; Held Gotfredsen, C.; Ostenfeld Larsen, T. Comparative Chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357). Metabolites 2012, 2, 39-56. https://doi.org/10.3390/metabo2010039

    AMA Style

    Rank C, Klejnstrup ML, Petersen LM, Kildgaard S, Frisvad JC, Held Gotfredsen C, Ostenfeld Larsen T. Comparative Chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357). Metabolites. 2012; 2(1):39-56. https://doi.org/10.3390/metabo2010039

    Chicago/Turabian Style

    Rank, Christian, Marie Louise Klejnstrup, Lene Maj Petersen, Sara Kildgaard, Jens Christian Frisvad, Charlotte Held Gotfredsen, and Thomas Ostenfeld Larsen. 2012. "Comparative Chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357)" Metabolites 2, no. 1: 39-56. https://doi.org/10.3390/metabo2010039

    APA Style

    Rank, C., Klejnstrup, M. L., Petersen, L. M., Kildgaard, S., Frisvad, J. C., Held Gotfredsen, C., & Ostenfeld Larsen, T. (2012). Comparative Chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357). Metabolites, 2(1), 39-56. https://doi.org/10.3390/metabo2010039

    Article Metrics

    Back to TopTop