EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

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Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In
Aspergillus nidulans
, light inhibits sexual reproduction as well as secondary metabolism. We identified
the heterotrimeric
velvet
complex VelB/VeA/LaeA connecting light-responding developmental
regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark,
physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the
nuclear master regulator of secondary metabolism, LaeA. Deletion of either
velB
or
veA
results in
defects in both sexual fruiting-body formation and the production of secondary metabolites.

VelB/VeA/LaeA Complex CoordinatesLight Signal with Fungal Developmentand Secondary Metabolism

Ochratoxins are a group of mycotoxins produced by a variety of moulds. Ochratoxin A (OTA), the most prominent member of this toxin family, was first described by van der Merwe et al. in Nature in 1965. Dietary exposure to OTA represents a serious health issue and has been associated with several human and animal diseases including poultry ochratoxicosis, porcine nephropathy, human endemic nephropathies and urinary tract tumours in humans. More than 30 years ago, OTA was shown to be carcinogenic in rodents and since then extensive research has been performed in order to investigate its mode of action, however, this is still under debate. OTA is regarded as the most toxic family member, however, other ochratoxins or their metabolites and, in particular, ochratoxin mixtures or combinations with other mycotoxins may represent serious threats to human and animal health. This review summarises and evaluates current knowledge about the differential and comparative toxicity of the ochratoxin group.

/pdf/comparative-ochratoxin-toxicity-a-review-of-the-available-vap1128d1b.pdf

Comparative Ochratoxin Toxicity: A Review of the Available Data.

This review presents an update on the current knowledge of the secondary metabolite potential of the major fungal species used in industrial biotechnology, i.e., Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei. These species have a long history of safe use for enzyme production. Like most microorganisms that exist in a challenging environment in nature, these fungi can produce a large variety and number of secondary metabolites. Many of these compounds present several properties that make them attractive for different industrial and medical applications. A description of all known secondary metabolites produced by these species is presented here. Mycotoxins are a very limited group of secondary metabolites that can be produced by fungi and that pose health hazards in humans and other vertebrates when ingested in small amounts. Some mycotoxins are species-specific. Here, we present scientific basis for (1) the definition of mycotoxins including an update on their toxicity and (2) the clarity on misclassification of species and their mycotoxin potential reported in literature, e.g., A. oryzae has been wrongly reported as an aflatoxin producer, due to misclassification of Aspergillus flavus strains. It is therefore of paramount importance to accurately describe the mycotoxins that can potentially be produced by a fungal species that is to be used as a production organism and to ensure that production strains are not capable of producing mycotoxins during enzyme production. This review is intended as a reference paper for authorities, companies, and researchers dealing with secondary metabolite assessment, risk evaluation for food or feed enzyme production, or considerations on the use of these species as production hosts.

/pdf/safety-of-the-fungal-workhorses-of-industrial-biotechnology-3kg2uk9zl3.pdf

Safety of the fungal workhorses of industrial biotechnology: update on the mycotoxin and secondary metabolite potential of Aspergillus niger , Aspergillus oryzae , and Trichoderma reesei

Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes of the biosynthetic pathway. We discuss recent information on molecular studies of OTA biosynthesis despite insufficient genetic background in detail. Accordingly, genetic regulation has also been explored with regard to the interaction between the regulators and the environmental factors. In this review, we focus on three aspects of OTA: OTA-producing strains, OTA biosynthetic pathway and the regulation mechanisms of OTA production. This can pave the way to assist in protecting food and feed from OTA contamination by understanding OTA biosynthetic pathway and regulatory mechanisms.

https://www.mdpi.com/2072-6651/8/3/83/pdf

Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms.

Ochratoxin A (OTA) is a toxic secondary metabolite produced by Aspergillus and Penicillium species that widely contaminates food and feed. We sequenced and assembled the complete ∼37-Mb genome of Aspergillusochraceus fc-1, a well-known producer of OTA. Key genes of the OTA biosynthetic pathway were identified by comparative genomic analyses with five other sequenced OTA-producing fungi: A. carbonarius, A. niger, A. steynii, A. westerdijkiae, and Penicillium nordicum. OTA production was completely inhibited in the deletion mutants (Δ otaA , Δ otaB , Δ otaC , Δ otaD , and Δ otaR1 ), and OTA biosynthesis was restored by feeding a postblock substrate to the corresponding mutant. The OTA biosynthetic pathway was unblocked in the Δ otaD mutant by the addition of heterologously expressed halogenase. OTA biosynthesis begins with a polyketide synthase (PKS), OtaA, utilizing acetyl coenzyme A (acetyl-CoA) and malonyl-CoA to synthesize 7-methylmellein, which is oxidized to OTβ by cytochrome P450 monooxygenase (OtaC). OTβ and l-β-phenylalanine are combined by a nonribosomal peptide synthetase (NRPS), OtaB, to form an amide bond to synthesize OTB. Finally, OTB is chlorinated by a halogenase (OtaD) to OTA. The otaABCD genes were expressed at low levels in the Δ otaR1 mutant. A second regulator, otaR2 , which is adjacent to the biosynthetic gene, could modulate only the expression of otaA , otaB , and otaD . Thus, we have identified a consensus OTA biosynthetic pathway that can be used to prevent and control OTA synthesis and will help us understand the variation and production of the intermediate components in the biosynthetic pathway. IMPORTANCE Ochratoxin A (OTA) is a significant mycotoxin that contaminates cereal products, coffee, grapes, wine, cheese, and meat. OTA is nephrotoxic, carcinogenic, teratogenic, and immunotoxic. OTA contamination is a serious threat to food safety, endangers human health, and can cause huge economic losses. At present, >20 species of the genera Aspergillus and Penicillium are known to produce OTA. Here we demonstrate that a consensus OTA biosynthetic pathway exists in all OTA-producing fungi and is encoded by a gene cluster containing four highly conserved biosynthetic genes and a bZIP transcription factor.

A Consensus Ochratoxin A Biosynthetic Pathway: Insights from the Genome Sequence of Aspergillus ochraceus and a Comparative Genomic Analysis.

In Aspergillus and Penicillium species, an essential pH-response transcription factor pacC is involved in growth, pathogenicity, and toxigenicity. To investigate the connection between ochratoxin A (OTA) biosynthesis and ambient pH, the AopacC in Aspergillus ochraceus was functionally characterized using a loss-of-function mutant. The mycelium growth was inhibited under pH 4.5 and 10.0, while the sporulation increased under alkaline condition. A reduction of mycelium growth and an elevation of sporulation was observed in ΔAopacC mutant. Compared to neutral condition, OTA contents were respectively reduced by 71.6 and 79.8% under acidic and alkaline conditions. The expression of AopacC increased with the elevated pH, and deleting AopacC dramatically decreased OTA production and biosynthetic genes Aopks expression. Additionally, the ΔAopacC mutant exhibited attenuated infection ability toward pear fruits. These results suggest that AopacC is an alkaline-induced regulator responsible for growth and OTA biosy...

pH-Signaling Transcription Factor AopacC Regulates Ochratoxin A Biosynthesis in Aspergillus ochraceus

Ochratoxin A (OTA), a potentially carcinogenic mycotoxin which contaminates grains, is produced by several Aspergillus species. A comparative sequence analysis of the OTA-producing Aspergillus ochraceus fc-1 strain and other Aspergillus species was performed. Two new OTA-related polyketide synthase (PKS) (AoOTApks) genes were identified. The predicted amino acid sequence of AoOTApks-1 displayed high similarity to previously identified PKSs from OTA-producing A. carbonarius ITEM 5010 (67%; [PI] No. 173482) and A. niger CBS 513.88 (62%; XP_001397313). However, the predicted amino acid sequence of AoOTApks-2 displayed lower homology with A. niger CBS 513.88 (38%) and A. carbonarius ITEM 5010 (28%). A phylogenetic analysis of the β-ketosynthase and acyl-transferase domains of the AoOTApks proteins indicated that they shared a common origin with other OTA-producing species, such as A. carbonarius, A. niger, and A. westerdijkiae. A real-time reverse-transcription PCR analysis showed that the expression of AoOTApks-1 and -2 was positively correlated with the OTA concentration. The pks gene deleted mutants ∆AoOTApks-1 and ∆AoOTApks-2 produced nil and lesser OTA than the wild-type strain, respectively. Our study suggests that AoOTApks-1 could be involved in OTA biosynthesis, while AoOTApks-2 might be indirectly involved in OTA production.

Fei Liu

Papers

Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms.

A Consensus Ochratoxin A Biosynthetic Pathway: Insights from the Genome Sequence of Aspergillus ochraceus and a Comparative Genomic Analysis.

pH-Signaling Transcription Factor AopacC Regulates Ochratoxin A Biosynthesis in Aspergillus ochraceus

Functional Characterization of New Polyketide Synthase Genes Involved in Ochratoxin A Biosynthesis in Aspergillus Ochraceus fc-1.