BackgroundThe fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus.ResultsWe have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli.ConclusionsMany aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1151-0) contains supplementary material, which is available to authorized users.

Bacillus cereus, responsible for food poisoning, and Clostridium difficile, the causative agent of Clostridium difficile-associated diarrhea (CDAD), are both spore-forming pathogens involved in food spoilage, food intoxication, and other infections in humans and animals. The proteinaceous coat and the exosporium layers from spores are important for their resistance and pathogenicity characteristics. The exosporium additionally provides an ability to adhere to surfaces eventually leading to spore survival in food. Thus, studying these layers and identifying suitable protein targets for rapid detection and removal of spores is of the utmost importance. In this study, we identified 100 proteins from B. cereus spore coat, exosporium and 54 proteins from the C. difficile coat insoluble protein fraction. In an attempt to define a universal set of spore outer layer proteins, we identified 11 superfamily domains common to the identified proteins from two Bacilli and one Clostridium species. The evaluated orthologue relationships of identified proteins across different spore formers resulted in a set of 13 coat proteins conserved across the spore formers and 12 exosporium proteins conserved in the B. cereus group, which could be tested for quick and easy detection or targeted in strategies aimed at removal of spores from surfaces.

BackgroundThe industrially relevant filamentous fungus Aspergillus niger is widely used in industry for its secretion capabilities of enzymes and organic acids. Biotechnologically produced organic acids promise to be an attractive alternative for the chemical industry to replace petrochemicals. Itaconic acid (IA) has been identified as one of the top twelve building block chemicals which have high potential to be produced by biotechnological means. The IA biosynthesis cluster (cadA, mttA and mfsA) has been elucidated in its natural producer Aspergillus terreus and transferred to A. niger to enable IA production. Here we report the rewiring of a secondary metabolite pathway towards further improved IA production through the overexpression of a putative cytosolic citrate synthase citB in a A. niger strain carrying the IA biosynthesis cluster.ResultsWe have previously shown that expression of cadA from A. terreus results in itaconic acid production in A. niger AB1.13, albeit at low levels. This low-level production is boosted fivefold by the overexpression of mttA and mfsA in itaconic acid producing AB1.13 CAD background strains. Controlled batch cultivations with AB1.13 CAD + MFS + MTT strains showed increased production of itaconic acid compared with AB1.13 CAD strain. Moreover, preliminary RNA-Seq analysis of an itaconic acid producing AB1.13 CAD strain has led to the identification of the putative cytosolic citrate synthase citB which was induced in an IA producing strain. We have overexpressed citB in a AB1.13 CAD + MFS + MTT strain and by doing so hypothesize to have targeted itaconic acid production to the cytosolic compartment. By overexpressing citB in AB1.13 CAD + MFS + MTT strains in controlled batch cultivations we have achieved highly increased titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h while no CA was produced.ConclusionsExpression of the IA biosynthesis cluster in Aspergillus niger AB1.13 strain enables IA production. Moreover, in the AB1.13 CAD strain IA production resulted in overexpression of a putative cytosolic citrate synthase citB. Upon overexpression of citB we have achieved titers of up to 26.2 g/L IA with a productivity of 0.35 g/L/h in controlled batch cultivations. By overexpressing citB we have also diminished side product formation and optimized the production pathway towards IA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0527-2) contains supplementary material, which is available to authorized users.

Besides enzymatic conversions, many eukaryotic metabolic pathways also involve transport proteins that shuttle molecules between subcellular compartments, or into the extracellular space. Fungal itaconate production involves two such transport steps, involving an itaconate transport protein (Itp), and a mitochondrial tricarboxylate transporter (Mtt). The filamentous actinomycete Aspergillus terreus and the unicellular basidiomycete Ustilago maydis both produce itaconate, but do so via very different molecular pathways, and under very different cultivation conditions. In contrast, the transport proteins of these two strains are assumed to have a similar function. This study aims to investigate the roles of both the extracellular and mitochondrial transporters from these two organisms by expressing them in the corresponding U. maydis knockouts and monitoring the extracellular product concentrations. Both transporters from A. terreus complemented their corresponding U. maydis knockouts in mediating itaconate production. Surprisingly, complementation with Mtt of A. terreus (At_MfsA) led to a partial switch from itaconate to (S)-2-hydroxyparaconate secretion. Apparently, the export protein from A. terreus has a higher affinity for (S)-2-hydroxyparaconate than for itaconate, even though this species is classically regarded as an itaconate producer. Complementation with At_MttA increased itaconate production by 2.3-fold compared to complementation with Um-Mtt1, indicating that the mitochondrial carrier from A. terreus supports a higher metabolic flux of itaconic acid precursors than its U. maydis counterpart. The biochemical implications of these differences are discussed in the context of the biotechnological application in U. maydis and A. terreus for the production of itaconate and (S)-2-hydroxyparaconate. Keywords Itaconate (S)-2-hydroxyparaconate Ustilago maydis Aspergillus terreus Transporter Metabolism

BackgroundItaconic acid (IA), a C5-dicarboxylic acid, has previously been identified as one of the top twelve biochemicals that can be produced by biotechnological means. IA is naturally produced by Aspergillus terreus, however, heterologous production in the related species Aspergillus niger has been proposed earlier. Remarkably, we observed that during high producing conditions and elevated titers A. niger detoxifies the extracellular medium of IA. In order to determine the genes responsible for this decline in IA titers a transcriptome analysis was performed.ResultsTranscriptome analysis has led to the identification of two novel and previously unknown IA bioconversion pathways in A. niger. One pathway is proposed to convert IA into pyruvate and acetyl-CoA through the action of itaconyl-CoA transferase (IctA), itaconyl-CoA hydratase (IchA) and citramalyl-CoA lyase, similar to the pathway identified in A. terreus. Another pathway putatively converts IA into 1-methyl itaconate through the action of trans-aconitate methyltransferase (TmtA). Upon deleting the key genes ictA and ichA we have observed increased IA production and titers and cessation of IA bioconversion. Surprisingly, deletion of tmtA lead to strong reduction of heterologous IA production.ConclusionHeterologous IA production in A. niger induces the expression of IA bioconversion pathways. These pathways can be inhibited by deleting the key genes ictA, ichA and tmtA. Deletion of ictA and ichA resulted in increased IA production. Deletion of tmtA, however, resulted in almost complete cessation of IA production.Electronic supplementary materialThe online version of this article (10.1186/s40694-018-0062-5) contains supplementary material, which is available to authorized users.