Revisiting a ‘simple’ fungal metabolic pathway reveals redundancy,complexity and diversity

Next to d -glucose, the pentoses l -arabinose and d -xylose are the main monosaccharide components of plant cell wall polysaccharides and are therefore of major importance in biotechnological applications that use plant biomass as a substrate. Pentose catabolism is one of the best-studied pathways of primary metabolism of Aspergillus niger, and an initial outline of this pathway with individual enzymes covering each step of the pathway has been previously established. However, although growth on l -arabinose and/or d -xylose of most pentose catabolic pathway (PCP) single deletion mutants of A. niger has been shown to be negatively affected, it was not abolished, suggesting the involvement of additional enzymes. Detailed analysis of the single deletion mutants of the known A. niger PCP genes led to the identification of additional genes involved in the pathway. These results reveal a high level of complexity and redundancy in this pathway, emphasizing the need for a comprehensive understanding of metabolic pathways before entering metabolic engineering of such pathways for the generation of more efficient fungal cell factories.     

Gene duplication,modularity and adaptation in the evolution of the aflatoxin gene cluster

Background  

The biosynthesis of aflatoxin (AF) involves over 20 enzymatic reactions in a complex polyketide pathway that converts acetate and malonate to the intermediates sterigmatocystin (ST) and O -methylsterigmatocystin (OMST), the respective penultimate and ultimate precursors of AF. Although these precursors are chemically and structurally very similar, their accumulation differs at the species level for Aspergilli. Notable examples are A. nidulans that synthesizes only ST, A. flavus that makes predominantly AF, and A. parasiticus that generally produces either AF or OMST. Whether these differences are important in the evolutionary/ecological processes of species adaptation and diversification is unknown. Equally unknown are the specific genomic mechanisms responsible for ordering and clustering of genes in the AF pathway of Aspergillus.     

Guppies,toadfish, lungfish,coelacanths and frogs: a scenario for the evolution of urea retention in fishes

Synopsis The question of how (and why) the ureosmotic strategy, characteristic of Latimeria chalumnae and the chondrichthians evolved is addressed. There are three requirements for ureosmotic regulation: urea synthesis via the ornithine-urea cycle, urea tolerance involving biochemical and physiological adjustments, and urea retention that requires renal, branchial, metabolic and reproductive adaptations. Several examples of lower vertebrates in which urea plays a physiological role are considered to see whether they might provide insight into the origin of ureosmotic regulation. The guppy shows high urea synthesis and retention during embryonic development, and it is possible that a developmental role of urea is a general phenomenon in fishes. The toadfish, thought to be an enigma with high urea synthesis in the absence of an obvious physiological role of urea, is ureotelic under some conditions. Its urea excretion is likely related to renal function and/or parental care. In lungfish high ureogenesis is associated with estivation in periodically dry habitats. The resultant hyperuremia prevents ammonia toxicity, inhibits water loss and may repress metabolism. Latimeria is a classic marine ureosmotic regulator in which urea is used as an osmolyte that allows osmotic equilibrium with sea water while maintaining low ion levels. Adults of the frog, Rana cancrivora, are also ureosmotic regulators in brackish water. A scenario is proposed that suggests how ureosmotic regulation could have evolved in Latimeria and other fishes. The ornithine-urea cycle (composed of an arginine synthetic pathway and a second pathway that splits arginine into urea) occurred in fossil anadromous agnathans. Here the first pathway functioned in the ammocoete-like larvae for the generation of arginine to supplement a protein-deficient diet of algae, whereas the arginase pathway was important in the embryo for vitellin catabolism. Gnathostome evolution was associated with trends towards large eggs and prolonged development, requiring a complete ornithine-urea cycle for ammonia detoxification in embryos. Retention of a complete ornithine-urea cycle throughout adult life (via paedomorphosis) would preadapt any relatively large, sluggish, euryhaline fish for ureosmotic regulation when it was exposed to sea water. It is suggested that ureosmotic regulators evolved from freshwater or anadromous ancestors that entered the marine habitat. Once early ureosmotic regulators were established in the sea there would have been strong selection for internal fertilization and development, as is seen in Latimeria and many elasmobranchs. It is suggested that ureosmotic regulation was a common strategy in Paleozoic marine gnathostomes.     

Cloning, characterization of the acyl-CoA : 6-amino penicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene

Summary The penDE gene encoding acyl-CoA:6-amino penicillanic acid acyltransferase (AAT), the last enzyme of the penicillin biosynthetic pathway, has been cloned from the DNA of Aspergillus nidulans. The gene contains three introns which are located in the 5 region of the open reading frame. It encodes a protein of 357 amino acids with a molecular weight of 39 240 Da. The penDE gene of A. nidulans shows 73% similarity at the nucleotide level with the penDE gene of Penicillium chrysogenum. The A. nidulans gene was expressed in P. chrysogenum and complemented the AAT deficiency of the non-producer mutants of P. chrysogenum, npe6 and npe8. The penDE gene of A. nidulans is linked to the pcbC gene, which encodes the isopenicillin N synthase, as also occurs in P. chrysogenum. Both genes show the same orientation and are separated by an intergenic region of 822 nucleotides.     

Heterologous and homologous expression of the arginine biosynthetic argC~H cluster from Corynebacterium crenatum for improvement of (L) -arginine production

The genes involved in l-arginine biosynthesis in Corynebacterium crenatum are organized as the argCJBDFRGH cluster like in Corynebacterium glutamicum. However, the argC~H cluster of the C. crenatum SYPA 5-5, which is an industrialized l-arginine producer, had a lethal mutation occurring in the ArgR repressor encoding gene. The argC~H cluster with an inactive argR was overexpressed in E. coli and C. crenatum. In the recombinant E. coli JM109 enzyme activities were increased, and more l-arginine was found in the supernatants from l-glutamine. When the argC~H cluster was overexpressed in C. crenatum under its native promoter Parg, l-arginine production was increased by 24.9%, but the presence of the recombinant plasmid pJC-9039 had a negative effect on cell growth. Surprisingly, the DO value of the recombinant strain dropped gently and stayed at a lower level from 24 h to the end of fermentation. The results demonstrated an increasing utilization of oxygen and the distinct enhancement of unit cell l-arginine yields with the cluster argC~H-bearing in C. crenatum SYPA-9039. This study provides a kind of Corynebacteria with improved l-arginine-producing ability and an efficient elevation for producing amino acid. Moreover, the promoter Parg would be used as a valid promoter to express objective genes for metabolic engineering in Corynebacteria.     

Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis

Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.     

Activation of Glutamate Receptors Stimulates the Formation of Nitrite in Synaptosomes from Rat Cerebellum

Abstract: Synaptosomes from rat cerebellum were used to investigate the involvement of different glutamate receptor subtypes in the control of the synthesis of nitric oxide (NO), measured as its breakdown product nitrite (NO₂^-). Synaptosomes incubated in the presence of NAD|PH and l -arginine produced measurable levels of NO₂^-, which were reduced by addition of Nω-nitro-l -arginine methyl ester, an inhibitor of nitric oxide synthase. The selective ionotropic glutamate receptor agonist N-methyl-d -aspartate (NMDA) induced a pronounced increase in NO₂^-formation, which was prevented by Nω-nitro-l -arginine methyl ester and by the specific NMDA receptor antagonist Dl -2-amino-5-phosphonovaleric acid (AP-5). The NMDA-induced increase in NO₂^-formation was blocked by chelation of extracellular Ca²⁺ with EGTA. Both l -glutamate and the selective agonist for the metabotropic glutamate receptors (β)-1-aminocyclopentane-trans-1,3-dicarboxylic acid raised NO₂^-production, which retumed to control levels after addition of Nω-nitro-l -arginine methyl ester. The selective glutamate ionotropic receptor agonist (R,S)-α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid did not cause any change in NO₂ formation. The stimulatory effect of l -glutamate was blocked by the metabotropic glutamate receptor antagonist Dl -2-amino-4-phosphonobutyric acid but was unaffected by the selective NMDA receptor blocker AP-5. Removal of extracellular Ca²⁺ by EGTA did not affect the action of l -glutamate; whereas W-7, an inhibitor of calmodulin, and dantrolene, a compound that blocks the mobilization of Ca²⁺ from intracellular stores, abolished the effect of l -glutamate on NO₂^-formation. It is suggested that stimulation of ionotropic NMDA receptors activates NO metabolism by causing an influx of Ca²⁺ from the extracellular space, whereas activation of metabotropic receptors by l -glutamate provokes a mobilization of Ca²⁺ from intracellular stores, which stimulates nitric oxide synthase activity by forning Ca²⁺/calmodulin complexes.     

Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn²⁺ and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.     

Spatial regulation of the spindle assembly checkpoint and anaphase‐promoting complex in Aspergillus nidulans

The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of γ‐tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time‐lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co‐localize at the spindle pole body. SAC activity is, thus, spatially regulated in A. nidulans. Likewise, Cdc20, an activator of the anaphase‐promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the γ‐tubulin mutation mipAD159 causes a nuclear‐specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2.     

Securing a furan-based biorefinery: disclosing the genetic basis of the degradation of hydroxymethylfurfural and its derivatives in the model fungus Aspergillus nidulans

Hydroxymethylfurfural (HMF) is a promising lignocellulosic-derived source for the generation of diverse chemical building blocks constituting an alternative to fossil fuels. However, it remains unanswered if ubiquitous fungi can ensure their efficient decay, similar to that observed in highly specialised fungi. To disclose the genetic basis of HMF degradation in aspergilli, we performed a comprehensive analysis of Aspergillus nidulans ability to tolerate and to degrade HMF and its derivatives (including an HMF-dimer). We identified the degradation pathway using a suite of metabolomics methods and showed that HMF was modified throughout sequential reactions, ultimately yielding derivatives subsequently channelled to the TCA cycle. Based on the previously revealed hmfFGH gene cluster of Cupriavidus basilensis, we combined gene expression of homologous genes in Aspergillus nidulans and functional analyses in single-deletion mutants. Results were complemented with orthology analyses across the genomes of twenty-five fungal species. Our results support high functional redundancy for the initial steps of the HMF degradation pathway in the majority of the analysed fungal genomes and the assignment of a single-copy furan-2,5-dicarboxylic acid decarboxylase gene in A. nidulans. Collectively our data made apparent the superior capacity of aspergilli to mineralise HMF, furthering the environmental sustainability of a furan-based chemistry.     

Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase,DnfB

Endocytosis and exocytosis are strictly segregated at the ends of hyphal cells of filamentous fungi, with a collar of endocytic activity encircling the growing cell tip, which elongates through directed membrane fusion. It has been proposed that this separation supports an endocytic recycling pathway that maintains polar localization of proteins at the growing apex. In a search for proteins in the filamentous fungus Aspergillus nidulans that possess an NPFxD motif, which signals for endocytosis, a Type 4 P‐Type ATPase was identified and named DnfA. Interestingly, NPFxD is at a different region of DnfA than the same motif in the Saccharomyces cerevisiae ortholog, although endocytosis is dependent on this motif for both proteins. DnfA is involved in asexual sporulation and polarized growth. Additionally, it is segregated within the Spitzenkörper from another Type 4 P‐type ATPase, DnfB. Next, the phosphatidylserine marker GFP::Lact‐C2 was expressed in growing hyphae, which revealed that this phospholipid is enriched on the cytosolic face of secretory vesicles. This distribution is affected by deleting either dnfA or dnfB. These findings provide evidence for the spatial and temporal segregation of Type4‐ATPases in filamentous fungi, and the asymmetric distribution of phosphatidylserine to the Spitzenkörper in A. nidulans.     

G‐protein coupled receptor‐mediated nutrient sensing and developmental control in Aspergillus nidulans

Nutrient sensing and utilisation are fundamental for all life forms. As heterotrophs, fungi have evolved a diverse range of mechanisms for sensing and taking up various nutrients. Despite its importance, only a limited number of nutrient receptors and their corresponding ligands have been identified in fungi. G‐protein coupled receptors (GPCRs) are the largest family of transmembrane receptors. The Aspergillus nidulans genome encodes 16 putative GPCRs, but only a few have been functionally characterised. Our previous study showed the increased expression of an uncharacterised putative GPCR, gprH, during carbon starvation. GprH appears conserved throughout numerous filamentous fungi. Here, we reveal that GprH is a putative receptor involved in glucose and tryptophan sensing. The absence of GprH results in a reduction in cAMP levels and PKA activity upon adding glucose or tryptophan to starved cells. GprH is pre‐formed in conidia and is increasingly active during carbon starvation, where it plays a role in glucose uptake and the recovery of hyphal growth. GprH also represses sexual development under conditions favouring sexual fruiting and during carbon starvation in submerged cultures. In summary, the GprH nutrient‐sensing system functions upstream of the cAMP‐PKA pathway, influences primary metabolism and hyphal growth, while represses sexual development in A. nidulans.     

Microtubule‐organizing centers of Aspergillus nidulans are anchored at septa by a disordered protein

Microtubule‐organizing centers (MTOCs) are large, multi‐subunit protein complexes. Schizosaccharomyces pombe harbors MTOCs at spindle pole bodies, transient MTOCs in the division plane (eMTOCs) and nuclear‐envelope associated MTOCs in interphase cells (iMTOCs). In the filamentous fungus Aspergillus nidulans SPBs and septum‐associated MTOCs were described. Although comparable to S. pombe eMTOCs, A. nidulans sMTOCS are permanent septum‐associated structures. The composition of sMTOCs is poorly understood and how they are targeted to septa was unknown. Here, we show that in A. nidulans several SPB outer plaque proteins also locate to sMTOCs while other SPB proteins do not, including SfiA, a protein required for SPB duplication in Saccharomyces cerevisiae and S. pombe and PcpA, the anchor for γ‐TuSCs at the SPB inner plaque. The A. nidulans disordered protein Spa18^Mto2 and the centrosomin‐domain containing protein ApsB^Mto1 were required for recruiting the γ‐TuRC component GcpC to sMTOCs and for seeding MT formation from septa. Testing different septum‐associated proteins for a role in sMTOC function, Spa10 was identified. It forms a septal pore disc structure, recruits Spa18 and ApsB to septa and is required for sMTOC activity. This is the first evidence for a septum‐specific protein, Spa10, as anchor for a specific class of MTOCs.