Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

ThePenicillium chrysogenum andAspergillus nidulans wetA developmental regulatory genes are functionally equivalent

Aspergillus nidulans andPenicillium chrysogenum are related fungi that reproduce asexually by forming multicellular conidiophores and uninucleate conidia. InA. nidulans, spore maturation is controlled by thewetA (AwetA) regulatory gene. We cloned a homologous gene (PwetA) fromP. chrysogenum to determine if spore maturation is regulated by a similar mechanism in this species. ThePwetA andAwetA genes are similar in structure and functional organization. The inferred polypeptides share 77% overall amino acid sequence similarity, with several regions having > 85% similarity. The genes also had significant, local sequence similarities in their 5′ flanking regions, including conserved binding sites for the product of the regulatory geneabaA.PwetA fully complemented anA. nidulans wetA deletion mutation, demonstrating thatPwetA and its 5′ regulatory sequences function normally inA. nidulans. These results indicate that the mechanisms controlling sporulation inA. nidulans andP. chrysogenum are evolutionarily conserved.     

Improvement of homologous GH10 xylanase production by deletion of genes with predicted function in the Aspergillus nidulans secretion pathway

Filamentous fungi are important cell factories for large-scale enzyme production. However, production levels are often low, and this limitation has stimulated research focusing on the manipulation of genes with predicted function in the protein secretory pathway. This pathway is the major route for the delivery of proteins to the cell exterior, and a positive relationship between the production of recombinant enzymes and the unfolded protein response (UPR) pathway has been observed. In this study, Aspergillus nidulans was exposed to UPR-inducing chemicals and differentially expressed genes were identified by RNA-seq. Twelve target genes were deleted in A. nidulans recombinant strains producing homologous and heterologous GH10 xylanases. The knockout of pbnA (glycosyltransferase), ydjA (Hsp40 co-chaperone), trxA (thioredoxin) and cypA (cyclophilin) improved the production of the homologous xylanase by 78, 171, 105 and 125% respectively. Interestingly, these deletions decreased the overall protein secretion, suggesting that the production of the homologous xylanase was specifically altered. However, the production of the heterologous xylanase and the secretion of total proteins were not altered by deleting the same genes. Considering the results, this approach demonstrated the possibility of rationally increase the production of a homologous enzyme, indicating that trxA, cypA, ydjA and pbnA are involved in protein production by A. nidulans.     

Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans

The use of microarrays in the analysis of gene expression is becoming widespread for many organisms, including yeast. However, although the genomes of a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms. Here, we describe the construction and validation of microarrays for the fungus Aspergillus nidulans using PCR products from a 4092 EST conidial germination library. An experiment was designed to validate these arrays by monitoring the expression profiles of known genes following the addition of 1% (w/v) glucose to wild-type A. nidulans cultures grown to mid-exponential phase in Vogel's minimal medium with ethanol as the sole carbon source. The profiles of genes showing statistically significant differential expression following the glucose up-shift are presented and an assessment of the quality and reproducibility of the A. nidulans arrays discussed.     

Design and optimization of new piperidines as renin inhibitors

The discovery of a new series of piperidine-based renin inhibitors is described herein. SAR optimization upon the P3 renin sub-pocket is described, leading to the discovery of 9 and 41, two bioavailable renin inhibitors orally active at low doses in a transgenic rat model of hypertension.     

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host .zntA gene product.     

High-yield secretion of multiple client proteins in Aspergillus

Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.     

New classes of potent and bioavailable human renin inhibitors

New classes of de novo designed renin inhibitors are reported. Some of these compounds display excellent in vitro and in vivo activities toward human renin in a TGR model. The synthesis of these new types of mono- and bicyclic scaffolds are reported, and properties of selected compounds discussed.     

Thermal adaptation strategies of the extremophile bacterium Thermus filiformis based on multi-omics analysis

Extremophiles - Thermus filiformis is an aerobic thermophilic bacterium isolated from a hot spring in New Zealand. The experimental study of the mechanisms of thermal adaptation is important to...