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This work was financially supported by Fundacao para a Ciencia e a Tecnologia (FCT), project MOSTMICRO ITQB with reference no. UIDB/04612/2020 and UIDP/04612/2020, and by project "PinusResina" no. PDR2020-101-031905, funded by PDR2020 through Portugal2020. This research was carried out through a partnership with the RESOLUTION LAB, an infrastructure at NOVA School of Science and Technology. The authors acknowledge and thank the support given by the Portuguese Foundation for Science and Technology (FCT) to CENSE through the strategic project UIDB/04085/2020. T.M. and C.M. are grateful to FCT for the working contract financed by national funds under Norma Transitoria D.L. no. 57/2016 and the fellowship SFRH/BD/118377/2016, respectively. In fungi, salicylate catabolism was believed to proceed only through the catechol branch of the 3-oxoadipate pathway, as shown, e.g., in Aspergillus nidulans. However, the observation of a transient accumulation of gentisate upon the cultivation of Aspergillus terreus in salicylate medium questions this concept. To address this, we have run a comparative analysis of the transcriptome of these two species after growth in salicylate using acetate as a control condition. The results revealed the high complexity of the salicylate metabolism in A. terreus with the concomitant positive regulation of several pathways for the catabolism of aromatic compounds. This included the unexpected joint action of two pathways—3-hydroxyanthranilate and nicotinate—possibly crucial for the catabolism of aromatics in this fungus. Importantly, the 3-hydroxyanthranilate catabolic pathway in fungi is described here for the first time, whereas new genes participating in the nicotinate metabolism are also proposed. The transcriptome analysis showed also for the two species an intimate relationship between salicylate catabolism and secondary metabolism. This study emphasizes that the central pathways for the catabolism of aromatic hydrocarbons in fungi hold many mysteries yet to be discovered. IMPORTANCE Aspergilli are versatile cell factories used in industry for the production of organic acids, enzymes, and pharmaceutical drugs. To date, bio-based production of organic acids relies on food substrates. These processes are currently being challenged to switch to renewable nonfood raw materials—a reality that should inspire the use of lignin-derived aromatic monomers. In this context, aspergilli emerge at the forefront of future bio-based approaches due to their industrial relevance and recognized prolific catabolism of aromatic compounds. Notwithstanding considerable advances in the field, there are still important knowledge gaps in the central catabolism of aromatic hydrocarbons in fungi. Here, we disclose a novel central pathway, 3-hydroxyanthranilate, defying previously established ideas on the central metabolism of the aromatic amino acid tryptophan in Ascomycota. We also observe that the catabolism of the aromatic salicylate greatly activated the secondary metabolism, furthering the significance of using lignin-derived aromatic hydrocarbons as a distinctive biomass source. publishersversion published

In fungi, salicylate catabolism was believed to proceed only through the catechol branch of the 3-oxoadipate pathway, as shown e.g. in Aspergillus nidulans. However, the observation of a transient accumulation of gentisate upon cultivation of Aspergillus terreus in salicylate media questions this concept. To address this we have run a comparative analysis of the transcriptome of these two species after growth in salicylate using acetate as a control condition. The results revealed the high complexity of the salicylate metabolism in A. terreus with the concomitant positive regulation of several pathways for the catabolism of aromatic compounds. This included the unexpected joint action of two pathways: the nicotinate and the 3-hydroxyanthranilate, possibly crucial for the catabolism of aromatics in this fungus. New genes participating in the nicotinate metabolism are here proposed, whereas the 3-hydroxyanthranilate catabolic pathway in fungi is described for the first time. The transcriptome analysis showed also for the two species an intimate relationship between salicylate catabolism and secondary metabolism. This study emphasizes that the central pathways for the catabolism of aromatic hydrocarbons in fungi hold many mysteries yet to be discovered.IMPORTANCEAspergilli are versatile cell factories used in industry for production of organic acids, enzymes and pharmaceutical drugs. To date, organic acids bio-based production relies on food substrates. These processes are currently being challenged to switch to renewable non-food raw materials; a reality that should inspire the use of lignin derived aromatic monomers. In this context, Aspergilli emerge at the forefront of future bio-based approaches due to their industrial relevance and recognized prolific catabolism of aromatic compounds. Notwithstanding considerable advances in the field, there are still important knowledge gaps in the central catabolism of aromatic hydrocarbons in fungi. Here, we disclosed a novel central pathway, defying previous established ideas on the central metabolism of the aromatic amino acid tryptophan in Ascomycota. We also observed that the catabolism of the aromatic salicylate greatly activated the secondary metabolism, furthering the significance of using lignin derived aromatic hydrocarbons as a distinctive biomass source.

Pentachlorophenol (PCP) is globally dispersed and contamination of soil with this biocide adversely affects its functional biodiversity, particularly of fungi - key colonizers. Their functional role as a community is poorly understood, although a few pathways have been already elucidated in pure cultures. This constitutes here our main challenge - elucidate how fungi influence the pollutant mitigation processes in forest soils. Circumstantial evidence exists that cork oak forests in N. W. Tunisia - economically critical managed forests are likely to be contaminated with PCP, but the scientific evidence has previously been lacking. Our data illustrate significant forest contamination through the detection of undefined active sources of PCP. By solving the taxonomic diversity and the PCP-derived metabolomes of both the cultivable fungi and the fungal community, we demonstrate here that most strains (predominantly penicillia) participate in the pollutant biotic degradation. They form an array of degradation intermediates and by-products, including several hydroquinone, resorcinol and catechol derivatives, either chlorinated or not. The degradation pathway of the fungal community includes uncharacterized derivatives, e.g. tetrachloroguaiacol isomers. Our study highlights fungi key role in the mineralization and short lifetime of PCP in forest soils and provide novel tools to monitor its degradation in other fungi dominated food webs.

Centaurea calcitrapa hairy root cultures were established by infection with Agrobacterium rhizogenes strain LBA 9402. The liquid medium hairy root cultures exhibited a biomass doubling time of approximately 1.5 days in the 20 days exponential growth phase. The effect of the initial sucrose and nitrogen concentrations in biomass and proteinase production of the liquid medium cultures was studied. The highest values for both proteolytic activity and fresh weight were attained between 30 and 50 g/l of sucrose. A low ammonium:nitrate ratio favoured the development of hairy roots and proteolytic activity. The best results for both parameters in terms of nitrogen nutrition were obtained with nitrate (24.7 mM) as the only nitrogen source. The maximum proteolytic activity was found to be at pH 4.0, within the pH range for aspartic proteinases (APs), and the inhibition studies showed that only pepstatin A, specific for that class of enzymes, revealed a significant inhibitory effect. The C. calcitrapa aspartic proteinase (cenprosin) gene was detected in hairy roots using specific polymerase chain reaction (PCR) primers. The specific proteolytic activity present in hairy roots seems to be lower than the reported for flowers but similar to the existent in the untransformed roots and cell suspension cultures.

A collective view of the degradation of monochlorocatechols in fungi is yet to be attained, though these compounds are recognised as key degradation intermediates of numerous chlorinated aromatic hydrocarbons, including monochlorophenols. In the present contribution we have analysed the degradation pathways of monochlorophenols in Aspergillus nidulans using essentially metabolomics. Degradation intermediates herein identified included those commonly reported (e.g. 3-chloro-cis,cis-muconate) but also compounds never reported before in fungi revealing for 4-chlorocatechol and for 3- chlorocatechol unknown degradation paths yielding 3-chlorodienelactone and catechol, respectively. A different 3-chlorocatechol degradation path led to accumulation of 2- chloromuconates (a potential dead-end), notwithstanding preliminary evidence of chloromuconolactones and protoanemonin simultaneous formation. In addition, some transformation intermediates, of which sulfate conjugates of monochlorophenols/ chlorocatechols were the most common, were also identified. This study provides critical information for understanding the role of fungi in the degradation of chlorinated aromatic hydrocarbons; furthering their utility in the development of innovative bioremediation strategies.

BbiCPL1 was the first papain-like cysteine protease from a piroplasm to be identified with proteolytic activity. Here we report the improved production of the active recombinant enzyme, and the biochemical characterization of this potential drug target. BbiCPL1 showed characteristic properties of its class, including hydrolysis of papain-family peptide substrates, an acidic pH optimum, requirement of a reducing environment for maximum activity, and inhibition by standard cysteine protease inhibitors such as E-64, leupeptin, ALLN and cystatin. The optimum pH for the protease activity against peptide substrates was 5.5, but enzymatic activity was observed between pH 4.0 and pH 9.0. At slightly basic pH 7.5, BbiCPL1 maintained 83% of maximum activity, suggesting a role in cytosol environment.

The rodent malaria parasite Plasmodium chabaudi encodes one food vacuole plasmepsin—the aspartic proteinases important in haemoglobin degradation. A recombinant form of this enzyme was found to cleave a variety of peptide substrates and was susceptible to a selection of naturally occurring and synthetic inhibitors, displaying an inhibition profile distinct from that of aspartic proteinases from other malaria parasites. In addition, inhibitors of HIV proteinase that kill P. chabaudi in vivo were also inhibitors of this new plasmepsin. P. chabaudi is a widely used model for human malaria species and, therefore, the characterisation of this plasmepsin is an important contribution towards understanding its biology.

Papain-like cysteine proteases (CP) have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. One gene was identified by sequence similarity search to be homologous to the CP family in the ongoing Babesia bigemina genome sequencing project database. The newly identified CP gene, called babesipain-1, was cloned and expressed as a fusion protein, and the effect of different inhibitors on proteolytic activity was tested. A series of new artemisinin-vinyl sulfone hybrid molecules were tested as inhibitors being effective on the range of 0.3-30 microm, depending on the core-containing molecule.