Background: Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genusHypocrea/Trichoderma.Results: Here we report an analysis of the genome sequences of the two biocontrol speciesTrichoderma atroviride(teleomorphHypocrea atroviridis) andTrichoderma virens(formerlyGliocladium virens, teleomorphHypocrea virens), and a comparison withTrichoderma reesei(teleomorphHypocrea jecorina). These threeTrichodermaspecies display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative toT. reeseior other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows thatT. reeseiandT. virensare derived relative toT. atroviride. The mycoparasitism-specific genes thus arose in a commonTrichodermaancestor but were subsequently lost inT. reesei.Conclusions: The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Global climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes. A hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization. This study revealed the existence of a variety of xylose transporters in the cell factories A. niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.

Current European (EU) policies, such as the Green Deal, envisage safe and sustainable by design (SSbD) practices for the management of chemicals, which cogently entail nanomaterials (NMs) and advanced materials (AdMa). These practices, applied at the earliest stages of innovation and throughout the life-cycle of chemicals, materials and products, could prevent and/or minimise their environmental, health and safety (EHS) and sustainability impacts. This requires a shift from the established risk control paradigms towards prevention-based approaches at the design stage that accelerate the development of safer and more sustainable chemicals, materials, products and processes, while promoting a transition towards a circular economy and a more sustainable future. The EU commission has funded several Horizon 2020 projects applying the concepts of SSbD to nanotechnologies, biotechnologies and advanced materials. This article is inspired from the answers and opinions shared during a stakeholders meeting arranged throughout the workshop entitled ‘Safe and Sustainable by Design Paradigms applied to NMs and AdMa’, held in Venice, Italy, in September 2022. The goal of the workshop was to identify differences and overlaps between the SSbD approaches and to provide common messages on the progress towards the implementation of concrete SSbD concepts, and to reveal challenges faced in their realistic and straightforward execution. In this article, we provide insights into the intersecting industrial domains, the technical and organisational challenges to the practical implementation of the SSbD, and future financial directions in supporting and maintaining the digital products currently under development within the H2020 projects, in order to ultimately enable their uptake by industry and regulators.

Background: Many filamentous fungal genomes contain complex groups of multicopper oxidase (MCO) coding genes that makes them a good source for new laccases with potential biotechnological interest. A bioinformatics analysis of the Aspergillus niger ATCC 1015 genome resulted in the identification of thirteen MCO genes. Ten of them were cloned and homologously overexpressed. Results: A bioinformatic analysis of the A. niger ATCC 1015 genome revealed the presence of 13 MCO genes belonging to three different subfamilies on the basis of their phylogenetic relationships: ascomycete laccases, fungal pigment MCOs and fungal ferroxidases. According to in silico amino acid sequence analysis, the putative genes encoding for functional extracellular laccases (mcoA, mcoB, mcoC, mcoD, mcoE, mcoF, mcoG, mcoI, mcoJ and mcoM) were placed under the control of the glaA promoter and overexpressed in A. niger N593. Enzyme activity plate assays with several common laccase substrates showed that all genes are actually expressed and code for active MCOs. Interestingly, expressed enzymes show different substrate specificities. In addition, optimization of fungal pigment MCOs extracellular production was investigated. The performance of the widely used glucoamylase signal sequence (ssGlaA) in McoA secretion was studied. Results obtained suggest that ssGlaA do not yield higher levels of secreted McoA when compared to its native secretion signal. Also, McoB synthesis was investigated using different nitrogen sources in minimal medium liquid cultures. Higher yields of extracellular McoB were achieved with (NH₄)₂ tartrate. Conclusions: Aspergillus niger is a good source of new laccases. The different substrate specificity observed in plate assays makes them interesting to be purified and biochemically compared. The homologous signal sequence of McoA has been shown to be a good choice for its extracellular overexpression. From the nitrogen sources tested (NH₄)₂ tartrate has been found to be the most appropriate for McoB production in A. niger.

The development of biological processes that replace the existing petrochemical-based industry is one of the biggest challenges in biotechnology. Aspergillus niger is one of the main industrial producers of lignocellulolytic enzymes, which are used in the conversion of lignocellulosic feedstocks into fermentable sugars. Both the hydrolytic enzymes responsible for lignocellulose depolymerisation and the molecular mechanisms controlling their expression have been well described, but little is known about the transport systems for sugar uptake in A. niger. Understanding the transportome of A. niger is essential to achieve further improvements at strain and process design level. Therefore, this study aims to identify and classify A. niger sugar transporters, using newly developed tools for in silico and in vivo analysis of its membrane-associated proteome. In the present research work, a hidden Markov model (HMM), that shows a good performance in the identification and segmentation of functionally validated glucose transporters, was constructed. The model (HMMgluT) was used to analyse the A. niger membrane-associated proteome response to high and low glucose concentrations at a low pH. By combining the abundance patterns of the proteins found in the A. niger plasmalemma proteome with their HMMgluT scores, two new putative high-affinity glucose transporters, denoted MstG and MstH, were identified. MstG and MstH were functionally validated and biochemically characterised by heterologous expression in a S. cerevisiae glucose transport null mutant. They were shown to be a high-affinity glucose transporter (K m = 0.5 ± 0.04 mM) and a very high-affinity glucose transporter (K m = 0.06 ± 0.005 mM), respectively. This study, focusing for the first time on the membrane-associated proteome of the industrially relevant organism A. niger, shows the global response of the transportome to the availability of different glucose concentrations. Analysis of the A. niger transportome with the newly developed HMMgluT showed to be an efficient approach for the identification and classification of new glucose transporters.