Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects.

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

Inopinatum lactosum gen. & comb. nov., the first yeast-like fungus in Leotiomycetes.

Sporobolomyces lactosus is a pink yeast-like fungus that is not congeneric with other members of Sporobolomyces (Basidiomycota, Microbotryomycetes, Sporidiobolales). During our ongoing studies of pink yeasts we determined that S. lactosus was most closely related to Pseudeurotium zonatum (Ascomycota, Leotiomycetes, Thelebolales). A molecular phylogenetic analysis using sequences of the ITS region and the small and large subunit (SSU, LSU) rRNA genes, indicated that four isolates of S. lactosus, including three ex-type isolates, were placed in Thelebolales with maximum support. A new genus is proposed to accommodate S. lactosus, Inopinatum. This is the first pink yeast reported in Leotiomycetes.

High-throughput proteomics and metabolomic studies guide re-engineering of metabolic pathways in eukaryotic microalgae: A review.

Eukaryotic microalgae are a rich source of commercially important metabolites including lipids, pigments, sugars, amino acids and enzymes. However, their inherent genetic potential is usually not enough to support high level production of metabolites of interest. In order to move on from the traditional approach of improving product yields by modification of the cultivation conditions, understanding the metabolic pathways leading to the synthesis of the bioproducts of interest is crucial. Identification of new targets for strain engineering has been greatly facilitated by the rapid development of high-throughput sequencing and spectroscopic techniques discussed in this review. Despite the availability of high throughput analytical tools, examples of gathering and application of proteomic and metabolomic data for metabolic engineering of microalgae are few and mainly limited to lipid production. The present review highlights the application of contemporary proteomic and metabolomic techniques in eukaryotic microalgae for redesigning pathways for enhanced production of algal metabolites.

Rapid optimisation of cellulolytic enzymes ratios in Saccharomyces cerevisiae using in vitro SCRaMbLE.

BACKGROUND: For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes, with the optimum combined activity ratio dependent on the composition of the substrate. RESULTS: In vitro SCRaMbLE generated a library of plasmids containing different ratios of a ß-glucosidase gene (CEL3A) from Saccharomycopsis fibuligera and an endoglucanase gene (CEL5A) from Trichoderma reesei. S. cerevisiae, transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that 4,6-O-(3-ketobutylidene)-4-nitrophenyl-ß-D-cellopentaoside (BPNPG5) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2, respectively (ratio of 1.6:1). CONCLUSIONS: Here, we describe a synthetic biology approach to rapidly optimise gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

Rapid Colorimetric Detection of Genome Evolution in SCRaMbLEd Synthetic Saccharomyces cerevisiae Strains.

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

Molecular tools and applications of Euglena gracilis: From biorefineries to bioremediation.

Euglena gracilis is a promising source of commercially important metabolites such as vitamins, wax esters, paramylon, and amino acids. However, the molecular tools available to create improved Euglena strains are limited compared to other microorganisms that are currently exploited in the biotechnology industry. The complex poly-endosymbiotic nature of the Euglena genome is a major bottleneck for obtaining a complete genome sequence and thus represents a notable shortcoming in gaining molecular information of this organism. Therefore, the studies and applications have been more focused on using the wild-type strain or its variants and optimizing the nutrient composition and cultivation conditions to enhance the production of biomass and valuable metabolites. In addition to producing metabolites, the E. gracilis biorefinery concept also provides means for the production of biofuels and biogas as well as residual biomass for the remediation of industrial and municipal wastewater. Using Euglena for bioremediation of environments contaminated with heavy metals is of special interest due to the strong ability of the organism to accumulate and sequester these compounds. The published draft genome and transcriptome will serve as a basis for further molecular studies of Euglena and provide a guide for the engineering of metabolic pathways of relevance for the already established as well as novel applications.

Probing the Role of the Chloroplasts in Heavy Metal Tolerance and Accumulation in Euglena gracilis.

The E. gracilis Zm-strain lacking chloroplasts, characterized in this study, was compared with the earlier assessed wild type Z-strain to explore the role of chloroplasts in heavy metal accumulation and tolerance. Comparison of the minimum inhibitory concentration (MIC) values indicated that both strains tolerated similar concentrations of mercury (Hg) and lead (Pb), but cadmium (Cd) tolerance of the Z-strain was twice that of the Zm-strain. The ability of the Zm-strain to accumulate Hg was higher compared to the Z-strain, indicating the existence of a Hg transportation and accumulation mechanism not depending on the presence of chloroplasts. Transmission electron microscopy (TEM) showed maximum accumulation of Hg in the cytosol of the Zm-strain and highest accumulation of Cd in the chloroplasts of the Z-strain indicating a difference in the ability of the two strains to deposit heavy metals in the cell. The highly abundant heavy metal transporter MTP2 in the Z-strain may have a role in Cd transportation to the chloroplasts. A multidrug resistance-associated protein highly increased in abundance in the Zm-strain could be a potential Hg transporter to either cytosol or mitochondria. Overall, the chloroplasts appear to have major role in the tolerance and accumulation of Cd in E. gracilis.

Effect of peptidases secreted by the opportunistic pathogen Scedosporium aurantiacum on human epithelial cells.

Peptidases secreted by a clinical high-virulence Scedosporium aurantiacum isolate (strain WM 06.482; CBS 136046) under normoxic and hypoxic conditions were separated via size-exclusion chromatography, and peptidase activities present in each fraction were determined using class-specific substrates. The fractions demonstrating peptidase activity were assessed for their effects on the attachment and viability of A549 human lung epithelial cells in vitro. Of the peptidases detected in the size-exclusion chromatography fractions, the elastase-like peptidase reduced cell viability, the chymotrypsin-like peptidase was associated with cell detachment, and the cysteine peptidases were able to abolish both cell attachment and viability. The loss of cell viability and attachment became more prominent with an increase in the peptidase activity and could also be specifically prevented by addition of class-specific peptidase inhibitors. Our findings indicate that peptidases secreted by S. aurantiacum can breach the human alveolar epithelial cell barrier and, thus, may have a role in the pathobiology of the organism.

Bioproducts From Euglena gracilis: Synthesis and Applications.

In recent years, the versatile phototrophic protist Euglena gracilis has emerged as an interesting candidate for application-driven research and commercialisation, as it is an excellent source of dietary protein, pro(vitamins), lipids, and the ß-1,3-glucan paramylon only found in euglenoids. From these, paramylon is already marketed as an immunostimulatory agent in nutraceuticals. Bioproducts from E. gracilis can be produced under various cultivation conditions discussed in this review, and their yields are relatively high when compared with those achieved in microalgal systems. Future challenges include achieving the economy of large-scale cultivation. Recent insights into the complex metabolism of E. gracilis have highlighted unique metabolic pathways, which could provide new leads for product enhancement by genetic modification of the organism. Also, development of molecular tools for strain improvement are emerging rapidly, making E. gracilis a noteworthy challenger for microalgae such as Chlorella spp. and their products currently on the market.

Interactions of an Emerging Fungal Pathogen Scedosporium aurantiacum with Human Lung Epithelial Cells.

Scedosporium fungi are found in various natural and host-associated environments, including the lungs of cystic fibrosis patients. However, their role in infection development remains underexplored. Here the attachment of conidia of a virulent S. aurantiacum strain WM 06.482 onto the human lung epithelial A549 cells in vitro was visualized using microscopy to examine the initial steps of infection. We showed that 75-80% of fungal conidia were bound to the A549 cells within four hours of co-incubation, and started to produce germ tubes. The germinating conidia seemed to invade the cells through the intercellular space, no intracellular uptake of fungal conidia by the airway epithelial cells after conidial attachment. Transcriptomic analysis of the A549 cells revealed that the up-regulated genes were mainly associated with cell repair and inflammatory processes indicating a protective response against S. aurantiacum infection. Network analysis of the differentially expressed genes showed activation of the innate immune system (NF-kB pathway) leading to the release of pro-inflammatory cytokines. We believe this is the first report showing the transcriptomic response of human alveolar epithelial cells exposed to S. aurantiacum conidia paving a way for better understanding of the mechanism of the infection process.

Development of screening strategies for the identification of paramylon-degrading enzymes.

Enzymatic degradation of the ß-1,3-glucan paramylon could enable the production of bioactive compounds for healthcare and renewable substrates for biofuels. However, few enzymes have been found to degrade paramylon efficiently and their enzymatic mechanisms remain poorly understood. Thus, the aim of this work was to find paramylon-degrading enzymes and ways to facilitate their identification. Towards this end, a Euglena gracilis-derived cDNA expression library was generated and introduced into Escherichia coli. A flow cytometry-based screening assay was developed to identify E. gracilis enzymes that could hydrolyse the fluorogenic substrate fluorescein di-ß-D-glucopyranoside in combination with time-saving auto-induction medium. In parallel, four amino acid sequences of potential E. gracilis ß-1,3-glucanases were identified from proteomic data. The open reading frame encoding one of these candidate sequences (light_m.20624) was heterologously expressed in E. coli. Finally, a Congo Red dye plate assay was developed for the screening of enzyme preparations potentially able to degrade paramylon. This assay was validated with enzymes assumed to have paramylon-degrading activity and then used to identify four commercial preparations with previously unknown paramylon degradation ability.

Growth and protease secretion of Scedosporium aurantiacum under conditions of hypoxia.

One of the micro-environmental stresses that fungal pathogens, such as Scedosporium aurantiacum, colonising human lungs encounter in vivo is hypoxia, or deficiency of oxygen. In this work, we studied the impacts of a hypoxic micro-environment (oxygen levels ≤1%) on the growth of a clinical S. aurantiacum isolate (WM 06.482; CBS 136046) and an environmental strain (S. aurantiacum WM 10.136; CBS 136049) on mucin-containing synthetic cystic fibrosis sputum medium. Additionally, profiles of secreted proteases were compared between the two isolates and protease activity was assessed using class-specific substrates and inhibitors. Overall, both isolates grew slower and produced less biomass under hypoxia compared to normoxic conditions. The pH of the medium decreased to 4.0 over the cultivation time, indicating that S. aurantiacum released acidic compounds into the medium. Accordingly, secreted proteases of the two isolates were dominated by acidic proteases, including aspartic and cysteine proteases, with optimal protease activity at pH 4.0 and 6.0 respectively. The clinical isolate produced higher aspartic and cysteine protease activities. Conversely, all serine proteases, including elastase-like, trypsin-like, chymotrypsin-like and subtilisin-like proteases had higher activities in the environmental isolate. Sequence similarities to 13 secreted proteases were identified by mass spectrometry (MS) by searching against other fungal proteases in the NCBI database. Results from MS analysis were consistent with those from activity assays. The clinical highly-virulent, and environmental low-virulence S. aurantiacum isolates responded differently to hypoxia in terms of the type of proteases secreted, which may reflect their different virulence properties.

Overview of Gene Expression Using Filamentous Fungi.

Filamentous fungi are lower eukaryotes increasingly used for expression of foreign proteins ranging from industrial enzymes originating from other fungi and bacteria to proteins of mammalian origin, such as antibodies and growth factors. Their strengths include an excellent capacity for protein secretion and their eukaryotic protein processing machinery. Proteins secreted from filamentous fungi are modified in the secretory pathway, with folding, proteolytic processing, and addition of glycans being the main modifications. Unlike from many other expression systems, however, plasmids and host strains for expression of gene products in filamentous fungi are not readily available commercially, and the expression system must thus be stitched together in the laboratory. In this overview, the key elements of fungal expression systems are discussed from a practical point of view and with a view towards the future. The principles and considerations presented here can be applied to a range of filamentous fungi. © 2018 by John Wiley & Sons, Inc.

Making a Bacterial Thermophilic Enzyme in a Fungal Expression System.

This unit describes production of a bacterial thermophilic xylanase enzyme in an industrially exploited filamentous fungus, Trichoderma reesei. Successful expression of a gene of interest in a heterologous host involves front-end design of the expression constructs using bioinformatics tools, making the constructs in the laboratory, and introducing them into the expression host. This is followed by synthesis and characterization of the gene product on a laboratory scale and optimization of the cultivation parameters in a controlled, scaled-up fermentation. The thermophilic xylanase B (XynB) enzyme from the bacterium Dictyoglomus thermophilum discussed here can be easily purified by heat-precipitation from the culture supernatant of the mesophilic host. A functional XynB can also be produced in Escherichia coli, but at a lower yield compared to that obtained in T. reesei. The protocol provided here can be adapted to various other proteins and filamentous fungal hosts. © 2018 by John Wiley & Sons, Inc.

Microwave pretreatment of paramylon enhances the enzymatic production of soluble ß-1,3-glucans with immunostimulatory activity.

A hydrothermal microwave pretreatment was established to facilitate the enzymatic production of soluble bioactive ß-1,3-glucans from the recalcitrant substrate paramylon. The efficacy of this pretreatment was monitored with a newly developed direct Congo Red dye-based assay over a range of temperatures. Microwave pretreatment at 170 °C for 2 min resulted in a significantly enhanced enzymatic hydrolysis of paramylon. The action of endo-ß-1,3- and exo- ß-1,3-glucanases on the microwave-pretreated paramylon produced soluble ß-1,3-glucans with degrees of polymerisation (DP) ranging from 2-59 and 2-7, respectively. In comparison, acid-mediated hydrolysis of untreated paramylon resulted in ß-1,3-glucans with a DP range of 2-38. The hydrolysates were assayed on their immunostimulatory effect on murine macrophages by measuring the production of the inflammation-linked marker tumour necrosis factor alpha (TNFα) using immunofluorescence. All of the tested hydrolysis products were shown to induce TNFα production, with the most significant immunostimulatory effect observed with the hydrolysate from the exo-ß-1,3-glucanase treatment.

Comparative assessment of the Euglena gracilis var. saccharophila variant strain as a producer of the ß-1,3-glucan paramylon under varying light conditions.

Euglena gracilis Z and a "sugar loving" variant strain E. gracilis var. saccharophila were investigated as producers of paramylon, a ß-1,3-glucan polysaccharide with potential medicinal and industrial applications. The strains were grown under diurnal or dark growth conditions on a glucose-yeast extract medium supporting high-level paramylon production. Both strains produced the highest paramylon yields (7.4-8 g · L-1 , respectively) while grown in the dark, but the maximum yield was achieved faster by E. gracilis var. saccharophila (48 h vs. 72 h). The glucose-to-paramylon yield coefficient Ypar/glu  = 0.46 ± 0.03 in the E. gracilis var. saccharophila cultivation, obtained in this study, is the highest reported to date. Proteomic analysis of the metabolic pathways provided molecular clues for the strain behavior observed during cultivation. For example, overexpression of enzymes in the gluconeogenesis/glycolysis pathways including fructokinase-1 and chloroplastic fructose-1,6-bisphosphatase (FBP) may have contributed to the faster rate of paramylon accumulation in E. gracilis var. saccharophila. Differentially expressed proteins in the early steps of chloroplastogenesis pathway including plastid uroporphyrinogen decarboxylases, photoreceptors, and a highly abundant (68-fold increase) plastid transketolase may have provided the E. gracilis var. saccharophila strain an advantage in paramylon production during diurnal cultivations. In conclusion, the variant strain E. gracilis var. saccharophila seems to be well suited for producing large amounts of paramylon. This work has also resulted in the identification of molecular targets for future improvement of paramylon production in E. gracilis, including the FBP and phosophofructokinase 1, the latter being a key regulator of glycolysis.

Pseudomonas aeruginosa Inhibits the Growth of Scedosporium and Lomentospora In Vitro.

In vitro bacterial-fungal interaction studies in cystic fibrosis (CF) have mainly focused on interactions between bacteria and Candida. Here we investigated the effect of Pseudomonas aeruginosa on the growth of Scedosporium/Lomentospora spp. Standard suspensions of P. aeruginosa (16 non-mucoid and nine mucoid isolates) were dropped onto paper disks, placed on lawns of Lomentospora prolificans (formerly Scedosporium prolificans) strain WM 14.140 or Scedosporium aurantiacum strain WM 11.78 on solid agar. The median inhibitory activity (mIz) was calculated for each fungal-bacterial combination. As a group, mIz values for non-mucoid phenotype P. aeruginosa strains were significantly lower than those for mucoid strains (P < 0.001); 14/16 (87.5%) non-mucoid strains had mIz <1.0 against both fungi versus just 3/9 mucoid strains (33.4%) (P = 0.01). One non-mucoid (PA14) and one mucoid (CIDMLS-PA-28) P. aeruginosa strain effecting inhibition were selected for further studies. Inhibition of both L. prolificans and S. aurantiacum by these strains was confirmed using the XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) reduction assay. Following incubation with XTT, inhibition of fungal growth was determined as the ratio of absorbance in liquid culture with Pseudomonas to that in control fungal cultures. An absorbance ratio of <1.0 consistent with bacterial inhibition of fungal growth was observed for all four P. aeruginosa-fungal combinations (P < 0.05). Fluorescence microscopy, subsequent to co-culture of either fungal isolate with P. aeruginosa strain PA14 or CIDMLS-PA-28 revealed poorly formed hyphae, compared with control fungal cultures. P. aeruginosa inhibits growth of L. prolificans and S. aurantiacum in vitro, with non-mucoid strains more commonly having an inhibitory effect. As P. aeruginosa undergoes phenotype transitions from non-mucoid to the mucoid form with progression of CF lung disease, this balance may influence the appearance of Scedosporium fungi in the airways.

Enhancing structural characterisation of glucuronidated O-linked glycans using negative mode ion trap higher energy collision-induced dissociation mass spectrometry.

RATIONALE: High protein production and secretion with eukaryotic glycosylation machinery make T. reesei RUT-C30 a suitable expression host for recombinant proteins. The N-glycosylation of secreted proteins of RUT-C30 is known to vary depending on culture nutrients but O-glycosylation has been less extensively studied. METHODS: O-Glycans and glycopeptides from secreted proteins were separated by porous graphitised carbon and C-18 liquid chromatography, respectively. O-Glycans were analysed in negative ion mode by electrospray ionisation linear ion trap mass spectrometry and glycopeptides in positive ion mode by electrospray ionisation hybrid quadrupole-orbitrap mass spectrometry. Tandem mass spectrometry was used on O-glycans and glycopeptides including ion trap higher energy collision-induced dissociation (tHCD) to detect glycan fragments not detectable with standard ion trap fragmentation. tHCD allowed targeted MS3 experiments to be performed on structures containing hexuronic acid, which was not possible with ion trap CID, validating this novel O-glycan composition. Positive mode C18-LC/ESI-MS/MS was used to identify and characterise glycopeptides found to be modified with this class of O-glycans, identifying cellobiohydrolase I as a carrier of these novel O-glycans. RESULTS: Negative mode ion trap higher energy collision-induced dissociation allowed detection and targeted MS3 experiments to be performed on the hexuronic acid substituent of O-glycan structures, which was not possible with ion trap CID, validating the novel O-glycan composition to include hexuronic acid. Using glycopeptide analysis, this novel O-glycan composition was found to be present on the catalytic domain of cellobiohydrolase I, the most abundant secreted protein by T. reesei. CONCLUSIONS: These are the first reported O-glycans to contain acidic sugars in fungi and they could have significant implications for cellobiohydrolase I structure and activity as well as the activity of recombinant proteins expressed in this host system. Copyright © 2017 John Wiley & Sons, Ltd.

Secretion of Proteases by an Opportunistic Fungal Pathogen Scedosporium aurantiacum.

Scedosporium aurantiacum is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate Scedosporium aurantiacum (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5-75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the S. aurantiacum proteases.