Functional analysis of the protocatechuate branch of the ß-ketoadipate pathway in Aspergillus niger.

Bacteria and fungi catabolize plant-derived aromatic compounds by funneling into one of seven dihydroxylated aromatic intermediates, which then undergo ring fission and conversion to TCA cycle intermediates. Two of these intermediates, protocatechuic acid and catechol, converge on ß-ketoadipate which is further cleaved to succinyl-CoA and acetyl-CoA. These ß-ketoadipate pathways have been well characterized in bacteria. The corresponding knowledge of these pathways in fungi is incomplete. Characterization of these pathways in fungi would expand our knowledge and improve the valorization of lignin-derived compounds. Here, we used homology to characterize bacterial or fungal genes to predict the genes involved in the ß-ketoadipate pathway for protocatechuate utilization in the filamentous fungus Aspergillus niger. We further used the following approaches to refine the assignment of the pathway genes: whole transcriptome sequencing to reveal genes upregulated in the presence of protocatechuic acid; deletion of candidate genes to observe their ability to grow on protocatechuic acid; determination by mass spectrometry of metabolites accumulated by deletion mutants; and enzyme assays of the recombinant proteins encoded by candidate genes. Based on the aggregate experimental evidence, we assigned the genes for the five pathway enzymes as follows: NRRL3_01405 (prcA) encodes protocatechuate 3,4-dioxygenase; NRRL3_02586 (cmcA) encodes 3-carboxy-cis,cis-muconate cyclase; NRRL3_01409 (chdA) encodes 3-carboxymuconolactone hydrolase/decarboxylase; NRRL3_01886 (kstA) encodes ß-ketoadipate:succinyl-CoA transferase; and NRRL3_01526 (kctA) encodes ß-ketoadipyl-CoA thiolase. Strain carrying ΔNRRL3_00837 could not grow on protocatechuic acid, suggesting that it is essential for protocatechuate catabolism. Its function is unknown as recombinant NRRL3_00837 did not affect the in vitro conversion of protocatechuic acid to ß-ketoadipate.

Expansion of Auxiliary Activity Family 5 sequence space via biochemical characterization of six new copper radical oxidases.

Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5_2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5_2 members from diverse fungi for recombinant expression in Komagataella pfaffii (syn. Pichia pastoris) and biochemical characterization in vitro. Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5_2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity.IMPORTANCEEnzyme discovery and characterization underpin advances in microbial biology and the application of biocatalysts in industrial processes. On one hand, oxidative processes are central to fungal saprotrophy and pathogenesis. On the other hand, controlled oxidation of small molecules and (bio)polymers valorizes these compounds and introduces versatile functional groups for further modification. The biochemical characterization of six new copper radical oxidases further illuminates the catalytic diversity of these enzymes, which will inform future biological studies and biotechnological applications.

Genome and secretome insights: unravelling the lignocellulolytic potential of Myceliophthora verrucosa for enhanced hydrolysis of lignocellulosic biomass.

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.

Transcriptional and secretome analysis of Rasamsonia emersonii lytic polysaccharide mono-oxygenases.

The current study is the first to describe the temporal and differential transcriptional expression of two lytic polysaccharide monooxygenase (LPMO) genes of Rasamsonia emersonii in response to various carbon sources. The mass spectrometry based secretome analysis of carbohydrate active enzymes (CAZymes) expression in response to different carbon sources showed varying levels of LPMOs (AA9), AA3, AA7, catalase, and superoxide dismutase enzymes pointing toward the redox-interplay between the LPMOs and auxiliary enzymes. Moreover, it was observed that cello-oligosaccharides have a negative impact on the expression of LPMOs, which has not been highlighted in previous reports. The LPMO1 (30 kDa) and LPMO2 (47 kDa), cloned and expressed in Pichia pastoris, were catalytically active with (kcat/Km) of 6.6×10-2 mg-1 ml min-1 and 1.8×10-2 mg-1 ml min-1 against Avicel, respectively. The mass spectrometry of hydrolysis products of Avicel/carboxy methyl cellulose (CMC) showed presence of C1/C4 oxidized oligosaccharides indicating them to be Type 3 LPMOs. The 3D structural analysis of LPMO1 and LPMO2 revealed distinct arrangements of conserved catalytic residues at their active site. The developed enzyme cocktails consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant LPMO1/LPMO2 resulted in significantly enhanced saccharification of steam/acid pretreated unwashed rice straw slurry from PRAJ industries (Pune, India). The current work indicates that LPMO1 and LPMO2 are catalytically efficient and have a high degree of thermostability, emphasizing their usefulness in improving benchmark enzyme cocktail performance. KEY POINTS: • Mass spectrometry depicts subtle interactions between LPMOs and auxiliary enzymes. • Cello-oligosaccharides strongly downregulated the LPMO1 expression. • Developed LPMO cocktails showed superior hydrolysis in comparison to CellicCTec3.

A thermostable and inhibitor resistant ß-glucosidase from Rasamsonia emersonii for efficient hydrolysis of lignocellulosics biomass.

The present study reports a highly thermostable ß-glucosidase (GH3) from Rasamsonia emersonii that was heterologously expressed in Pichia pastoris. Extracellular ß-glucosidase was purified to homogeneity using single step affinity chromatography with molecular weight of ~ 110 kDa. Intriguingly, the purified enzyme displayed high tolerance to inhibitors mainly acetic acid, formic acid, ferulic acid, vanillin and 5-hydroxymethyl furfural at concentrations exceeding those present in acid steam pretreated rice straw slurry used for hydrolysis and subsequent fermentation in 2G ethanol plants. Characteristics of purified ß-glucosidase revealed the optimal activity at 80 °C, pH 5.0 and displayed high thermostability over broad range of temperature 50-70 °C with maximum half-life of ~ 60 h at 50 °C, pH 5.0. The putative transglycosylation activity of ß-glucosidase was appreciably enhanced in the presence of methanol as an acceptor. Using the transglycosylation ability of ß-glucosidase, the generated low cost mixed glucose disaccharides resulted in the increased induction of R. emersonii cellulase under submerged fermentation. Scaling up the recombinant protein production at fermenter level using temporal feeding approach resulted in maximal ß-glucosidase titres of 134,660 units/L. Furthermore, a developed custom made enzyme cocktail consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant ß-glucosidase resulted in significantly enhanced hydrolysis of pretreated rice straw slurry from IOCL industries (India). Our results suggest multi-faceted ß-glucosidase from R. emersonii can overcome obstacles mainly high cost associated enzyme production, inhibitors that impair the sugar yields and thermal inactivation of enzyme.

Developing endophytic Penicillium oxalicum as a source of lignocellulolytic enzymes for enhanced hydrolysis of biorefinery relevant pretreated rice straw.

Endophytic fungi, as plant symbionts, produce an elaborate array of enzymes for efficient disintegration of lignocellulosic biomass into constituent monomeric sugars, making them novel source of lignocellulolytic CAZymes with immense potential in future biorefineries. The present study reports lignocellulolytic enzymes production potential of an endophytic halotolerant Penicillium oxalicum strain isolated from Citrus limon, under submerged and solid-state fermentation (SmF & SSF, respectively), in the presence and absence of salt (1 M NaCl). The comparative QTOF-LC/MS-based exoproteome analysis of the culture extracts unveiled differential expression of CAZymes, with the higher abundance of GH6 and GH7 family cellobiohydrolase in the presence of 1 M salt. The strain improvement program, employing cyclic mutagenesis and diploidization, was utilized to develop hyper-cellulase producing mutant strains of P. oxalicum. The enzyme production of the developed strain (POx-M35) was further enhanced through statistical optimization of the culture conditions utilizing glucose mix disaccharides (GMDs) as an inducer. This optimization process resulted in the lignocellulolytic cocktail that contained high titers (U/mL) of endoglucanase (EG) (146.16), cellobiohydrolase (CBHI) (6.99), ß-glucosidase (ß-G) (26.21), xylanase (336.05) and FPase (2.02 U/mL), which were 5.47-, 5.54-, 8.55-, 4.96-, and 4.39-fold higher when compared to the enzyme titers obtained in wild HP1, respectively. Furthermore, the lignocellulolytic cocktails designed by blending secretome produced by mutant POx-M35 with xylanases (GH10 and GH11) derived from Malbranchea cinnamomea resulted in efficient hydrolysis of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw. This study underscores the potential of bioprospecting novel fungus and developing an improved strain for optimized production and constitution of lignocellulolytic cocktails that can be an important determinant in advancing biomass conversion technologies.

Improving candidate Biosynthetic Gene Clusters in fungi through reinforcement learning.

MOTIVATION: Precise identification of Biosynthetic Gene Clusters (BGCs) is a challenging task. Performance of BGC discovery tools is limited by their capacity to accurately predict components belonging to candidate BGCs, often overestimating cluster boundaries. To support optimizing the composition and boundaries of candidate BGCs, we propose reinforcement learning approach relying on protein domains and functional annotations from expert curated BGCs. RESULTS: The proposed reinforcement learning method aims to improve candidate BGCs obtained with state-of-the-art tools. It was evaluated on candidate BGCs obtained for two fungal genomes, Aspergillus niger and Aspergillus nidulans. The results highlight an improvement of the gene precision by above 15% for TOUCAN, fungiSMASH and DeepBGC; and cluster precision by above 25% for fungiSMASH and DeepBCG, allowing these tools to obtain almost perfect precision in cluster prediction. This can pave the way of optimizing current prediction of candidate BGCs in fungi, while minimizing the curation effort required by domain experts. AVAILABILITY AND IMPLEMENTATION: https://github.com/bioinfoUQAM/RL-bgc-components. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparative analysis of functional diversity of rumen microbiome in bison and beef heifers.

IMPORTANCE: Ruminants play a key role in the conversion of cellulolytic plant material into high-quality meat and milk protein for humans. The rumen microbiome is the driver of this conversion, yet there is little information on how gene expression within the microbiome impacts the efficiency of this conversion process. The current study investigates gene expression in the rumen microbiome of beef heifers and bison and how transplantation of ruminal contents from bison to heifers alters gene expression. Understanding interactions between the host and the rumen microbiome is the key to developing informed approaches to rumen programming that will enhance production efficiency in ruminants.

Xylan glucuronic acid side chains fix suberin-like aliphatic compounds to wood cell walls.

Wood is the most important repository of assimilated carbon in the biosphere, in the form of large polymers (cellulose, hemicelluloses including glucuronoxylan, and lignin) that interactively form a composite, together with soluble extractives including phenolic and aliphatic compounds. Molecular interactions among these compounds are not fully understood. We have targeted the expression of a fungal α-glucuronidase to the wood cell wall of aspen (Populus tremula L. × tremuloides Michx.) and Arabidopsis (Arabidopsis thaliana (L.) Heynh), to decrease contents of the 4-O-methyl glucuronopyranose acid (mGlcA) substituent of xylan, to elucidate mGlcA's functions. The enzyme affected the content of aliphatic insoluble cell wall components having composition similar to suberin, which required mGlcA for binding to cell walls. Such suberin-like compounds have been previously identified in decayed wood, but here, we show their presence in healthy wood of both hardwood and softwood species. By contrast, γ-ester bonds between mGlcA and lignin were insensitive to cell wall-localized α-glucuronidase, supporting the intracellular formation of these bonds. These findings challenge the current view of the wood cell wall composition and reveal a novel function of mGlcA substituent of xylan in fastening of suberin-like compounds to cell wall. They also suggest an intracellular initiation of lignin-carbohydrate complex assembly.

A novel 5-ring multifocal electroretinography stimulus for detecting hydroxychloroquine retinal toxicity.

PURPOSE: Multifocal electroretinogram (mfERG) shows great utility as a screening tool to detect early hydroxychloroquine (HCQ) retinopathy, but its widespread use is limited by the lack of accessibility and long test duration. In this study, we evaluated a novel concentric 5-ring mfERG stimulus to provide a simplified and rapid protocol for screening HCQ toxicity. METHODS: Patients referred for HCQ retinopathy screening were consented to this observational cross-sectional study. Patients with amblyopia, high refractive error (more than 8 diopters), other retinal diseases precluding appropriate evaluation or history of retinal surgery were excluded. The data were collected from patients undergoing HCQ screening at a single center from July 2019 to March 2020. Patients were tested with the new concentric 5-ring mfERG stimulus, standard 61-hexagon mfERG stimulus, spectral domain optical coherence tomography and automated 10-2 visual fields. For the main outcome, the 5-ring mfERG was compared to 61-hexagon stimulus to determine the time-to-test completion and assess the association between ring (R1-R5) amplitude and ring ratio compared against cumulative dose, dose by real body weight and duration of therapy using Pearson correlation. RESULTS: In total, 52 patients (104 eyes; 5 males and 47 females) were recruited with a mean age of 59 years (range 23-85 years). The 5-ring protocol was markedly quicker to perform (1.3 ± 0.2 min; mean (SD)) compared to the 61-hexagon protocol (5.2 ± 0.6 min), p < 0.0001; n = 10 patients. The new R2/R5 ring ratio showed a moderate correlation with daily dose (r = - 0.640), cumulative dose (r = - 0.581) and duration of therapy (r = - 0.417). Similar correlations were observed with the new R2/R4 ring ratio which were not significantly different from the new R2/R5 correlation coefficients. The new R2/R5 ring ratio demonstrated a stronger correlation with daily (p = 0.002) and cumulative dose (p = 0.0001) compared to the 61-hexagon stimulus. CONCLUSIONS: In this exploratory study, our novel 5-ring mfERG protocol significantly shortened data acquisition time while providing comparable results to the standard 61-hexagon stimulus for detecting HCQ-induced electrophysiological changes that are correlated with HCQ dosages and treatment duration. Our protocol has the potential to be more clinically practical by simplifying routine screening.

The chimeric GaaR-XlnR transcription factor induces pectinolytic activities in the presence of D-xylose in Aspergillus niger.

Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin. KEY POINTS: • Chimeric transcription factors were generated by on-site mutations using CRISPR/Cas9. • PpgaX-hph reporter strain allowed for the screening of functional GaaR-XlnR mutants. • Chimeric GaaR-XlnR induced pectinolytic activities in the presence of D-xylose.

Loss of function of the carbon catabolite repressor CreA leads to low but inducer-independent expression from the feruloyl esterase B promoter in Aspergillus niger.

OBJECTIVE: With the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB, a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter. RESULT: PfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively. The genetic screen on the non-inducing carbon source D-fructose yielded in total 111 trans-acting mutants. The genome of one of the mutants was sequenced and revealed several SNPs, including a point mutation in the creA gene encoding a transcription factor known to be involved in carbon catabolite repression. Subsequently, all mutants were analyzed for defects in carbon catabolite repression by determining sensitivity towards allyl alcohol. All except four of the 111 mutants were sensitive to allyl alcohol, indicating that the vast majority of the mutants are defective in carbon catabolite repression. The creA gene of 32 allyl alcohol sensitive mutants was sequenced and 27 of them indeed contained a mutation in the creA gene. Targeted deletion of creA in the reporter strain confirmed that the loss of CreA results in constitutive expression from the faeB promoter. CONCLUSION: Loss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.

Multiparametric MR Investigation of Proteoglycan Diffusivity, T2 Relaxation, and Concentration in an Ex Vivo Model of Intervertebral Disc Degeneration.

BACKGROUND: Proteoglycan (PG) is a major component of the intervertebral disc extracellular matrix (ECM) that acts to hydrate the disc nucleus. Early detection of PG degradation is valuable for both diagnosis and preclinical research of intervertebral disc degeneration (IVDD). PURPOSE: To compare different MR techniques for detecting early degradative changes of PG in IVDD. STUDY TYPE: Prospective. PHANTOM/SPECIMEN: Glycosaminoglycan (GAG) phantom/bovine discs with papain injection and human cadaveric discs. FIELD STRENGTH/SEQUENCES: 7T/diffusion-weighted MR spectroscopy (DW-MRS), T2 -weighted MRS (T2 W-MRS), and chemical exchange saturation transfer (CEST) imaging. ASSESSMENT: DW-MRS, T2 W-MRS, and CEST imaging were applied longitudinally to measure PG diffusivity, T2 value, overall content, and spatial distribution in the disc nucleus with enzyme-induced proteolytic ECM degradation (n = 8). Similar MR measurements were applied in GAG phantom and human cadaveric discs with different levels of degeneration (n = 6). STATISTICAL TESTS: T-tests were conducted to measure the differences of PG properties between pre- and post-enzyme injection. Linear regression and mixed-effects models were used to assess the associations among different PG properties as well as the degeneration grades in human cadaveric discs. RESULTS: In bovine discs, PG diffusivity increased most rapidly after the enzyme was injected into the disc nucleus (12 hours postinjection, t = 5.76, P = 0.0007). The PG T2 value did not change significantly (t < 1.54, P > 0.17 for all timepoints) during ECM degradation and was not associated with PG diffusivity (t = 0.06, P = 0.95). PG distribution change was more rapid than overall PG content and was strongly associated with PG diffusivity increase (t = -9.25, P < 1 × 10-8 ). In severely degenerated human cadaveric discs, the PG ADCs and T2 values were both associated with degeneration grades. DATA CONCLUSION: PG diffusivity is a direct biomarker for early ECM degradation, while PG distribution can be an indirect biomarker for early IVDD. LEVEL OF EVIDENCE: 2 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2020;51:1390-1400.

The effects of external Mn2+ concentration on hyphal morphology and citric acid production are mediated primarily by the NRAMP-family transporter DmtA in Aspergillus niger.

BACKGROUND: Citric acid, a commodity product of industrial biotechnology, is produced by fermentation of the filamentous fungus Aspergillus niger. A requirement for high-yield citric acid production is keeping the concentration of Mn2+ ions in the medium at or below 5 µg L-1. Understanding manganese metabolism in A. niger is therefore of critical importance to citric acid production. To this end, we investigated transport of Mn2+ ions in A. niger NRRL2270. RESULTS: we identified an A. niger gene (dmtA; NRRL3_07789), predicted to encode a transmembrane protein, with high sequence identity to the yeast manganese transporters Smf1p and Smf2p. Deletion of dmtA in A. niger eliminated the intake of Mn2+ at low (5 µg L-1) external Mn2+ concentration, and reduced the intake of Mn2+ at high (> 100 µg L-1) external Mn2+ concentration. Compared to the parent strain, overexpression of dmtA increased Mn2+ intake at both low and high external Mn2+ concentrations. Cultivation of the parent strain under Mn2+ ions limitation conditions (5 µg L-1) reduced germination and led to the formation of stubby, swollen hyphae that formed compact pellets. Deletion of dmtA caused defects in germination and hyphal morphology even in the presence of 100 µg L-1 Mn2+, while overexpression of dmtA led to enhanced germination and normal hyphal morphology at limiting Mn2+ concentration. Growth of both the parent and the deletion strains under citric acid producing conditions resulted in molar yields (Yp/s) of citric acid of > 0.8, although the deletion strain produced ~ 30% less biomass. This yield was reduced only by 20% in the presence of 100 µg L-1 Mn2+, whereas production by the parent strain was reduced by 60%. The Yp/s of the overexpressing strain was 17% of that of the parent strain, irrespective of the concentrations of external Mn2+. CONCLUSIONS: Our results demonstrate that dmtA is physiologically important in the transport of Mn2+ ions in A. niger, and manipulation of its expression modulates citric acid overflow.

Glucose-Mediated Repression of Plant Biomass Utilization in the White-Rot Fungus Dichomitus squalens.

The extent of carbon catabolite repression (CCR) at a global level is unknown in wood-rotting fungi, which are critical to the carbon cycle and are a source of biotechnological enzymes. CCR occurs in the presence of sufficient concentrations of easily metabolizable carbon sources (e.g., glucose) and involves downregulation of the expression of genes encoding enzymes involved in the breakdown of complex carbon sources. We investigated this phenomenon in the white-rot fungus Dichomitus squalens using transcriptomics and exoproteomics. In D. squalens cultures, approximately 7% of genes were repressed in the presence of glucose compared to Avicel or xylan alone. The glucose-repressed genes included the essential components for utilization of plant biomass-carbohydrate-active enzyme (CAZyme) and carbon catabolic genes. The majority of polysaccharide-degrading CAZyme genes were repressed and included activities toward all major carbohydrate polymers present in plant cell walls, while repression of ligninolytic genes also occurred. The transcriptome-level repression of the CAZyme genes observed on the Avicel cultures was strongly supported by exoproteomics. Protease-encoding genes were generally not glucose repressed, indicating their likely dominant role in scavenging for nitrogen rather than carbon. The extent of CCR is surprising, given that D. squalens rarely experiences high free sugar concentrations in its woody environment, and it indicates that biotechnological use of D. squalens for modification of plant biomass would benefit from derepressed or constitutively CAZyme-expressing strains.IMPORTANCE White-rot fungi are critical to the carbon cycle because they can mineralize all wood components using enzymes that also have biotechnological potential. The occurrence of carbon catabolite repression (CCR) in white-rot fungi is poorly understood. Previously, CCR in wood-rotting fungi has only been demonstrated for a small number of genes. We demonstrated widespread glucose-mediated CCR of plant biomass utilization in the white-rot fungus Dichomitus squalens This indicates that the CCR mechanism has been largely retained even though wood-rotting fungi rarely experience commonly considered CCR conditions in their woody environment. The general lack of repression of genes encoding proteases along with the reduction in secreted CAZymes during CCR suggested that the retention of CCR may be connected with the need to conserve nitrogen use during growth on nitrogen-scarce wood. The widespread repression indicates that derepressed strains could be beneficial for enzyme production.

Enzymes of early-diverging, zoosporic fungi.

The secretome, the complement of extracellular proteins, is a reflection of the interaction of an organism with its host or substrate, thus a determining factor for the organism's fitness and competitiveness. Hence, the secretome impacts speciation and organismal evolution. The zoosporic Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, and Cryptomycota represent the earliest diverging lineages of the Fungal Kingdom. The review describes the enzyme compositions of these zoosporic fungi, underscoring the enzymes involved in biomass degradation. The review connects the lifestyle and substrate affinities of the zoosporic fungi to the secretome composition by examining both classical phenotypic investigations and molecular/genomic-based studies. The carbohydrate-active enzyme profiles of 19 genome-sequenced species are summarized. Emphasis is given to recent advances in understanding the functional role of rumen fungi, the basis for the devastating chytridiomycosis, and the structure of fungal cellulosome. The approach taken by the review enables comparison of the secretome enzyme composition of anaerobic versus aerobic early-diverging fungi and comparison of enzyme portfolio of specialized parasites, pathogens, and saprotrophs. Early-diverging fungi digest most major types of biopolymers: cellulose, hemicellulose, pectin, chitin, and keratin. It is thus to be expected that early-diverging fungi in its entirety represents a rich and diverse pool of secreted, metabolic enzymes. The review presents the methods used for enzyme discovery, the diversity of enzymes found, the status and outlook for recombinant production, and the potential for applications. Comparative studies on the composition of secretome enzymes of early-diverging fungi would contribute to unraveling the basal lineages of fungi.

Discovery and characterization of family 39 glycoside hydrolases from rumen anaerobic fungi with polyspecific activity on rare arabinosyl substrates.

Enzyme activities that improve digestion of recalcitrant plant cell wall polysaccharides may offer solutions for sustainable industries. To this end, anaerobic fungi in the rumen have been identified as a promising source of novel carbohydrate active enzymes (CAZymes) that modify plant cell wall polysaccharides and other complex glycans. Many CAZymes share insufficient sequence identity to characterized proteins from other microbial ecosystems to infer their function; thus presenting challenges to their identification. In this study, four rumen fungal genes (nf2152, nf2215, nf2523, and pr2455) were identified that encode family 39 glycoside hydrolases (GH39s), and have conserved structural features with GH51s. Two recombinant proteins, NF2152 and NF2523, were characterized using a variety of biochemical and structural techniques, and were determined to have distinct catalytic activities. NF2152 releases a single product, ß1,2-arabinobiose (Ara2) from sugar beet arabinan (SBA), and ß1,2-Ara2 and α-1,2-galactoarabinose (Gal-Ara) from rye arabinoxylan (RAX). NF2523 exclusively releases α-1,2-Gal-Ara from RAX, which represents the first description of a galacto-(α-1,2)-arabinosidase. Both ß-1,2-Ara2 and α-1,2-Gal-Ara are disaccharides not previously described within SBA and RAX. In this regard, the enzymes studied here may represent valuable new biocatalytic tools for investigating the structures of rare arabinosyl-containing glycans, and potentially for facilitating their modification in industrial applications.

The obligate alkalophilic soda-lake fungus Sodiomyces alkalinus has shifted to a protein diet.

Sodiomyces alkalinus is one of the very few alkalophilic fungi, adapted to grow optimally at high pH. It is widely distributed at the plant-deprived edges of extremely alkaline lakes and locally abundant. We sequenced the genome of S. alkalinus and reconstructed evolution of catabolic enzymes, using a phylogenomic comparison. We found that the genome of S. alkalinus is larger, but its predicted proteome is smaller and heavily depleted of both plant-degrading enzymes and proteinases, when compared to its closest plant-pathogenic relatives. Interestingly, despite overall losses, S. alkalinus has retained many proteinases families and acquired bacterial cell wall-degrading enzymes, some of them via horizontal gene transfer from bacteria. This fungus has very potent proteolytic activity at high pH values, but slowly induced low activity of cellulases and hemicellulases. Our experimental and in silico data suggest that plant biomass, a common food source for most fungi, is not a preferred substrate for S. alkalinus in its natural environment. We conclude that the fungus has abandoned the ancestral plant-based diet and has become specialized in a more protein-rich food, abundantly available in soda lakes in the form of prokaryotes and small crustaceans.

Inducer-independent production of pectinases in Aspergillus niger by overexpression of the D-galacturonic acid-responsive transcription factor gaaR.

The transcription factor GaaR is needed for the expression of genes required for pectin degradation and transport and catabolism of the main degradation product, D-galacturonic acid (GA) in Aspergillus niger. In this study, we used the strong constitutive gpdA promoter of Aspergillus nidulans to overexpress gaaR in A. niger. Overexpression of gaaR resulted in an increased transcription of the genes encoding pectinases, (putative) GA transporters, and catabolic pathway enzymes even under non-inducing conditions, i.e., in the absence of GA. Exoproteome analysis of a strain overexpressing gaaR showed that this strain secretes highly elevated levels of pectinases when grown in fructose. The genes encoding exo-polygalacturonases were found to be subjected to CreA-mediated carbon catabolite repression, even in the presence of fructose. Deletion of creA in the strain overexpressing gaaR resulted in a further increase in pectinase production in fructose. We showed that GaaR localizes mainly in the nucleus regardless of the presence of an inducer, and that overexpression of gaaR leads to an increased concentration of GaaR in the nucleus.

The molecular response of the white-rot fungus Dichomitus squalens to wood and non-woody biomass as examined by transcriptome and exoproteome analyses.

The ability to obtain carbon and energy is a major requirement to exist in any environment. For several ascomycete fungi, (post-)genomic analyses have shown that species that occupy a large variety of habitats possess a diverse enzymatic machinery, while species with a specific habitat have a more focused enzyme repertoire that is well-adapted to the prevailing substrate. White-rot basidiomycete fungi also live in a specific habitat, as they are found exclusively in wood. In this study, we evaluated how well the enzymatic machinery of the white-rot fungus Dichomitus squalens is tailored to degrade its natural wood substrate. The transcriptome and exoproteome of D. squalens were analyzed after cultivation on two natural substrates, aspen and spruce wood, and two non-woody substrates, wheat bran and cotton seed hulls. D. squalens produced ligninolytic enzymes mainly at the early time point of the wood cultures, indicating the need to degrade lignin to get access to wood polysaccharides. Surprisingly, the response of the fungus to the non-woody polysaccharides was nearly as good a match to the substrate composition as observed for the wood polysaccharides. This indicates that D. squalens has preserved its ability to efficiently degrade plant biomass types not present in its natural habitat.