Even if the ocean represents a large part of Earth's surface, only a few studies describe marine-derived fungi compared to their terrestrial homologues. In this ecosystem, marine-derived fungi have had to adapt to the salinity and to the plant biomass composition. This articles studies the growth of five marine isolates and the tuning of lignocellulolytic activities under different conditions, including the salinity. A de novo transcriptome sequencing and assembly were used in combination with a proteomic approach to characterize the Carbohydrate Active Enzymes (CAZy) repertoire of one of these strains. Following these approaches, Stemphylium lucomagnoense was selected for its adapted growth on xylan in saline conditions, its high xylanase activity, and its improved laccase activities in seagrass-containing cultures with salt. De novo transcriptome sequencing and assembly indicated the presence of 51 putative lignocellulolytic enzymes. Its secretome composition was studied in detail when the fungus was grown on either a terrestrial or a marine substrate, under saline and non-saline conditions. Proteomic analysis of the four S. lucomagnoense secretomes revealed a minimal suite of extracellular enzymes for plant biomass degradation and highlighted potential enzyme targets to be further studied for their adaptation to salts and for potential biotechnological applications.
BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.
Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.
The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter(-1), i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 x 10(6) M(-1) s(-1) toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme.
The thermal stability of four molecular forms (native, refolded, glycosylated, non-glycosylated) of feruloyl esterase A (FAEA) was studied. From the most to the least thermo-resistant, the four molecular species ranked as follows: (i) glycosylated form produced native, (ii) non-glycosylated form produced native, (iii) non-glycosylated form produced as inclusion bodies and refolded, and (iv) glycosylated form produced native chemically denatured and then refolded. On the basis of these results and of crystal structure data, we discuss the respective importance of protein folding and glycosylation in the thermal stability of recombinant FAEA.
BACKGROUND: There have been many claims of adaptive molecular evolution, but what role does positive selection play in functional divergence? The aim of this study was to test the relationship between evolutionary and functional shifts with special emphasis on the role of the environment. For this purpose, we studied the fungal lipase/feruloyl esterase A family, whose functional diversification makes it a very promising candidate. RESULTS: The results suggested functional shift following a duplication event where neofunctionalisation of feruloyl esterase A had occurred with conservation of the ancestral lipase function. Evolutionary shift was detected using the branch-site model for testing positive selection on individual codons along specific lineages. Positively selected amino acids were detected. Furthermore, biological data obtained from site-directed mutagenesis experiments clearly demonstrated that certain amino acids under positive selection were involved in the functional shift. We reassessed evolutionary history in terms of environmental response, and hypothesized that environmental changes such as colonisation by terrestrial plants might have driven adaptation by functional diversification in Euascomycetes (Aspergilli), thus conferring a selective advantage on this group. CONCLUSION: The results reported here illustrate a rare example of connection between fundamental events in molecular evolution. We demonstrated an unequivocal connection between evolutionary and functional shifts, which led us to conclude that these events were probably linked to environmental change.
Title: Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme Levasseur A, Benoit I, Asther M, Record E Ref: Protein Expr Purif, 37:126, 2004 : PubMed
The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeB gene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of the gpd promoter and trpC terminator and expressed in a protease deficient A. niger strain. Homologous overproduction allows to reach an esterase activity of 18 nkat mL(-1) against MCA as substrate. The improvement factor was 16-fold higher as compared to the production level obtained with non-transformed A. niger strain induced by sugar beet pulp. The corresponding secretion yield was estimated to be around 100 mg L(-1). Recombinant FAEB was purified 14.6-fold to homogeneity from an 8-day-old culture by a single affinity chromatographic step with a recovery of 64%. SDS-PAGE revealed a single band with a molecular mass of 75 kDa, while under non-denatured conditions, native enzyme has a molecular mass of around 150 kDa confirming that the recombinant FAEB is a homodimer. The recombinant and native FAEB have the same characteristics concerning temperature and pH optima, i.e., 50 degrees C and 6, respectively. In addition, the recombinant FAEB was determined to be quite stable up to 50 degrees C for 120 min. Kinetic constants for MCA, MpCA, and chlorogenic acid (5-O-caffeoyl quinic acid) were as follows: Km: 0.13, 0.029, and 0.16 mM and Vmax: 1101, 527.6, and 28.3 nkat mg(-1), respectively. This is the first report on the homologous overproduction of feruloyl esterase B in A. niger.
