High-yield production of aryl alcohol oxidase under limited growth conditions in small-scale systems using a mutant Aspergillus nidulans strain.

Aryl alcohol oxidase (MtGloA) is an enzyme that belongs to the ligninolytic consortium and can play an important role in the bioenergy industry. This study investigated production of an MtGloA client enzyme by a mutant strain of Aspergillus nidulans unable to synthesize its own pyridoxine. Pyridoxine limitation can be used to control cell growth, diverting substrate to protein production. In agitated culture, enzyme production was similar when using media with 1 mg/L and without pyridoxine (26.64 ± 6.14 U/mg mycelia and 26.14 ± 8.39 U/mg mycelia using media with and without pyridoxine, respectively). However, the treatment lacking pyridoxine had to be supplemented with pyridoxine after 156 h of fermentation to sustain continued enzyme production. Use of extremely diluted pyridoxine levels allowed reduced fungal growth while maintaining steady enzyme production. Concentrations of 9 and 13.5 µg/L pyridoxine allowed MtGloA production with a growth rate of only 5% of that observed when using the standard 1 mg/L pyridoxine media.

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg(203)-Ala(230) loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation.

High-yield recombinant xylanase production by Aspergillus nidulans under pyridoxine limitation.

The present study investigated the limitation of pyridoxine on an Aspergillus nidulans culture that produces xylanase B (XynB) as a client enzyme and was unable to synthesize pyridoxine. This technique was used to limit cell growth and divert substrate to product formation for a surface grown culture that could be used in trickle bed reactors. It was observed that growth was limited when pyridoxine was absent, while enzyme production was unaffected. Enzyme production was 1,026 U after 480 h of continuous fermentation, which was similar to a culture that grew on medium with pyridoxine. Furthermore, the present study investigated the growth rate of A. nidulans with pyridoxine in the medium and determined the productivity of XynB production with and without pyridoxine. A maximum growth rate of 0.311/h was observed. The maximum XynB productivity of 21.14 U/g h was achieved when pyridoxine was not added to the medium.

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-ß-1,4-glucanase (GH12) from Aspergillus niveus.

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-ß-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan ß-1,3 or ß-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in ß-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.

Transcriptional profiling of Neurospora crassa Δmak-2 reveals that mitogen-activated protein kinase MAK-2 participates in the phosphate signaling pathway.

The filamentous fungus Neurospora crassa is an excellent model system for examining molecular responses to ambient signals in eukaryotic microorganisms. Inorganic phosphate (Pi) is an essential growth-limiting nutrient in nature and is crucial for the synthesis of nucleic acids and the flow of genetic information. The genetic and molecular mechanisms controlling the response to Pi starvation in N. crassa include at least four genes (nuc-2, preg, pogv, and nuc-1), which are involved in a hierarchical regulatory activation network. In a previous work, we identified a number of genes modulated by NUC-2 protein, including the mak-2 gene, which codes for a mitogen-activated protein kinase (MAPK), suggesting its participation in the phosphate signaling pathway. Thus, to identify other genes involved in metabolic responses to exogenous phosphate sensing and the functioning of the MAPK MAK-2, we performed microarray experiments using a mak-2 knockout strain (Δmak-2) grown under phosphate-shortage conditions by comparing its transcription profile to that of a control strain grown in low- and high-phosphate cultures. These experiments revealed 912 unique differentially expressed genes involved in a number of physiological processes related to phosphate transport, metabolism, and regulation as well as posttranslational modification of proteins, and MAPK signaling pathways. Quantitative Real-time PCR gene expression analysis of 18 selected genes, using independent RNA samples, validated our microarray results. A high Pearson correlation between microarray and quantitative Real-time PCR data was observed. The analysis of these differentially expressed genes in the Δmak-2 strain provide evidence that the mak-2 gene participates in the hierarchical phosphate-signaling pathway in N. crassa in addition to its involvement in other metabolic routes such as the isoprenylation pathway, thus revealing novel aspects of the N. crassa phosphorus-sensing network.

The genome of the anaerobic fungus Orpinomyces sp. strain C1A reveals the unique evolutionary history of a remarkable plant biomass degrader.

Anaerobic gut fungi represent a distinct early-branching fungal phylum (Neocallimastigomycota) and reside in the rumen, hindgut, and feces of ruminant and nonruminant herbivores. The genome of an anaerobic fungal isolate, Orpinomyces sp. strain C1A, was sequenced using a combination of Illumina and PacBio single-molecule real-time (SMRT) technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large noncoding intergenic regions (73.1%), proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in early-branching fungal lineages and/or nonfungal Opisthokonta. These included genes for posttranslational fucosylation, the production of specific intramembrane proteases and extracellular protease inhibitors, the formation of a complete axoneme and intraflagellar trafficking machinery, and a near-complete focal adhesion machinery. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments. This capability, acquired during its separate evolutionary trajectory in the rumen, along with its resilience and invasiveness compared to prokaryotic anaerobes, renders anaerobic fungi promising agents for consolidated bioprocessing schemes in biofuels production.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-ß-1,3-glucanase from Thermotoga petrophila.

1,3-ß-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal ß-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90°C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules.

Comparison of transcriptional and translational changes caused by long-term menadione exposure in Aspergillus nidulans.

Under long-term oxidative stress caused by menadione sodium bisulfite, genome-wide transcriptional and proteome-wide translational changes were compared in Aspergillus nidulans vegetative cells. The comparison of proteomic and DNA microarray expression data demonstrated that global gene expression changes recorded with either flip-flop or dendrimer cDNA labeling techniques supported proteome changes moderately with 40% and 34% coincidence coefficients, respectively. Enzyme levels in the glycolytic pathway were alternating, which was a direct consequence of fluctuating gene expression patterns. Surprisingly, enzymes in the vitamin B2 and B6 biosynthetic pathways were repressed concomitantly with the repression of some protein folding chaperones and nuclear transport elements. Under long-term oxidative stress, the peroxide-detoxifying peroxiredoxins and cytochrome c peroxidase were replaced by thioredoxin reductase, a nitroreductase and a flavohemoprotein, and protein degradation became predominant to eliminate damaged proteins.

Exploring lignin depolymerization by a bi-enzyme system containing aryl alcohol oxidase and lignin peroxidase in aqueous biocompatible ionic liquids.

Enzymatic depolymerization of lignin to produce low molecular weight products requires mild reaction conditions and exhibits higher selectivity compared to thermochemical lignin depolymerization. However, it remains challenging to depolymerize lignin enzymatically, partially due to the low solubility of lignin in aqueous phase. This study aimed to develop a novel approach to combine aqueous lignin extraction with enzymatic lignin depolymerization in biocompatible ionic liquids. A bi-enzyme system containing aryl alcohol oxidase (AAO) and lignin peroxidase (LiP) was developed to depolymerize lignin. Temperature and pH profiles for LiP and AAO were determined. Biocompatibilities of LiP and AAO in different deep eutectic solvents and ionic liquids were investigated. Aqueous cholinium glycinate was found to be an efficient and suitable solvent to solubilize lignin and serve as a biocompatible medium for enzymes to work. LiP and AAO together reduced lignin molecular weight in both solid and liquid phase after enzymatic lignin depolymerization.

Substrate cleavage pattern, biophysical characterization and low-resolution structure of a novel hyperthermostable arabinanase from Thermotoga petrophila.

Arabinan is a plant structural polysaccharide degraded by two enzymes; alpha-l-arabinofuranosidase and endo-1,5-alpha-l-arabinanase. These enzymes are highly diversified in nature, however, little is known about their biochemical and biophysical properties. We have characterized a novel arabinanase (AbnA) isolated from Thermotoga petrophila with unique thermostable properties such as the insignificant decrease of residual activity after incubation up to 90 degrees C. We determined the AbnA mode of operation through capillary zone electrophoresis, which accumulates arabinotriose and arabinobiose as end products after hydrolysis of arabinan-containing polysaccharides. Spectroscopic analyses by Far-UV circular dichroism and intrinsic tryptophan fluorescence emission demonstrated that AbnA is folded and formed mainly by beta-sheet structural elements. In silico molecular modeling showed that the AbnA structure encompasses a five-bladed beta-propeller catalytic core juxtaposed by distorted up-and-down beta-barrel domain. The low-resolution structure determined by small angle X-ray scattering indicated that AbnA is monomeric in solution and its molecular shape is in full agreement with the model.

AtfA bZIP-type transcription factor regulates oxidative and osmotic stress responses in Aspergillus nidulans.

The aim of the study was to demonstrate that the bZIP-type transcription factor AtfA regulates different types of stress responses in Aspergillus nidulans similarly to Atf1, the orthologous 'all-purpose' transcription factor of Schizosaccharomyces pombe. Heterologous expression of atfA in a S. pombe Deltaatf1 mutant restored the osmotic stress tolerance of fission yeast in surface cultures to the same level as recorded in complementation studies with the atf1 gene, and a partial complementation of the osmotic and oxidative-stress-sensitive phenotypes was also achieved in submerged cultures. AtfA is therefore a true functional ortholog of fission yeast's Atf1. As demonstrated by RT-PCR experiments, elements of both oxidative (e.g. catalase B) and osmotic (e.g. glycerol-3-phosphate dehydrogenase B) stress defense systems were transcriptionally regulated by AtfA in a stress-type-specific manner. Deletion of atfA resulted in oxidative-stress-sensitive phenotypes while the high-osmolarity stress sensitivity of the fungus was not affected significantly. In A. nidulans, the glutathione/glutathione disulfide redox status of the cells as well as apoptotic cell death and autolysis seemed to be controlled by regulatory elements other than AtfA. In conclusion, the orchestrations of stress responses in the aspergilli and in fission yeast share several common features, but further studies are needed to answer the important question of whether a fission yeast-like core environmental stress response also operates in the euascomycete genus Aspergillus.

Functional and structural characterization of an α-ʟ-arabinofuranosidase from Thermothielavioides terrestris and its exquisite domain-swapped ß-propeller fold crystal packing.

The fungus Thermothielavioides terrestris plays an important role in the global carbon cycle with enzymes capable of degrading polysaccharides from biomass, therefore an attractive source of proteins to be investigated and understood. From cloning to a three-dimensional structure, we foster a deeper characterization of an α-ʟ-arabinofuranosidase, a glycoside hydrolase from the family 62 (TtAbf62), responsible to release arabinofuranose from non-reducing ends of polysaccharides. TtAbf62 was tested with synthetic (pNP-Araf) and polymeric substrates (arabinan and arabinoxylan), showing optimal temperature and pH (for pNP-Araf) of 30 °C and 4.5-5.0, respectively. Kinetic parameters revealed different specific activity for the three substrates, with a higher affinity for pNP-Araf (KM: 4 ± 1 mM). The hydrolyzing activity of TtAbf62 on sugarcane bagasse suggests high efficiency in the decomposition of arabinoxylan, abundant hemicellulose presented in the sugarcane cell wall. The crystal packing of TtAbf62 reveals an exquisite domain swapping, located at the supramolecular arrangement through a disulfide bond. All crystallographic behaviors go against its monomeric state in solution, indicating a crystal-induced artifact. Structural information will form the basis for further studies aiming the development of optimized enzymatic properties to be used in biotechnological applications.

Improvement of homologous GH10 xylanase production by deletion of genes with predicted function in the Aspergillus nidulans secretion pathway.

Filamentous fungi are important cell factories for large-scale enzyme production. However, production levels are often low, and this limitation has stimulated research focusing on the manipulation of genes with predicted function in the protein secretory pathway. This pathway is the major route for the delivery of proteins to the cell exterior, and a positive relationship between the production of recombinant enzymes and the unfolded protein response (UPR) pathway has been observed. In this study, Aspergillus nidulans was exposed to UPR-inducing chemicals and differentially expressed genes were identified by RNA-seq. Twelve target genes were deleted in A. nidulans recombinant strains producing homologous and heterologous GH10 xylanases. The knockout of pbnA (glycosyltransferase), ydjA (Hsp40 co-chaperone), trxA (thioredoxin) and cypA (cyclophilin) improved the production of the homologous xylanase by 78, 171, 105 and 125% respectively. Interestingly, these deletions decreased the overall protein secretion, suggesting that the production of the homologous xylanase was specifically altered. However, the production of the heterologous xylanase and the secretion of total proteins were not altered by deleting the same genes. Considering the results, this approach demonstrated the possibility of rationally increase the production of a homologous enzyme, indicating that trxA, cypA, ydjA and pbnA are involved in protein production by A. nidulans.

Xylan decomposition by Aspergillus clavatus endo-xylanase.

Agricultural and forest waste products are abundant and low-cost biomass sources useful in renewable fuel energy and feedstock preparation. Hydrolysis of a major biomass component, hemicellulose, is accomplished by the action of endo-xylanases. Reaction products vary in composition and degree of polymerization as a function of both feedstock and the enzyme activities utilized, ranging from monomeric sugars to complex branched polysaccharides. The study herein describes heterologous expression in Aspergillus awamori of a betabeta-(1-4) endo-xylanase isolated from the whole-genome DNA sequence of A. clavatus along with a comprehensive biochemical and functional analysis of the enzyme, including substrate preference and hydrolysis patterns. The A. clavatus xylanase promotes incomplete hydrolysis of xylan substrates resulting in xylobiose, xylotriose and xylotetraose. Incomplete degradation resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitating digestion and absorption of nutrients.

Biochemical and structural insights into a thermostable cellobiohydrolase from Myceliophthora thermophila.

Cellobiohydrolases hydrolyze cellulose, a linear polymer with glucose monomers linked exclusively by ß-1,4 glycosidic linkages. The widespread hydrogen bonding network tethers individual cellulose polymers forming crystalline cellulose, which prevent the access of hydrolytic enzymes and water molecules. The most abundant enzyme secreted by Myceliophthora thermophila M77 in response to the presence of biomass is the cellobiohydrolase MtCel7A, which is composed by a GH7-catalytic domain (CD), a linker, and a CBM1-type carbohydrate-binding module. GH7 cellobiohydrolases have been studied before, and structural models have been proposed. However, currently available GH7 crystal structures only define separate catalytic domains and/or cellulose-binding modules and do not include the full-length structures that are involved in shaping the catalytic mode of operation. In this study, we determined the 3D structure of catalytic domain using X-ray crystallography and retrieved the full-length enzyme envelope via small-angle X-ray scattering (SAXS) technique. The SAXS data reveal a tadpole-like molecular shape with a rigid linker connecting the CD and CBM. Our biochemical studies show that MtCel7A has higher catalytic efficiency and thermostability as well as lower processivity when compared to the well-studied TrCel7A from Trichoderma reesei. Based on a comparison of the crystallographic structures of CDs and their molecular dynamic simulations, we demonstrate that MtCel7A has considerably higher flexibility than TrCel7A. In particular, loops that cover the active site are more flexible and undergo higher conformational fluctuations, which might account for decreased processivity and enhanced enzymatic efficiency. Our statistical coupling analysis suggests co-evolution of amino acid clusters comprising the catalytic site of MtCel7A, which correlate with the steps in the catalytic cycle of the enzyme. DATABASE: The atomic coordinates and structural factors of MtCel7A have been deposited in the Protein Data Bank with accession number 5W11.

Continuous aryl alcohol oxidase production under growth-limited conditions using a trickle bed reactor.

An A. nidulans strain with a pyridoxine marker was used for continuous production of aryl alcohol oxidase (AAO) in a trickle bed reactor (TBR). Modified medium with reduced zinc, no copper, and 5 g/L ascorbic acid that reduced melanin production and increased AAO productivity under growth limited conditions was used. Two air flow rates, 0.11 L/min (0.1 vvm) and 1.1 L/min (1.0 vvm) were tested. More melanin formation and reduced protein productivity were observed with air flow rate of 1.1 L/min. Three random packings were used as support for the fungus inside the TBR column, two of which were hydrophobic and one which was hydrophilic, and three different dilution rates were tested. The use of GEA BCN 030 hydrophobic packing resulted in greater AAO yield and productivity than the other packings. Increasing dilution rates favored melanin formation and citric, lactic and succinic acid accumulation, which decreased AAO yield and productivity.

The Genome of a Thermo Tolerant, Pathogenic Albino Aspergillus fumigatus.

Biotechnologists are interested in thermo tolerant fungi to manufacture enzymes active and stable at high temperatures, because they provide improved catalytic efficiency, strengthen enzyme substrate interactions, accelerate substrate enzyme conversion rates, enhance mass transfer, lower substrate viscosity, lessen contamination risk and offer the potential for enzyme recycling. Members of the genus Aspergillus live a wide variety of lifestyles, some embrace GRAS status routinely employed in food processing while others such as Aspergillus fumigatus are human pathogens. A. fumigatus produces melanins, pyomelanin protects the fungus against reactive oxygen species and DHN melanin produced by the pksP gene cluster confers the gray-greenish color. pksP mutants are attenuated in virulence. Here we report on the genomic DNA sequence of a thermo tolerant albino Aspergillus isolated from rain forest composted floors. Unexpectedly, the nucleotide sequence was 95.7% identical to the reported by Aspergillus fumigatus Af293. Genome size and predicted gene models were also highly similar, however differences in DNA content and conservation were observed. The albino strain, classified as Aspergillus fumigatus var. niveus, had 160 gene models not present in A. fumigatus Af293 and A. fumigatus Af293 had 647 not found in the albino strain. Furthermore, the major pigment generating gene cluster pksP appeared to have undergone genomic rearrangements and a key tyrosinase present in many aspergilli was missing from the genome. Remarkably however, despite the lack of pigmentation A. fumigatus var. niveus killed neutropenic mice and survived macrophage engulfment at similar rates as A. fumigatus Af293.

Functional characterization of a lytic polysaccharide monooxygenase from the thermophilic fungus Myceliophthora thermophila.

Thermophilic fungi are a promising source of thermostable enzymes able to hydrolytically or oxidatively degrade plant cell wall components. Among these enzymes are lytic polysaccharide monooxygenases (LPMOs), enzymes capable of enhancing biomass hydrolysis through an oxidative mechanism. Myceliophthora thermophila (synonym Sporotrichum thermophile), an Ascomycete fungus, expresses and secretes over a dozen different LPMOs. In this study, we report the overexpression and biochemical study of a previously uncharacterized LPMO (MtLPMO9J) from M. thermophila M77 in Aspergillus nidulans. MtLPMO9J is a single-domain LPMO and has 63% sequence similarity with the catalytic domain of NcLPMO9C from Neurospora crassa. Biochemical characterization of MtLPMO9J revealed that it performs C4-oxidation and is active against cellulose, soluble cello-oligosaccharides and xyloglucan. Moreover, biophysical studies showed that MtLPMO9J is structurally stable at pH above 5 and at temperatures up to 50°C. Importantly, LC-MS analysis of the peptides after tryptic digestion of the recombinantly produced protein revealed not only the correct processing of the signal peptide and methylation of the N-terminal histidine, but also partial autoxidation of the catalytic center. This shows that redox conditions need to be controlled, not only during LPMO reactions but also during protein production, to protect LPMOs from oxidative damage.

Prevention of melanin formation during aryl alcohol oxidase production under growth-limited conditions using an Aspergillus nidulans cell factory.

An Aspergillus nidulans cell factory was genetically engineered to produce an aryl alcohol oxidase (AAO). The cell factory initiated production of melanin when growth-limited conditions were established using stationary plates and shaken flasks. This phenomenon was more pronounced when the strain was cultured in a trickle bed reactor (TBR). This study investigated different approaches to reduce melanin formation in fungal mycelia and liquid medium in order to increase the enzyme production yield. Removal of copper from the medium recipe reduced melanin formation in agar cultures and increased enzyme activities by 48% in agitated liquid cultures. Copper has been reported as a key element for tyrosinase, an enzyme responsible for melanin production. Ascorbic acid (0.44g/L) stopped melanin accumulation, did not affect growth parameters and resulted in AAO activity that was more than two-fold greater than a control treatment with no ascorbic acid.

PipeOnline 2.0: automated EST processing and functional data sorting.

Expressed sequence tags (ESTs) are generated and deposited in the public domain, as redundant, unannotated, single-pass reactions, with virtually no biological content. PipeOnline automatically analyses and transforms large collections of raw DNA-sequence data from chromatograms or FASTA files by calling the quality of bases, screening and removing vector sequences, assembling and rewriting consensus sequences of redundant input files into a unigene EST data set and finally through translation, amino acid sequence similarity searches, annotation of public databases and functional data. PipeOnline generates an annotated database, retaining the processed unigene sequence, clone/file history, alignments with similar sequences, and proposed functional classification, if available. Functional annotation is automatic and based on a novel method that relies on homology of amino acid sequence multiplicity within GenBank records. Records are examined through a function ordered browser or keyword queries with automated export of results. PipeOnline offers customization for individual projects (MyPipeOnline), automated updating and alert service. PipeOnline is available at http://stress-genomics.org.