Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation.

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.     

Carbon catabolite repression of the Aspergillus nidulans xlnA gene

Expression of the Aspergillus nidulans 22 kDa endoxylanase gene, xlnA , is controlled by at least three mechanisms: specific induction by xylan or xylose; carbon catabolite repression (CCR); and regulation by ambient pH. Deletion analysis of xlnA upstream sequences has identified two positively acting regions: one that mediates specific induction by xylose; and another that mediates the influence of ambient pH and contains two PacC consensus binding sites. The extreme derepressed mutation creA^d 30 results in considerable, although not total, loss of xlnA glucose repressibility, indicating a major role for CreA in its CCR. Three consensus CreA binding sites are present upstream of the structural gene. Point mutational analysis using reporter constructs has identified a single site, xlnA .C1, that is responsible for direct CreA repression in vivo . Using the creA^d 30 derepressed mutant background, our results indicate the existence of indirect repression by CreA.     

Efficient hydrolysis of hemicellulose by a Fusarium graminearum xylanase blend produced at high levels in Escherichia coli

A Fusarium graminearum-based enzyme blend for the efficient hydrolysis of hemicellulose, a crucial step for competitive bioethanol production, is described. The heretofore-uncharacterized endo-1,4-beta-xylanase (XylD), 1,4-beta-xylosidase (XyloA), and bifunctional xylosidase/arabinofuranosidase (Xylo/ArabA) were produced at high levels in Escherichia coli (10-38 mg/l). They displayed compatible pH and temperature-dependences, allowing their utilization for simultaneous substrate digestions. Monosaccharide analysis indicated a strong positive synergism between the enzymes during the degradation of oat spelt xylan. Two units of each protein catalyzed the release of 61% and 15% of the total amount of available d-xylose and l-arabinose, respectively, in only 4 h. The detailed cooperative mechanism of the three hydrolases was elucidated by polysaccharide analysis using carbohydrate gel electrophoresis (PACE) and the enzymes were shown to be suitable for the partial hydrolysis of pretreated crude plant biomass.     

Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding beta-xylosidase from the thermophilic fungus Talaromyces emersonii

The gene coding for beta-xylosidase, bxl1, has been cloned from the thermophilic filamentous fungus, Talaromyces emersonii. This is the first report of a hemicellulase gene from this novel source. At the genomic level, bxl1 consists of an open reading frame of 2388 nucleotides with no introns that encodes a putative protein of 796 amino acids. The bxl1 translation product contains a signal peptide of 21 amino acids that yields a mature protein of 775 amino acids, with a predicted molecular mass of 86.8 kDa. The deduced amino acid sequence of bxl1 exhibits considerable homology with the primary structures of the Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, and Trichoderma reesei beta-xylosidase gene products, and with some beta-glucosidases, all of which have been classified as Family 3 glycosyl hydrolases. Northern blot analysis of the bxl1 gene indicates that it is induced by xylan and methyl-beta-D-xylopyranoside. D-Xylose induced expression of bxl1 but was shown to repress induction of the gene at high concentrations. The presence of six CreA binding sites in the upstream regulatory sequence (URS) of the bxl1 gene indicates that the observed repression by D-glucose may be mediated, at least partly, by this catabolite repressor.     

Engineering the central pathways in Lactococcus lactis: functional expression of the phosphofructokinase (pfk) and alternative oxidase (aox1) genes from Aspergillus niger in Lactococcus lactis facilitates improved carbon conversion rates under oxidizing conditions

The present work describes a novel central pathway engineering method that has been designed with the aim to increase the carbon conversion rates under oxidizing conditions in L. lactis fermentations. The nisin producer L. lactis ATCC11454 strain has been genetically engineered by cloning a truncated version of the phosphofructokinase gene (pfk13), along with the pkaC, encoding for the catalytic subunit of cAMP-dependent protein kinase, and the alternative oxidase (aox1) genes of A. niger. Functional expression of the above genes resulted in enhanced PFK activity and the introduction of AOX activity and alternative respiration in the presence of a source of heme in the substrate, under fully aerobic growth conditions. The constructed strain is capable of fermenting high concentrations of glucose as was demonstrated in a series of glucostat fed-batch fermentations with glucose levels maintained at 55, 138 and 277 mM. The high maximum specific uptake rate of glucose of 1.8 mMs(-1)gCDW(-1) at 277 mM glucose is characteristic of the improved ability of the microorganism to handle elevated glucose concentrations under conditions otherwise causing severe reduction of PFK activity. The increased carbon flow through glycolysis led to increased protein synthesis that was reflected in increased biomass and nisin levels. The pfk 13-pkaC-aox1-transformant strain's fermentation at 277 mM glucose gave a final biomass concentration of 7.5 g/l and nisin activity of 14,000 IU/ml which is, compared to the parental strain's production levels at its optimal 55 mM glucose, increased by a factor of 2.34 for biomass and 4.37 for nisin.     

Induction of cellulose in Schizophyllum commune: thiocellobiose as a new inducer.

Several mono-, di, tetra-, and polysaccharides were screened for their ability to induced cellulase production by the tetrapolar hymenomycete Schizophyllum commune. Out of 21 carbohydrates screened, 4 (thiocellobiose, carboxymethylcellulose, cellobiose, and xylan) induced all three enzymes tested (carboxymethylcellulase, beta-glucosidase, and xylanase). The inducing effect increased with rising concentrations of the inducers up to a certain value, beyond which there was either a leveling off or a decrease of the enzymatic activities. The most powerful inducer, thiocellobiose, showed the highest activity at 0.5 mM. Cellobiose, carboxymethylcellulose, and xylan showed their highest activities at 1 mM and 1%, respectively. Surprisingly, sophorose did not enhance enzyme production. The enzymatic activities were monitored over a period of 24 h. Thiocelloboise elicited a response immediately after incubation, but with all other inducers there was a latency period before their effect could be measured. High-performance liquid chromatography showed no hydrolysis of thiocellobiose when incubated in the presence of S. commune extracellular enzymes.     

Utilization of Xylan by Yeasts and Its Conversion to Ethanol by Pichia stipitis Strains

Yeasts able to grow on d-xylose were screened for the ability to hydrolyze xylan. Xylanase activity was found to be rare; a total of only 19 of more than 250 strains yielded a positive test result. The activity was localized largely in the genus Cryptococcus and in Pichia stipitis and its anamorph Candida shehatae. The ability to hydrolyze xylan was generally uncoupled from that to hydrolyze cellulose; only three of the xylan-positive strains also yielded a positive test for cellulolytic activity. Of the 19 xylanolytic strains, 2, P. stipitis CBS 5773 and CBS 5775, converted xylan into ethanol, with about 60% of a theoretical yield computed on the basis of the amount of d-xylose present originally that could be released by acid hydrolysis.     

CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA.

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.     

Exploration of members of Aspergillus sections Nigri, Flavi, and Terrei for feruloyl esterase production

The ability of members of Aspergillus sections Nigri, Flavi, and Terrei to produce feruloyl esterases was studied according to their substrate specificity against synthetic methyl esters of hydroxycinnamic acids. Type A feruloyl esterases (FAEA), induced during growth on cereal-derived products, show a preference for the phenolic moiety of substrates that contain methoxy substitutions, as found in methyl sinapinate, whereas type B feruloyl esterases (FAEB) show a preference for the phenolic moiety of substrates that contain hydroxyl substitutions, as occurs in methyl caffeate. All the strains of Aspergillus section Nigri (e.g., A. niger and A. foetidus) were able to produce feruloyl esterases with activity profiles similar to those reported for FAEA and FAEB of A. niger when grown on oat-spelt xylan and sugar beet pulp, respectively. The two genes encoding these proteins, faeA and faeB, were identified by Southern blot analysis. The strains of Aspergillus sections Flavi (e.g., A. flavus, A. flavo-furcatus, and A. tamarii) and Terrei (e.g., A. terreus) were able to produce type A and type B enzymes. faeA was revealed in genomic DNA of these strains, and FAEA was determined by immunodetection in cultures grown in oat-spelt xylan. In addition, type B enzymes, not related to faeB, were efficiently induced by oat-spelt xylan and exhibited very original activity profiles on sugar beet pulp. This work confirms that the members of the genus Aspergillus are good feruloyl esterase producers.     

Expression profiling of pectinolytic genes from Aspergillus niger

The expression of 26 pectinolytic genes from Aspergillus niger was studied in a wild type strain and a CreA derepressed strain, under 16 different growth conditions, to obtain an expression profile for each gene. These expression profiles were then submitted to cluster analysis to identify subsets of genes with similar expression profiles. With the exception of the feruloyl esterase encoding genes, all genes were expressed in the presence of D-galacturonic acid, polygalacturonate, and/or sugar beet pectin. Despite this general observation five distinct groups of genes were identified. The major group consisted of 12 genes of which the corresponding enzymes act on the pectin backbone and for which the expression, in general, is higher after 8 and 24 h of incubation, than after 2 or 4 h. Two other groups of genes encoding pectin main chain acting enzymes were detected. Two additional groups contained genes encoding L-arabinose and D-galactose releasing enzymes, and ferulic acid releasing enzymes, respectively. The genes encoding beta-galactosidase and the L-arabinose releasing enzymes were not only expressed in the presence of D-galacturonic acid, but also in the presence of L-arabinose, suggesting that they are under the control of two regulatory systems. Similarly, the rhamnogalacturonan acetylesterase encoding gene was not only expressed in the presence of D-galacturonic acid, polygalacturonate and sugar beet pectin, but also in the presence of L-rhamnose. The data presented provides indications for a general pectinolytic regulatory system responding to D-galacturonic acid or a metabolite derived from it. In addition, subsets of pectinolytic genes are expressed in response to the presence of L-arabinose, L-rhamnose or ferulic acid.