The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised.

A classification of glycosyl hydrolases based on amino acid sequence similarities.

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.

Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen causing disease in a wide range of plants. This review summarizes current knowledge of mechanisms employed by the fungus to parasitize its host with emphasis on biology, physiology and molecular aspects of pathogenicity. In addition, current tools for research and strategies to combat S. sclerotiorum are discussed. Taxonomy: Sclerotinia sclerotiorum (Lib.) de Bary: kingdom Fungi, phylum Ascomycota, class Discomycetes, order Helotiales, family Sclerotiniaceae, genus Sclerotinia. Identification: Hyphae are hyaline, septate, branched and multinucleate. Mycelium may appear white to tan in culture and in planta. No asexual conidia are produced. Long-term survival is mediated through the sclerotium; a pigmented, multi-hyphal structure that can remain viable over long periods of time under unfavourable conditions for growth. Sclerotia can germinate to produce mycelia or apothecia depending on environmental conditions. Apothecia produce ascospores, which are the primary means of infection in most host plants. Host range: S. sclerotiorum is capable of colonizing over 400 plant species found worldwide. The majority of these species are dicotyledonous, although a number of agriculturally significant monocotyledonous plants are also hosts. Disease symptoms: Leaves usually have water-soaked lesions that expand rapidly and move down the petiole into the stem. Infected stems of some species will first develop dark lesions whereas the initial indication in other hosts is the appearance of water-soaked stem lesions. Lesions usually develop into necrotic tissues that subsequently develop patches of fluffy white mycelium, often with sclerotia, which is the most obvious sign of plants infected with S. sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen.

Aspergillus niger is one of the most important microorganisms used in biotechnology. It has been in use already for many decades to produce extracellular (food) enzymes and citric acid. In fact, citric acid and many A. niger enzymes are considered GRAS by the United States Food and Drug Administration. In addition, A. niger is used for biotransformations and waste treatment. In the last two decades, A. niger has been developed as an important transformation host to over-express food enzymes. Being pre-dated by older names, the name A. niger has been conserved for economical and information retrieval reasons and there is a taxonomical consensus based on molecular data that the only other common species closely related to A. niger in the Aspergillus series Nigri is A. tubingensis. A. niger, like other filamentous fungi, should be treated carefully to avoid the formation of spore dust. However, compared with other filamentous fungi, it does not stand out as a particular problem concerning allergy or mycopathology. A few medical cases, e.g. lung infections, have been reported, but always in severely immunocompromised patients. In tropical areas, ear infections (otomycosis) do occur due to A. niger invasion of the outer ear canal but this may be caused by mechanical damage of the skin barrier. A. niger strains produce a series of secondary metabolites, but it is only ochratoxin A that can be regarded as a mycotoxin in the strict sense of the word. Only 3-10% of the strains examined for ochratoxin A production have tested positive under favourable conditions. New and unknown isolates should be checked for ochratoxin A production before they are developed as production organisms. It is concluded, with these restrictions, that A. niger is a safe production organism.

https://link.springer.com/content/pdf/10.1007%2Fs00253-002-1032-6.pdf

On the safety of Aspergillus niger - a review

Plant cell wall consists mainly of the large biopolymers cellulose, hemicellulose, lignin and pectin. These biopolymers are degraded by many microorganisms, in particular filamentous fungi, with the aid of extracellular enzymes. Filamentous fungi have a key role in degradation of the most abundant biopolymers found in nature, cellulose and hemicelluloses, and therefore are essential for the maintenance of the global carbon cycle. The production of plant cell wall degrading enzymes, cellulases, hemicellulases, ligninases and pectinases, is regulated mainly at the transcriptional level in filamentous fungi. The genes are induced in the presence of the polymers or molecules derived from the polymers and repressed under growth conditions where the production of these enzymes is not necessary, such as on glucose. The expression of the genes encoding the enzymes is regulated by various environmental and cellular factors, some of which are common while others are more unique to either a certain fungus or a class of enzymes. This review summarises our current knowledge on the transcriptional regulation, focusing on the recently characterized transcription factors that regulate genes coding for enzymes involved in the breakdown of plant cell wall biopolymers.

Transcriptional regulation of plant cell wall degradation by filamentous fungi

Aspergillusniger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage λEMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western-blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A. nidulans transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pgaC gene. This gene was sequenced and the protein-coding region was found to be interrupted by three introns; the different intron/exon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383-amino-acid prepro-protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N-terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A. niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to represent a binding site for a regulatory protein as it shows a high similarity to the yeast CYC1 upstream activation site recognized by the HAP2/3/4 activation complex.

The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes

The structure and expression of the polygalacturonase-encoding pgaII genes of two recently recognized species, Aspergillus niger and Aspergillus tubigensis, was investigated. While the structure of the pgaII genes is very similar, showing 83% DNA sequence identity and 94% identity at the amino acid level, they have diverged significantly. The NH2-terminal sequence suggests that these PGs are made as pre pro-proteins and the secretory propeptide of the PGII precursors shows sequence homology with some other fungal pro-peptides. The expression of the pgaII genes is strongly regulated by the carbon source and the A. tubigensis gene is expressed and regulated in A. niger transformants. The low similarity of the fungal PGs with those of bacterial and plant origin is discussed in relation to the possible functional role of specific amino acids.

Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2890%2981066-W

Molecular cloning, nucleotide sequence and expression of the gene encoding prepro‐polygalacturonasell of Aspergillus niger

The filamentous fungus Aspergillus niger produces several endopolygalacturonases that are involved in the degradation of pectin. PGI, the enzyme representing the second most abundant activity in a commercial enzyme preparation, was further characterized and the corresponding gene was isolated. The nucleotide sequence of the pgaI gene was determined and the protein coding region was found to be interrupted by two short introns, one of which has a unusual donor splice site. The deduced 368 amino acids long protein with a putative prepropeptide of 31 amino acids shows 60% sequence identity to PGII in the mature protein. PGI overproducing A. niger strains were obtained by cotransformation with the cloned gene.

Identification and characterization of a second polygalacturonase gene of Aspergillus niger

Aspergillus niger pyrA co-transformants with multiple copies of the gene (pgaII) encoding the major endopolygalacturonase were characterized in detail. Typically, these transformants produced tenfold or more polygalacturonase from plasmids that had integrated in most cases at ectopic sites, in comparison to the untransformed strain. Some mitotic instability was observed upon application of a positive selection procedure for reversion of the pyrA marker. Analysis of these strains indicated that the most frequent event involved is the excision of part of the array of tandemly integrated plasmids, without “scrambling” of the plasmids remaining in the chromosome. From promoter deletion analysis it was concluded that the pgaII gene is subject to positive control. The putative positive regulatory protein appears not to be limiting for overexpression of the pgaII gene.

Hendrik J. D. Bussink

Papers

The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes

Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II.

Molecular cloning, nucleotide sequence and expression of the gene encoding prepro‐polygalacturonasell of Aspergillus niger

Identification and characterization of a second polygalacturonase gene of Aspergillus niger

Characterization of polygalacturonase-overproducing Aspergillus niger transformants