Carsten Mailand Hjort

Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes

Expression of several Aspergillus niger genes encoding major secreted, but not vacuolar, protease genes including the major acid protease gene pepA, was shown to be affected in the previously isolated A. niger protease mutant, AB1.13 [Mattern, I.E., van Noort, J.M., van den Berg, P., Archer, D.A., Roberts, I.N., Hondel, C.A.M.J.J., 1992. Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Molecular & General Genetics 2, 332-336]. Complementation cloning of the putative protease-regulatory gene affected in this mutant was accomplished using a functional selection approach based on the use of the A. nidulans amdS selection marker driven by the A. niger pepA promoter. As expected the PpepA::amdS selection marker is not expressed in the mutant. Introduction of a self-replicating cosmid library into the mutant strain carrying the PpepA::amdS marker allowed selection of AmdS+ transformants functionally complementing the proposed regulatory mutation. Analysis of complementing cosmid clones revealed that the complementing sequences contained a gene encoding a member of the fungal-specific Zn2Cys6-binuclear cluster protein family. Sequence comparison of the encoded protein, PrtT, showed that it has homologues among different Aspergillus species. The A. oryzae homologue was shown to govern expression of the major alkaline protease AlpA and neutral protease Np1 in this species. In contrast to several other pathway specific regulators, such as AmyR and XlnR, no PrtT orthologues could be found in any other non-Aspergillus (or related) species and surprisingly, also not in Aspergillus nidulans. Interestingly, in all Aspergillus species carrying a prtT orthologue the gene is tightly clustered to a completely syntenous region carrying an amylolytic gene cluster including another Zn2Cys6-binuclear cluster protein, AmyR. Northern analysis of the A. niger prtT gene showed (constitutive) expression from two upstream promoters about 700 bp apart. The presence of several short upstream open reading frames downstream of both the distal and the proximal transcription start point of the prtT gene suggests regulation at the post-translational level. Also regulation at the level of differential splicing is suggested by the fact that several Aspergillus EST databases carry a considerable fraction of clones in which in frame intron sequences are retained.

Cloning and characterization of oah, the gene encoding oxaloacetate hydrolase in Aspergillus niger.

Abstract The enzyme oxaloacetate hydrolase (EC 3.7.1.1), which is involved in oxalate formation, was purified from Aspergillus niger. The native enzyme has a molecular mass of 360–440 kDa, and the denatured enzyme has a molecular mass of 39 kDa, as determined by gel electrophoresis. Enzyme activity is maximal at pH 7.0 and 45 °C. The fraction containing the enzyme activity contained at least five proteins. The N-terminal amino acid sequences of four of these proteins were determined. The amino acid sequences were aligned with EST sequences from A. niger, and an EST sequence that showed 100% identity to all four sequences was identified. Using this EST sequence the gene encoding oxaloacetate hydrolase (oah) was cloned by inverse PCR. It consists of an ORF of 1227 bp with two introns of 92 and 112 bp, respectively. The gene encodes a protein of 341 amino acids with a molecular mass of 37 kDa. Under the growth conditions tested, the highest oah expression was found for growth on acetate as carbon source. The gene was expressed only at pH values higher than 4.0.

Altering the expression of two chitin synthase genes differentially affects the growth and morphology of Aspergillus oryzae

In Aspergillus oryzae, one full-length chitin synthase (chsB) and fragments of two other chitin synthases (csmA and chsC) were identified. The deduced amino acid sequence of chsB was similar (87% identity) to chsB from Aspergillus nidulans, which encodes a class III chitin synthase. The sequence obtained for csmA indicated that it had high similarity to class V chitin synthases. chsB and csmA disruption strains and a strain in which chsB transcription was controlled were constructed using the nitrite reductase (niiA) promoter. The strains were examined during hyphal growth by Northern analysis, analysis of the cell-wall composition and growth in the presence of Calcofluor white (CFW). The chsB disrupted strain and the uninduced p(niiA)-chsB strain exhibited hyperbranching, they had a lower level of conidiation than the wild-type and were sensitive to CFW at 50 mg l(-1). When chsB transcription was induced in the strain containing the p(niiA)-chsB construct, the strain displayed wild-type morphology on solid medium and at sub-maximum growth rates but the wild-type morphology was not fully restored during rapid growth in batch cultivation. The csmA disruption strain displayed morphological abnormalities, such as ballooning cells, intrahyphal hyphae and conidial scars. The growth was severely inhibited in the presence of 10 mg CFW l(-1). In none of the constructed strains did the cell-wall composition differ from the wild-type. Northern analysis indicated no change in the transcription of the chitin synthase genes csmA and chsC when chsB expression was altered, and there was no change in the transcription of chsB and chsC when csmA was disrupted.

Genome-scale analysis of the high-efficient protein secretion system of Aspergillus oryzae

BackgroundThe koji mold, Aspergillus oryzae is widely used for the production of industrial enzymes due to its particularly high protein secretion capacity and ability to perform post-translational modifications. However, systemic analysis of its secretion system is lacking, generally due to the poorly annotated proteome.ResultsHere we defined a functional protein secretory component list of A. oryzae using a previously reported secretory model of S. cerevisiae as scaffold. Additional secretory components were obtained by blast search with the functional components reported in other closely related fungal species such as Aspergillus nidulans and Aspergillus niger. To evaluate the defined component list, we performed transcriptome analysis on three α-amylase over-producing strains with varying levels of secretion capacities. Specifically, secretory components involved in the ER-associated processes (including components involved in the regulation of transport between ER and Golgi) were significantly up-regulated, with many of them never been identified for A. oryzae before. Furthermore, we defined a complete list of the putative A. oryzae secretome and monitored how it was affected by overproducing amylase.ConclusionIn combination with the transcriptome data, the most complete secretory component list and the putative secretome, we improved the systemic understanding of the secretory machinery of A. oryzae in response to high levels of protein secretion. The roles of many newly predicted secretory components were experimentally validated and the enriched component list provides a better platform for driving more mechanistic studies of the protein secretory pathway in this industrially important fungus.

High level Aspergillus production of proteins

on host physiology The organisers would like to thank Novozymes Delta Ltd who generously supported the meeting. Meeting

Industrial enzyme production for food applications

Publisher Summary This chapter highlights that enzymes have been used for food processing for as long as man has processed foods. However, deliberate use of enzymes is relatively new. As it has been learned how to use enzymes for food more deliberately, the need for controlled methods of production of the enzymes has become more and more necessary. A very significant number of the food enzymes produced today are based on recombinant production strains. However, acceptance of food products based on recombinant DNA technology is still not absolute. For many food segments, the food producers and their customers still demand products based on non-recombinant raw materials, additives, and processing aids. As long as non-recombinant products are needed to serve some segments, it is hard to anticipate any significant development of the product range and of the technology used to produce enzymes for the segments. Enzyme producers address the concerns of the regulatory authorities, the food producers, and the end consumers in a number of ways. In relation to construction of production organisms and production processes, a number of changes have already occurred.