C. Muller; C. M. Hjort; K. Hansen; J. Nielsen.
"Altering the expression of two chitin synthase genes differentially affects the growth and morphology of Aspergillus oryzae."
Microbiology (Reading), 148 (12), 4025-4033 (2002)
Abstract
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.
H. Pedersen; B. Christensen; C. Hjort; J. Nielsen.
"Construction and characterization of an oxalic acid nonproducing strain of Aspergillus niger."
Metab Eng, 2(1), 34-41 (2000)
Abstract
Aspergillus niger produces oxalic acid as a by-product which causes problems with downstream processing of industrial enzymes. To overcome this problem the oah gene encoding oxaloacetate hydrolase (EC 3.7.1.1) was disrupted in a glucoamylase-producing strain of A. niger and the resulting strain was incapable of producing oxalic acid. The strain with the disrupted gene was compared with the wild-type strain producing oxalic acid in batch cultivations. The specific growth rate of both strains was 0.20 h(-1). The citric acid yields were identical, but the glucoamylase yield was only 50% in the disruptant compared with the wild-type strain. Batch experiments with 13C-labeled glucose as substrate were carried out to determine the metabolic fluxes through the central metabolism. The two strains had almost identical metabolic fluxes, which suggested that it was possible to disrupt the oah gene without pleiotropic consequences. The flux through the pentose phosphate pathway was around 60% of the glucose uptake for both strains, which suggested that a sufficient supply of NADPH was available for biosynthesis.
P.L. Jørgense n; M. Tangney; P.E. Pedersen; S. Hastrup ; B. Diderichsen; S.T. Jørgensen.
"Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique."
Appl. Environ. Microbiol. 66, 825-827 (2000)
Abstract
A gene encoding an alkaline proteinase (subtilisin), commercially known as Savinase®, was cloned from alkalophilic Bacillus lentus NCIB 10309 into B. subtilis DN497, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the proteinase gene on the chromosome. From these studies, an improved genetic technique for integration and gene amplification was developed using the temp. sensitive plasmid pE194.
K. L. Petersen; J Lehmbeck; T. Christensen.
"A new transcriptional activator for amylase genes in Aspergillus."
Mol Gen Genet, 262, 668-676 (1999)
Abstract
We have cloned a regulatory gene for amylase synthesis in Aspergillus oryzae. This gene, amyR, encodes a 604-amino acid transcriptional activator with a Cys6 zinc cluster, that shows extensive homology to the DNA binding domain of GAL4 from Saccharomyces cerevisiae. The DNA binding domain of amyR binds to two types of sequences found in a number of promoters from Aspergillus genes coding for starch-degrading enzymes. One type of binding site is characterized by two CGG triplets separated by eight nucleotides. The other type has only one CGG trip let, which is followed by the sequence AAATTTAA.