Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.

Identification of regulatory mutants of Aspergillus aculeatus affected in rhamnogalacturonan hydrolase expression.

Rhamnogalacturonan hydrolase expression in A. aculeatus can be induced by pectin, but also by a combination of two constituent monosaccharides of pectin, rhamnose and galacturonic acid. The rhgA promoter was fused to the A. niger glucose oxidase coding sequence and a single copy of the hybrid gene was integrated at the rhgA locus in the genome of A. aculeatus. The gene product was subsequently used as reporter in a screening assay for the selection of rhamnogalacturonan hydrolase-overproducing mutant strains. At least four of the mutations were recessive and could be assigned to different loci. One mutation (rgr25) showed linkage with the rhgA locus. Inducible rhamnogalacturonan hydrolase expression levels of about 5-10 times that in the wild-type were found in the mutants rgr48, rgr25 and rgr34 after growth on a combination of rhamnose and galacturonic acid with or without fructose as a carbon source. In mutant rgr48 elevated levels of rhgA transcription were found.

Cloning, sequence and expression of the gene coding for rhamnogalacturonase of Aspergillus aculeatus; a novel pectinolytic enzyme.

Rhamnogalacturonase was purified from culture filtrate of Aspergillus aculeatus after growth in medium with sugar-beet pulp as carbon source. Purified protein was used to raise antibodies in mice and with the antiserum obtained a gene coding for rhamnogalacturonase (rhgA) was isolated from a lambda cDNA expression library. The cloned rhgA gene has an open-reading frame of 1320 base pairs encoding a protein of 440 amino acids with a predicted molecular mass of 45 962 Da. The protein contains a potential signal peptidase cleavage site behind Gly-18 and three potential sites for N-glycosylation. Limited homology with A. niger polygalacturonase amino acid sequences is found. A genomic clone of rhgA was isolated from a recombinant phage lambda genomic library. Comparison of the genomic and cDNA sequences revealed that the coding region of the gene is interrupted by three introns. Furthermore, amino acid sequences of four different peptides, derived from purified A. aculeatus rhamnogalacturonase, were also found in the deduced amino acid sequence of rhgA. A. aculeatus strains overexpressing rhamnogalacturonase were obtained by cotransformation using either the A. niger pyrA gene or the A. aculeatus pyrA gene as selection marker. For expression of rhamnogalacturonase in A. awamori the A. awamori pyrA gene was used as selection marker. Degradation patterns of modified hairy regions, determined by HPLC, show the recombinant rhamnogalacturonase to be active, and the enzyme was found to have a positive effect in the apple hot-mash liquefaction process.

Cloning, characterization and overexpression of the phytase-encoding gene (phyA) of Aspergillus niger.

Phytase catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. A gene (phyA) of Aspergillus niger NRRL3135 coding for extracellular, glycosylated phytase was isolated using degenerate oligodeoxyribonucleotides deduced from phytase amino acid (aa) sequences. Nucleotide (nt) sequence analysis of the cloned region revealed the presence of an open reading frame coding for 467 aa and interrupted once by an intron of 102 bp in the 5' part of the gene. The start codon is followed by a sequence coding for a putative signal peptide. Expression of phyA is controlled at the level of mRNA accumulation in response to inorganic phosphate levels. After cell growth in low-phosphate medium, a transcript of about 1.8 kb was visualized. Transcription of phyA initiates at at least seven start points within a region located 45-25 nt upstream from the start codon. In transformants of A. niger, expression of multiple copies of phyA resulted in up to more than tenfold higher phytase levels than in the wild-type strain.

A major immunogenic 36,000-molecular-weight antigen from Mycobacterium leprae contains an immunoreactive region of proline-rich repeats.

The 36,000-molecular-weight antigen (36K antigen) of Mycobacterium leprae is a major immunogenic protein carrying common and specific antigenic determinants recognized by antibodies and T cells in leprosy patients. Recombinant DNA clones containing the complete gene coding for the 36 K antigen, designated in this paper as PRA, were isolated from both lambda gt11 and cosmid libraries of the M. leprae genome. The DNA sequence of the pra gene coded for a polypeptide of 249 amino acids with a predicted molecular mass of 26,299 daltons. The deduced amino acid sequence revealed a proline-rich (42%) amino-terminal region containing a number of repeated sequences similar or identical to the sequence PGGSYPPPPP. The reactivity of four monoclonal antibodies (F47-9, F67-1, F67-5, and F126-5) was directed to this proline-rich region of the PRA protein. DNA sequence and immunological data indicated that the lambda gt11 recombinant Y3180, which was previously isolated by using antibody F47-9 (R. A. Young, V. Mehra, D. Sweetser, T. Buchanan, J. Clark-Curtiss, R. W. Davis, and B. R. Bloom, Nature (London) 316:450-452, 1985), specifies a fusion protein unrelated to PRA but containing a similar epitope recognized by F47-9.