Characterization and expression of a genomic pectin methyl esterase-encoding gene in Aspergillus niger
Related Experiment Videos
N Q Khanh1, E Ruttkowski, K Leidinger
1Institut für Biochemie, Technische Hochschule Darmstadt, F.R.G.
Abstract:
The genomic pectin methylesterase (PME)-encoding gene (pmeA) from Aspergillus niger strain RH5344 was cloned by probing a genomic DNA library with a cDNA coding for PME. The recombinant phage clone was isolated and a 6-kb HindIII fragment was subcloned and characterized. The gene consists of seven exons and six introns. The nucleotide sequences of the coding regions were identical to those found in the pmeA cDNA. Cotransformation of A. niger was achieved with the vector, pAN7-1, and transformants were then tested for PME production. Transformants which produced more PME than the untransformed recipient strain were subjected to Southern-blot and Northern-blot analysis. The results show that there is a reasonable correlation between gene copy number, mRNA levels and PME production. PME was produced by A. niger transformants in an active 43-kDa form, which is similar to that of the mature protein isolated from the strain, RH5344. On the basis of the results of affinity labeling of PME with sugar-specific lectins and the amino acid sequence data, it has been revealed that PME is a glycoprotein and the protein-bound glycans are oligosaccharides with a high mannose content.
Insights
Researchers cloned the pectin methylesterase (PME) gene from Aspergillus niger. Increased gene copy number and mRNA levels correlated with higher PME production in transformants.
Area of Science:
- Molecular Biology
- Biochemistry
- Mycology
Background:
- Pectin methylesterase (PME) plays a crucial role in plant cell wall modification.
- Understanding the genetic regulation of PME in filamentous fungi like Aspergillus niger is important for biotechnological applications.
Purpose of the Study:
- To clone and characterize the genomic pectin methylesterase (PME)-encoding gene (pmeA) from Aspergillus niger strain RH5344.
- To investigate the relationship between gene copy number, mRNA levels, and PME production in A. niger transformants.
Main Methods:
- Genomic DNA library screening using PME cDNA probe.
- Subcloning and characterization of the pmeA gene.
- Cotransformation of A. niger with the pmeA gene and analysis of PME production.
- Southern-blot and Northern-blot analysis to assess gene copy number and mRNA levels.
Main Results:
- The pmeA gene consists of seven exons and six introns, with coding regions identical to the cDNA sequence.
- A. niger transformants showed increased PME production compared to the wild-type strain.
- A positive correlation was observed between gene copy number, mRNA levels, and PME production.
- The produced PME is an active 43-kDa glycoprotein with high mannose content.
Conclusions:
- The genomic pmeA gene from A. niger has been successfully cloned and characterized.
- Gene amplification and increased transcription lead to enhanced PME production in A. niger.
- PME from A. niger is a glycoprotein, suggesting potential post-translational modifications affecting its function.