In Aspergillus niger, one of the most important industrial micro-organisms, strong anaplerotic reactions were found to be responsible for high productivity of primary metabolites. Two enzymes, 6-phosphofructo-1-kinase (PFK), a key regulatory enzyme of glycolytic pathway, and alternative oxidase, a membrane protein that uncouples NADH re-oxidation from ATP formation, have been recognised to play a key role in replenishing citric acid cycle intermediates. After a transfer of genes coding for both enzymes into Aspergillus terreus cells, a similar effect on the level of tricarboxylyc acid cycle intermediates was expected to be achieved and concomitantly increased productivity and/or yield of specific A. terreus bio-products. The genes pfkA coding for 6-phosphofructo-1-kinase and aox1 carrying the information for synthesis of alternative oxidase were isolated from A. niger gene library by the aid of the known sequence. Namely, both genes have been previously isolated, cloned and sequenced by other laboratories. The genes together with their native promoter and terminator regions have been integrated into appropriate vectors. Heterologous pfkA gene was introduced into A. terreus protoplasts by co-transformation with a plasmid carrying hygB gene as a marker for hygromycine resistance, while double transformants were isolated after additional co-insertion of a plasmids carrying aox1 and amdS gene. The integration of genes was confirmed by standard recombinant DNA techniques. In cell free extract of the modified strain higher specific activity of PFK was detected by comparing to the parental strain and significantly higher sensitivity of double transformant was recorded toward SHAM, a specific inhibitor of alternative oxidase. The obtained results indicate that A. niger genes could be efficiently expressed in other Aspergillus species, while their effect on primary metabolism will have to await further studies.
Full conference title:
23rd Fungal Genetics Conference
- Fungal Genetics Conference 23rd (2002)