1 Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark2 Department of Systems Biology, Technical University of Denmark3 unknown4 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark
Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Y-xp (total)), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7 +/- 0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3 +/- 0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (r(p total), the sum of extracellular and intracellular lipase productivity) was found to be 1.60 +/- 0.81 KU/g DW/h in maltose-limited chemostats at a dilution rate of 0.08 h(-1), compared with a total specific lipase productivity of 1.10 +/- 0.41 KU/g DW/h in glucose-limited chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.
Applied Microbiology and Biotechnology, 2004, Vol 65, p. 714-719