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Filamentous fungi are well known in the industry as producers of large amounts of extracellular proteins. However, production levels of heterologous proteins are often disappointing low. In this paper it is shown that increasing glycosylation is a powerful strategy for increasing production levels of chymosin in filamentous fungi. Two different concepts based on glycosylation were tested. First, we improved a poorly used N-glycosylation site within the prochymosin molecule. The resulting highly glycosylated chymosin molecule was expressed in Aspergillus niger. It was shown that production of the glycosylated protein was much more efficient, giving a yield increase of more than 100% compared to production of the native chymosin molecule. In an alternative strategy the N-glycosylation site was located outside of the native chymosin molecule, on a linker separating prochymosin from its carrier molecule. Also in this case significantly increased production levels were obtained. This strategy might offer a powerful tool for increasing production levels of other heterologous proteins as well.
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