Abstract
BackgroundThe secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway.ResultsUsing the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells.ConclusionsIn summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.
Highlights
The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway
Effect of casein on extracellular bovine chymosin production using five different recombinant strains In initial studies, we observed that casein, which is partially hydrolyzed in the A. awamori cultures, exerts a strong stimulatory effect on chymosin production
In the third strain (TAPL-4), the chy gene is expressed from the amyB promoter, but the pre sequence of chymosin has been replaced by the leader peptide (21 amino acids) of the A. oryzae AmyB protein in such a way that the sequence and all transcriptional signals of the amyB gene are retained together with the promoter
Summary
The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Filamentous fungi are very attractive host organisms for the production of heterologous proteins, since they have several advantages for protein expression compared to bacterial hosts. These advantages include i) the ability to produce large amounts of extracellular proteins, ii) the GRAS status in the food industry of several filamentous fungi such as Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Penicillium roqueforti among others [1,2], iii) rapid growth compared to other eukaryotic cells, iv) the secretion of correctly folded functional proteins and v) post-translational modifications, such as glycosylation.
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