Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

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.

Use of combined UV and chemical mutagenesis treatment of Aspergillus terreus D34 for hyper-production of cellulose-degrading enzymes and enzymatic hydrolysis of mild-alkali pretreated rice straw

“…Supplementation of 0.5% casein enhanced the total cellulase activity by 1.5-fold, while sorbose and cheese whey were found to be detrimental for cellulase and xylanase production (Additional file 1: Table S2). Recently, Kosalkova et al (2012) reported that casein or its biopolymers significantly increased the secretion of extracellular proteins up to 40-to 80-fold in A. awamori. Similarly Kawamori et al (1986) also tested l-sorbose for induction and production of CMCases in T. reesi, where avicel and glucose supplemented with 0.3% sorbose enhanced the CMCase activity by twofold.…”

Omics Approaches Applied to Penicillium chrysogenum and Penicillin Production: Revealing the Secrets of Improved Productivity

“…Finally, the functional proteomics that provides information about protein functions through the identification of interacting protein partners. Expression proteomics analyses have been carried out in penicillin-producer strains to compare between different them or between different culture conditions [ 64 , 65 , 83 , 87 , 88 , 89 , 90 ]. However, structural proteomics have been used to achieve reference maps of different sub-proteomes (e.g.…”

“…The addition of calcium salts in the culture medium has a positive effect in penicillin production and it also plays an important role in the growth of P. chrysogenum, whereas casein phosphopeptides are able to chelate Ca 2+ increasing the proteins secretion in Aspergillus strains [ 88 , 122 , 123 ]. The combination of both additives (Ca 2+ and casein phosphopeptides) increased 10–12-fold penicillin production in P. chrysogenum as described Domínguez-Santos and co-workers in 2017 [ 89 ].…”

Amino Acid Supplementation Improves the Production of Extracellular Peptidases by Aspergillus Section Flavi and their Ionic Immobilization

“…Fungi are efficient producers of enzymes with biotechnological potential and have advantages, such as production in large amounts, rapid growth and the ability to secrete functional proteins with correct folding and post-translational modifications [6].The genus Aspergillus is a great source for commercial enzymes, and is known for its ability to secrete a high level of enzymes into the environment [7,8]. Aspergillus section Flavi comprise 22 species known as Aspergillus flavus, A. oryzae, A. sojae, A. tamarii, A. parasiticus and recently two new species were identified: A. pseudocaelatus sp.…”