Protein profile in Aspergillus nidulans recombinant strains overproducing heterologous enzymes

Abstract

Filamentous fungi are robust cell factories and have been used for the production of large quantities of industrially relevant enzymes. However, the production levels of heterologous proteins still need to be improved. Therefore, this article aimed to investigate the global proteome profiling of Aspergillus nidulans recombinant strains in order to understand the bottlenecks of heterologous enzymes production. About 250, 441 and 424 intracellular proteins were identified in the control strain Anid_pEXPYR and in the recombinant strains Anid_AbfA and Anid_Cbhl respectively. In this context, the most enriched processes in recombinant strains were energy pathway, amino acid metabolism, ribosome biogenesis, translation, endoplasmic reticulum and oxidative stress, and repression under secretion stress (RESS). The global protein profile of the recombinant strains Anid_AbfA and Anid_Cbhl was similar, although the latter strain secreted more recombinant enzyme than the former. These findings provide insights into the bottlenecks involved in the secretion of recombinant proteins in A. nidulans, as well as in regard to the rational manipulation of target genes for engineering fungal strains as microbial cell factories.

Figure 1

Secretion of total proteins by Aspergillus nidulans strains. The A. nidulans strains A773, Anid_pEXPYR (carrying an empty pEXPYR vector), Anid_AbfA and Anid_Cbhl expressing α‐L‐arabinofuranosidase and cellobiohydrolase, respectively, were grown on minimum media containing 2% maltose for 24 h and 72 h at 37 °C. (A) Ten micrograms of secreted proteins was resolved by Coomassie blue‐staining SDS‐PAGE gel. The strains A773 and Anid_pEXPYR were used as a control in this experiment. Asterisks (*) indicated the recombinant proteins. (B) qPCR of the recombinant genes was calculated by the relative standard curve method. The expression of genes abfA and cbhl was normalized using the gene tubC (tubulin) as reference. MM: molecular marker.

Figure 2

Analysis of Aspergillus nidulans growth. Spores solution was inoculated in 30 ml of minimum medium (MM) supplemented with 2% (m/v) maltose. (A) After various time points at 37 °C, the supernatant was separated from the culture medium by gauze filtration and maltose content was measured by HPLC. (B) The mycelia of A. nidulans strains were dried overnight at 105 °C for measure of the dry weight. Each bar represents the mean and the standard deviation of values from three independent experiments.

Figure 3

Abundance and functional analysis of intracellular proteins. The strains were grown on 2% maltose minimum medium for 24 h. (A) Venn diagrams represent the number of total proteins found in the intracellular proteome of each strain as well as the overlaps among groups. (B) A heat map of the 480 proteins categorized by MIPS FunCat (see Table S2) and the scale indicates the number of proteins found in each category. The intracellular proteomes were clustered based on their total spectra profiles.

Figure 4

Abundance and functional analysis of intracellular proteins. The strains were grown on minimum medium and 2% maltose for 24 h. (A) Venn diagrams represent the number of more and less abundant proteins relative to A. nidulans Anid_pEXPYR strain. (B) A heat map of all proteins (see Table S3). MIPS FunCat categorization of the 308 more abundant proteins and 19 less abundant proteins common to Anid_AbfA and Anid_Cbhl strains. The scale indicates the number of proteins found in each category. The intracellular proteomes were clustered based on their total spectra profiles.

Figure 5

Evidence of repression under secretion stress (RESS) in Aspergillus nidulans recombinant strains. Transcriptional level of amyR and xlnR genes in Anid_AbfA and Anid_Cbhl was compared to the expression level in control strain (Anid_pEXPYR). Gene expression levels were normalized (ΔΔCT analysis) to the endogenous gene tubC (tubulin). Data were analysed using one‐way‐ANOVA with Bonferroni's post hoc test (****P < 0.0001).

Figure 6

Overview of biological process overrepresented in Aspergillus nidulans recombinant strains. Heterologous protein production remains a complex process with some bottlenecks. Generally, the recombinant gene contains strong promoter for high level expression of the target mRNA. Large quantities of mRNAs overload the translational pathway, which increase misfolded proteins amounts in ER, inducing an ER stress (1). The homeostasis maintenance is achieved by UPR that induces genes coding to chaperones, amino acid metabolism, ribosome biogenesis, translation, among others (2). Furthermore, energy demand required for heterologous protein production increased (3), resulting in high levels of reactive oxygen species in the cell (4). The secretome analysis of recombinant strains showed the downregulation of biomass‐degrading enzymes and their genes, suggesting the presence of the RESS mechanism. Associated with the overload of misfolded proteins in the ER, this mechanism downregulates transcriptional activators, such as amyR and xlnR that regulates expression of several amylases and CAZymes respectively (5). The heat maps (at the bottom) represent the protein abundance in the recombinant (Anid_AbfA; Anid_Cbhl) and control strains (Anid_pEXPYR) in each biological process categorized by MIPS FunCat. The scale indicates the number of proteins found in each category.