Improvementof Foreign-Protein Production in Aspergillus niger var. awamori by Constitutive Induction of the Unfolded-ProteinResponse

Abstract

Unfolded-protein response (UPR) denotes the upregulation of endoplasmic reticulum (ER)-resident chaperone and foldase genes and numerous other genes involved in secretory functions during the accumulation of unfolded proteins into the ER. Overexpression of individual foldases and chaperones has been used in attempts to improve protein production in different production systems. We describe here a novel strategy to improve foreign-protein production. We show that the constitutive induction of the UPR pathway in Aspergillus niger var. awamori can be achieved by expressing the activated form of the transcription factor hacA. This induction enhances the production of Trametes versicolor laccase by up to sevenfold and of bovine preprochymosin by up to 2.8-fold in this biotechnically important fungus. The regulatory range of UPR was studied by analyzing the mRNA levels of novel A. niger var. awamori genes involved in different secretory functions. This revealed both similarities and differences to corresponding studies in Saccharomyces cerevisiae.

The virulence of the opportunistic fungal pathogenAspergillus fumigatusrequires cooperation between the endoplasmic reticulum-associated degradation pathway (ERAD) and the unfolded protein response (UPR)

“…[13][14][15][16] The HacA protein translocates to the nucleus, where it increases the transcription of genes that encode proteins with functions in protein folding and secretion, The entire derA open reading frame was replaced with the hygromycin resistance cassette (hYG) as shown. southern blot analysis of NsiIdigested genomic DNa using a probe located in the 5' flanking region (p1) revealed the predicted truncation of a 9 kb wt NsiI fragment by the replacement of derA with hYG.…”

Heterologous laccase production and its role in industrial applications

“…Laccase heterologous expression systems have also been employed as model to measure the effectiveness of different promoters, 29 besides other more specific aims, such as (i) to study the effect of constitutive activation of the Unfolded-protein response (UPR) pathway on the production of heterologous protein in A. niger; 153 (ii) to examine the protein quality control mechanism; 83 (iii) to select strains with higher production levels of heterologous proteins; 156 (iv) to evaluate the physiological state of an organism in biotechnical processes; 124,125 (v) to establish the effects of reduced protease activity on the stabilities of secreted recombinant proteins. 154 Development of applications of recombinant laccases from filamentous fungi is discussed in a next section.…”

Peroxicretion, a Novel Tool for Engineering Membrane Trafficking

“…This UPR is a well studied mechanism [13][14][15][16][17] which is also applied to increase folding capacity. By upregulating the transcription factors needed, or the resulting chaperones and folding enzymes, the folding capacity of the cell is enhanced [18][19][20][21] .…”