The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins

103

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.

Section: Discussionmentioning

confidence: 99%

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

Valkonen

Ward²,

Wang³

et al. 2003

103

“…Probes used for Northern analysis were a 1.6-kb EcoRIHindIII fragment from pABiP1-22, a pABiP1 subclone (43), containing the A. niger bipA gene; a 2-kb BspHI-EcoRV fragment from pGLACLX (calnexin probe); a 1.1-kb NcoI-HindIII fragment from pMnP1.I (mnp1 probe) (10); and a 1.5-kb HindIII fragment from pAB5-2 (gpdA probe) (47). Northern hybridization signals were measured with Cyclone Storage Phosphor (Packard) and quantified with OptiQuant 3.0 software (Packard Instruments Co.).…”

Section: Strainsmentioning

confidence: 99%

“…The expression of chaperone-and foldase-encoding genes is induced by situations of stress in the ER that result in the accumulation of unfolded proteins through the so-called unfolded protein response (UPR) pathway (for reviews, see references 7 and 49). In filamentous fungi, UPR-inducing agents have been shown to provoke a rapid and strong increase in the expression of the major ER-resident chaperone binding protein (BiP) (28,43), whereas induction of the expression of foldases of the protein disulfide isomerase (PDI) family occurred in a delayed and/or less intense fashion (27,28,37).…”

mentioning

confidence: 99%

Calnexin Overexpression Increases Manganese Peroxidase Production in Aspergillus niger

Conesa

Jeenes

Archer

et al. 2002

Heme-containing peroxidases from white rot basidiomycetes, in contrast to most proteins of fungal origin, are poorly produced in industrial filamentous fungal strains. Factors limiting peroxidase production are believed to operate at the posttranslational level. In particular, insufficient availability of the prosthetic group which is required for peroxidase biosynthesis has been proposed to be an important bottleneck. In this work, we analyzed the role of two components of the secretion pathway, the chaperones calnexin and binding protein (BiP), in the production of a fungal peroxidase. Expression of the Phanerochaete chrysosporium manganese peroxidase (MnP) in Aspergillus niger resulted in an increase in the expression level of the clxA and bipA genes. In a heme-supplemented medium, where MnP was shown to be overproduced to higher levels, induction of clxA and bipA was also higher. Overexpression of these two chaperones in an MnP-producing strain was analyzed for its effect on MnP production. Whereas bipA overexpression seriously reduced MnP production, overexpression of calnexin resulted in a four-to fivefold increase in the extracellular MnP levels. However, when additional heme was provided in the culture medium, calnexin overexpression had no synergistic effect on MnP production. The possible function of these two chaperones in MnP maturation and production is discussed.

“…These methods have identified genes involved with protein folding, such as tigA (52) and cypB (7). Secretion blockers have also been used in combination with green fluorescent protein fusions to visualize unfolded protein and locate potential bottlenecks (15).…”

mentioning

confidence: 99%

Transcriptome Analysis of Recombinant Protein Secretion by Aspergillus nidulans and the Unfolded-Protein Response In Vivo

Sims

Gent

Lanthaler³

et al. 2005

Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.