Modulation of Aspergillus awamori Thaumatin Secretion by Modification of bipA Gene Expression

The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.

Section: Discussionmentioning

confidence: 99%

Modulation of Chaperone Gene Expression in Mutagenized Saccharomyces cerevisiae Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins

Payne

Finnis

Evans

et al. 2008

“…In aspergilli, the expression of human protein tissue plasminogen activator or a fused form of ␣-amylase-prochymosin triggered the unfolded protein response (UPR) due to ER stress (9,10). The overexpression of molecular chaperones like BiP and protein disulfide isomerase to assist protein folding improves the yield of heterologous proteins (11,12), a response that may be attributable to the alleviation of ER stress resulting from increased folding activity. When the UPR is triggered, the expression of a set of UPR-associated proteins, including molecular chaperones, vesicular traffic components, and ERassociated degradation (ERAD) proteins, is upregulated in an attempt to resolve the ER stress by increasing the folding, transport, and degradation of proteins (13).…”

mentioning

confidence: 99%

Modulating Endoplasmic Reticulum-Golgi Cargo Receptors for Improving Secretion of Carrier-Fused Heterologous Proteins in the Filamentous Fungus Aspergillus oryzae

Hoang

Maruyama

Kitamoto

2015

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme ␣-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of ␣-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi. Filamentous fungi possess a prominent secretory capacity and, thus, are excellent hosts for recombinant protein production. Numerous approaches and attempts have been made to produce industrially valuable proteins in filamentous fungi, such as Aspergillus and Trichoderma (1). However, higher eukaryotic proteins are generally inefficiently produced and secreted in these fungi compared to the production and secretion of fungal or endogenous proteins. Several bottlenecks in the heterologous protein production process have been identified to date, and a few limiting factors have been overcome by genetically modifying the expression host. In particular, reducing protease activity is necessary to limit the degradation of heterologous proteins, as was demonstrated by the 3-fold increase in the level of heterologous proteins in the culture supernatant of an Aspergillus oryzae strain with 10 protease genes deleted (2). Heterologous protein production by A. oryzae was also effectively improved by the repression of vacuolar protein sorting and autophagy (3, 4). The genetic fusion of a target protein with an endogenous protein carrier is a commonly used strategy to increase he...

“…In addition to homologous protein production, Aspergillus nidulans, A. niger, and Aspergillus oryzae have offered an attractive alternative to Escherichia coli and yeasts, such as Saccharomyces cerevisiae, Kluyveromyces lactis, or Pichia pastoris, for the expression of recombinant, heterologous proteins, and there have been a number of attempts to program filamentous fungi for the production of such heterologous proteins (13,14,20,27,29).…”

mentioning

confidence: 99%

N-Glycan Modification in Aspergillus Species

Kainz

Gallmetzer

Hatzl

et al. 2008

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional ␣-1,2-mannosidase producing increased amounts of N-glycans of the Man 5 GlcNAc 2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan 5 GlcNac 2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man 3 GlcNAc 2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.