scholarly journals Enhanced Production and Secretion of Heterologous Proteins by the Filamentous Fungus Aspergillus oryzae via Disruption of Vacuolar Protein Sorting Receptor Gene Aovps10

2010 ◽  
Vol 76 (17) ◽  
pp. 5718-5727 ◽  
Author(s):  
Jaewoo Yoon ◽  
Tuerxun Aishan ◽  
Jun-ichi Maruyama ◽  
Katsuhiko Kitamoto
Keyword(s):  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
Protein Production ◽  
Heterologous Protein ◽  
Protein Sorting ◽  
Heterologous Protein Production ◽  
Heterologous Proteins ◽  
Extracellular Production ◽  
Sorting Receptor ◽  
Vacuolar Protein

ABSTRACT Filamentous fungi have received attention as hosts for heterologous protein production because of their high secretion capability and eukaryotic posttranslational modifications. However, despite these positive attributes, a bottleneck in posttranscriptional processing limits protein yields. The vacuolar protein sorting gene VPS10 encodes a sorting receptor for the recognition and delivery of several yeast vacuolar proteins. Although it can also target recombinant and aberrant proteins for vacuolar degradation, there is limited knowledge of the effect of its disruption on heterologous protein production. In this study, cDNA encoding AoVps10 from the filamentous fungus Aspergillus oryzae was cloned and sequenced. Microscopic observation of the transformant expressing AoVps10 fused with enhanced green fluorescent protein showed that the fusion protein localized at the Golgi and prevacuolar compartments. Moreover, disruption of the Aovps10 gene resulted in missorting and secretion of vacuolar carboxypeptidase AoCpyA into the medium, indicating that AoVps10 is required for sorting of vacuolar proteins to vacuoles. To investigate the extracellular production levels of heterologous proteins, ΔAovps10 mutants expressing either bovine chymosin (CHY) or human lysozyme (HLY) were constructed. Interestingly, the ΔAovps10 mutation increased the maximum extracellular production levels of CHY and HLY by 3- and 2.2-fold, respectively. Western blot analysis of extracellular heterologous proteins also demonstrated an improvement in productivity. These results suggest that AoVps10 plays a role in the regulation of heterologous protein secretion in A. oryzae and may be involved in the vacuolar protein degradation through the Golgi apparatus.

scholarly journals Induction by Hypoxia of Heterologous-Protein Production with the KlPDC1 Promoter in Yeasts

2006 ◽  
Vol 73 (3) ◽  
pp. 922-929 ◽  
Author(s):  
Andrea Camattari ◽  
Michele M. Bianchi ◽  
Paola Branduardi ◽  
Danilo Porro ◽  
Luca Brambilla
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Gene Expression ◽  
Kluyveromyces Lactis ◽  
Expression System ◽  
Promoter Sequence ◽  
Heterologous Protein Production ◽  
Heterologous Proteins ◽  
Hypoxic Induction ◽  
Production Ratio

ABSTRACT The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1β was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get β-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.

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

2014 ◽  
Vol 81 (2) ◽  
pp. 533-543 ◽  
Author(s):  
Huy-Dung Hoang ◽  
Jun-ichi Maruyama ◽  
Katsuhiko Kitamoto
Keyword(s):  
Endoplasmic Reticulum ◽  
Filamentous Fungi ◽  
Protein Secretion ◽  
Filamentous Fungus ◽  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Proteins ◽  
Content Type ◽  
Heterologous Protein Secretion ◽  
Golgi Compartment

ABSTRACTFilamentous 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, usingAspergillus oryzaeas a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors,A. oryzaeVip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-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 α-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.

Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant

2009 ◽  
Vol 82 (6) ◽  
pp. 1105-1114 ◽  
Author(s):  
Takashi Nemoto ◽  
Taisuke Watanabe ◽  
Yutaka Mizogami ◽  
Jun-ichi Maruyama ◽  
Katsuhiko Kitamoto
Keyword(s):  
Aspergillus Oryzae ◽  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Protein Production

Using DNA-Tagged Mutagenesis to Improve Heterologous Protein Production in Aspergillus oryzae

2000 ◽  
Vol 29 (1) ◽  
pp. 28-37 ◽  
Author(s):  
Debbie S Yaver ◽  
Michael Lamsa ◽  
Rebecca Munds ◽  
Stephen H Brown ◽  
Suzie Otani ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Protein Production

scholarly journals A perfusion-capable microfluidic bioreactor for assessing microbial heterologous protein production

Lab on a Chip ◽  
2015 ◽  
Vol 15 (14) ◽  
pp. 2918-2922 ◽  
Author(s):  
Nicholas J. Mozdzierz ◽  
Kerry R. Love ◽  
Kevin S. Lee ◽  
Harry L. T. Lee ◽  
Kartik A. Shah ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Protein Production ◽  
Microbial Cell ◽  
Perfusion Bioreactor ◽  
Heterologous Proteins ◽  
Microfluidic Perfusion

This work presents an integrated microfluidic perfusion bioreactor for the continuous expression of heterologous proteins from suspended microbial cell cultures.

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