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.

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 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.

New Promoters to Improve Heterologous Protein Production in Aspergillus vadensis

2014 ◽  
Vol 3 (3) ◽  
pp. 244-251 ◽  
Author(s):  
Helena Culleton ◽  
Ourdia Bouzid ◽  
Vincent McKie ◽  
Ronald Vries
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Protein Production

scholarly journals Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

2021 ◽  
Vol 7 (3) ◽  
pp. 179
Author(s):  
Kai P. Hussnaetter ◽  
Magnus Philipp ◽  
Kira Müntjes ◽  
Michael Feldbrügge ◽  
Kerstin Schipper
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Ustilago Maydis ◽  
Downstream Processing ◽  
Culture Broth ◽  
Yeast Cells ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Regulatory Systems ◽  
Unconventional Secretion

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

Absence of See1p, a widely conservedSaccharomyces cerevisiaeprotein, confers both deficient heterologous protein production and endocytosis

Yeast ◽  
2008 ◽  
Vol 25 (12) ◽  
pp. 871-877 ◽  
Author(s):  
Cristina Martín-Granados ◽  
Sean-Patrick Riechers ◽  
Ulf Stahl ◽  
Christine Lang
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Protein Production

scholarly journals State and specific growth estimation in heterologous protein production by Pichia pastoris

AIChE Journal ◽  
2011 ◽  
Vol 58 (10) ◽  
pp. 2966-2979 ◽  
Author(s):  
José M. Barrigón ◽  
Ramon Ramon ◽  
Isabel Rocha ◽  
Francisco Valero ◽  
Eugénio C. Ferreira ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Protein Production ◽  
Heterologous Protein ◽  
Specific Growth ◽  
Heterologous Protein Production ◽  
Growth Estimation

Heterologous protein production using thePichia pastoris expression system

Yeast ◽  
2005 ◽  
Vol 22 (4) ◽  
pp. 249-270 ◽  
Author(s):  
Sue Macauley-Patrick ◽  
Mariana L. Fazenda ◽  
Brian McNeil ◽  
Linda M. Harvey
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Expression System ◽  
Heterologous Protein Production ◽  
Pastoris Expression System

scholarly journals Secretion of Human Serum Albumin by Kluyveromyces lactis Overexpressing KlPDI1 and KlERO1

2005 ◽  
Vol 71 (8) ◽  
pp. 4359-4363 ◽  
Author(s):  
Tiziana Lodi ◽  
Barbara Neglia ◽  
Claudia Donnini
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Protein Conformation ◽  
Protein Production ◽  
Heterologous Protein ◽  
Kluyveromyces Lactis ◽  
Heterologous Proteins ◽  
Disulfide Bridges ◽  
Genes Encoding

ABSTRACT The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1β, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.

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