Special Issue "Genetic and Process Engineering Tools for Next Generation Cell Factories"

A special issue of Bioengineering (ISSN 2306-5354).

Deadline for manuscript submissions: closed (15 January 2019)

Special Issue Editors

Guest Editor
Dr. Neil Dixon

University of Manchester, Manchester Institute of Biotechnology, Manchester, United Kingdom
Website | E-Mail
Interests: chemical biology; molecular detection/recognition; synthetic biology
Guest Editor
Dr. Gerald Striedner

Department of Biotechnology, Universitat fur Bodenkultur Wien, Vienna, Austria
Website | E-Mail
Interests: chemical biology; molecular detection/recognition; synthetic biology

Special Issue Information

Dear Colleagues,

This Special Issue is on the production and processing of therapeutic proteins and industrial enzymes from microbial hosts, with a focus on host cell engineering strategies to minimize host synthetic burden, reduce the impact on down-stream purification processes and to maximize the productivity of manufacturing processes. Integrated genetic engineering and process engineering approaches, for instance, where genetic modification of the host allows for a new processing strategy, or indeed where a problem identified in the process environment, is solved via genetic engineering.

This would include genetic engineering strategies to enhance product functionality/activity/quality, and to facilitate isolation and purification, examples could include: i) expression control tools to permit temporal, tuneable, and/or growth phase dependent induction of gene expression; ii) host engineering to introduce novel function or enhanced capability, e.g., the use of folding modulators, optimization of host secretion systems, expression of heterologous secretion system; iii) host engineering to remove or reduce unwanted characteristics, e.g., knock-out of proteases, membrane engineering for product release, depletion of common host contaminants, e.g., co-purifying proteins/DNA/RNA/lipopolysaccharides; and iv) product engineering approaches for enhanced expression, functionality or purification, e.g., purification tags, cross-linking modalities for conjugation and immobilization.

The field of process engineering could include: i) HTP small-scale techniques for process optimization, characterization, clone and process condition screening; ii) advanced automation concepts for process operation, data processing, storage and retrieval; iii) methodologies for process monitoring, modelling and model predictive process control; iv) bioprocess integration and intensification along scales and unit operations and across disciplines; v) issues related to batch or continuous process operation mode; and vi) media design concepts.

Dr. Neil Dixon
Dr. Gerald Striedner
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Bioengineering is an international peer-reviewed open access quarterly journal published by MDPI.

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Published Papers (2 papers)

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Research

Open AccessArticle QbD Based Media Development for the Production of Fab Fragments in E. coli
Bioengineering 2019, 6(2), 29; https://doi.org/10.3390/bioengineering6020029
Received: 26 January 2019 / Revised: 20 March 2019 / Accepted: 23 March 2019 / Published: 28 March 2019
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Abstract
Ranibizumab is a biotherapeutic Fab fragment used for the treatment of age-related macular degeneration and macular oedema. It is currently expressed in the gram-negative bacterium, Escherichia coli. However, low expression levels result in a high manufacturing cost. The protein expression can be [...] Read more.
Ranibizumab is a biotherapeutic Fab fragment used for the treatment of age-related macular degeneration and macular oedema. It is currently expressed in the gram-negative bacterium, Escherichia coli. However, low expression levels result in a high manufacturing cost. The protein expression can be increased by manipulating nutritional requirements (carbon source, nitrogen source, buffering agent), process parameters (pH, inducer concentration, agitation, temperature), and the genetic make-up of the producing strain. Further, understanding the impact of these factors on product quality is a requirement as per the principles of Quality by Design (QbD). In this paper, we examine the effect of various media components and process parameters on the expression level and quality of the biotherapeutic. First, risk analysis was performed to shortlist different media components based on the literature. Next, experiments were performed to screen these components. Eight components were identified for further investigation and were examined for their effect and interactions using a Fractional Factorial experimental design. Sucrose, biotin, and pantothenate were found to have the maximum effect during Fab production. Furthermore, cyanocobalamin glutathione and biotin-glutathione were the most significant interactions observed. Product identification was performed with Liquid Chromatography–Mass Spectrometry (LC-MS), the expression level was quantified using Bio-layer Interferometry, Reverse Phase-HPLC, and SDS-PAGE, and product quality were measured by RP-HPLC. Overall, a five-fold enhancement of the target protein titer was obtained (from 5 mg/L to 25 mg/L) using the screened medium components vis-a-vis the basal medium, thereby demonstrating the efficacy of the systematic approach purported by QbD. Full article
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Open AccessArticle Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli
Bioengineering 2019, 6(1), 27; https://doi.org/10.3390/bioengineering6010027
Received: 29 January 2019 / Revised: 14 March 2019 / Accepted: 19 March 2019 / Published: 21 March 2019
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Abstract
Escherichia coli strains have been modified in a variety of ways to enhance the production of different recombinant proteins, targeting membrane protein expression, proteins with disulphide bonds, and more recently, proteins which require N-linked glycosylation. The addition of glycans to proteins remains [...] Read more.
Escherichia coli strains have been modified in a variety of ways to enhance the production of different recombinant proteins, targeting membrane protein expression, proteins with disulphide bonds, and more recently, proteins which require N-linked glycosylation. The addition of glycans to proteins remains a relatively inefficient process and here we aimed to combine genetic modifications within central carbon metabolic pathways in order to increase glycan precursor pools, prior to transfer onto polypeptide backbones. Using a lectin screen that detects cell surface representation of glycans, together with Western blot analyses using an O-antigen ligase mutant strain, the enhanced uptake and phosphorylation of sugars (ptsA) from the media combined with conservation of carbon through the glyoxylate shunt (icl) improved glycosylation efficiency of a bacterial protein AcrA by 69% and over 100% in an engineered human protein IFN-α2b. Unexpectedly, overexpression of a gene involved in the production of DXP from pyruvate (dxs), which was previously seen to have a positive impact on glycosylation, was detrimental to process efficiency and the possible reasons for this are discussed. Full article
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