Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
4.2
4.6
Journals
Bioengineering
Special Issues
Genetic and Process Engineering Tools for Next Generation Cell Factories
share announcement

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) | Viewed by 33880

Special Issue Editors

Dr. Neil Dixon

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

E-Mail Website
Guest Editor
Department of Biotechnology, Universitat fur Bodenkultur Wien, Vienna, Austria
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

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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 monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Published Papers (5 papers)

Download All Papers
Order results
Result details
Show export options expand_more Show export options expand_less
Select all
Export citation of selected articles as:

Research

12 pages, 1122 KiB  
Article
The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System
by Johanna Hausjell, Regina Kutscha, Jeannine D. Gesson, Daniela Reinisch and Oliver Spadiut
Bioengineering 2020, 7(1), 8; https://doi.org/10.3390/bioengineering7010008 - 06 Jan 2020
Cited by 13 | Viewed by 6377
Abstract
Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts [...] Read more.
Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity. Full article
(This article belongs to the Special Issue Genetic and Process Engineering Tools for Next Generation Cell Factories)
►▼ Show Figures

Figure 1

23 pages, 3165 KiB  
Article
The Statistical Optimisation of Recombinant β-glucosidase Production through a Two-Stage, Multi-Model, Design of Experiments Approach
by Albert Uhoraningoga, Gemma K. Kinsella, Jesus M. Frias, Gary T. Henehan and Barry J. Ryan
Bioengineering 2019, 6(3), 61; https://doi.org/10.3390/bioengineering6030061 - 18 Jul 2019
Cited by 2 | Viewed by 6007
Abstract
β-glucosidases are a class of enzyme that are widely distributed in the living world, with examples noted in plants, fungi, animals and bacteria. They offer both hydrolysis and synthesis capacity for a wide range of biotechnological processes. However, the availability of native, or [...] Read more.
β-glucosidases are a class of enzyme that are widely distributed in the living world, with examples noted in plants, fungi, animals and bacteria. They offer both hydrolysis and synthesis capacity for a wide range of biotechnological processes. However, the availability of native, or the production of recombinant β-glucosidases, is currently a bottleneck in the widespread industrial application of this enzyme. In this present work, the production of recombinant β-glucosidase from Streptomyces griseus was optimised using a Design of Experiments strategy, comprising a two-stage, multi-model design. Three screening models were comparatively employed: Fractional Factorial, Plackett-Burman and Definitive Screening Design. Four variables (temperature, incubation time, tryptone, and OD600 nm) were experimentally identified as having statistically significant effects on the production of S.griseus recombinant β-glucosidase in E. coli BL21 (DE3). The four most influential variables were subsequently used to optimise recombinant β-glucosidase production, employing Central Composite Design under Response Surface Methodology. Optimal levels were identified as: OD600 nm, 0.55; temperature, 26 °C; incubation time, 12 h; and tryptone, 15 g/L. This yielded a 2.62-fold increase in recombinant β-glucosidase production, in comparison to the pre-optimised process. Affinity chromatography resulted in homogeneous, purified β-glucosidase that was characterised in terms of pH stability, metal ion compatibility and kinetic rates for p-nitrophenyl-β-D-glucopyranoside (pNPG) and cellobiose catalysis. Full article
(This article belongs to the Special Issue Genetic and Process Engineering Tools for Next Generation Cell Factories)
►▼ Show Figures

Graphical abstract

16 pages, 1216 KiB  
Article
Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy
by Olusegun Folarin, Darren Nesbeth, John M. Ward and Eli Keshavarz-Moore
Bioengineering 2019, 6(2), 54; https://doi.org/10.3390/bioengineering6020054 - 19 Jun 2019
Cited by 5 | Viewed by 8182
Abstract
There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From [...] Read more.
There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of −0.022, and pSVβ-Gal −0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing. Full article
(This article belongs to the Special Issue Genetic and Process Engineering Tools for Next Generation Cell Factories)
►▼ Show Figures

Figure 1

17 pages, 2966 KiB  
Article
QbD Based Media Development for the Production of Fab Fragments in E. coli
by Deepak Kumar, Jyoti Batra, Claire Komives and Anurag S. Rathore
Bioengineering 2019, 6(2), 29; https://doi.org/10.3390/bioengineering6020029 - 28 Mar 2019
Cited by 14 | Viewed by 6874
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
(This article belongs to the Special Issue Genetic and Process Engineering Tools for Next Generation Cell Factories)
►▼ Show Figures

Graphical abstract

13 pages, 1747 KiB  
Article
Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve N-Type Glycosylation of Recombinant Proteins in E. coli
by Benjamin Strutton, Stephen RP Jaffe, Caroline A Evans, Gregory JS Fowler, Paul D Dobson, Jagroop Pandhal and Phillip C Wright
Bioengineering 2019, 6(1), 27; https://doi.org/10.3390/bioengineering6010027 - 21 Mar 2019
Cited by 9 | Viewed by 5887
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
(This article belongs to the Special Issue Genetic and Process Engineering Tools for Next Generation Cell Factories)
►▼ Show Figures

Figure 1

Show export options expand_more Show export options expand_less
Select all
Export citation of selected articles as:
 
Displaying articles 1-5
Back to TopTop