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Investigation of the impact of Tat export pathway enhancement on E. coli culture, protein production and early stage recovery

Branston, Steven D., Matos, Cristina F.R.O., Freedman, Robert B., Robinson, Colin, Keshavarz-Moore, Eli (2011) Investigation of the impact of Tat export pathway enhancement on E. coli culture, protein production and early stage recovery. Biotechnology and Bioengineering, 109 (4). pp. 983-991. ISSN 0006-3592. (doi:10.1002/bit.24384) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided)

The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided. (Contact us about this Publication)
Official URL
http://dx.doi.org/10.1002/bit.24384

Abstract

The twin arginine translocation (Tat) pathway occurs naturally in E. coli and has the distinct ability to translocate folded proteins across the inner membrane of the cell. It has the potential to export commercially useful proteins that cannot be exported by the ubiquitous Sec pathway. To better understand the bioprocess potential of the Tat pathway, this article addresses the fermentation and downstream processing performances of E. coli strains with a wild-type Tat system exporting the over-expressed substrate protein FhuD. These were compared to strains cell-engineered to over-express the Tat pathway, since the native export capacity of the Tat pathway is low. This low capacity makes the pathway susceptible to saturation by over-expressed substrate proteins, and can result in compromised cell integrity. However, there is concern in the literature that over-expression of membrane proteins, like those of the Tat pathway, can impact negatively upon membrane integrity itself. Under controlled fermentation conditions E. coli cells with a wild-type Tat pathway showed poor protein accumulation, reaching a periplasmic maximum of only 0.5?mg?L?1 of growth medium. Cells over-expressing the Tat pathway showed a 25% improvement in growth rate, avoided pathway saturation, and showed 40-fold higher periplasmic accumulation of FhuD. Moreover, this was achieved whilst conserving the integrity of cells for downstream processing: experimentation comparing the robustness of cells to increasing levels of shear showed no detrimental effect from pathway over-expression. Further experimentation on spheroplasts generated by the lysozyme/osmotic shock method—a scaleable way to release periplasmic protein—showed similar robustness between strains. A scale-down mimic of continuous disk-stack centrifugation predicted clarifications in excess of 90% for both intact cells and spheroplasts. Cells over-expressing the Tat pathway performed comparably to cells with the wild-type system. Overall, engineering E. coli cells to over-express the Tat pathway allowed for greater periplasmic yields of FhuD at the fermentation scale without compromising downstream processing performance.

Item Type: Article
DOI/Identification number: 10.1002/bit.24384
Uncontrolled keywords: Tat pathway;cell engineering;fermentation;robustness;clarification
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Colin Robinson
Date Deposited: 06 Jan 2014 12:22 UTC
Last Modified: 29 May 2019 11:42 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/37729 (The current URI for this page, for reference purposes)
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