Technologies

The Quiescent Cell Expression System (Q-Cells)

A fundamental problem associated with the use of micro-organisms as cell factories is that a substantial proportion of energy and nutritional resources is channeled away from product formation towards the production of unwanted biomass. In a novel solution to this problem, Cambridge Microbial has developed a method of generating non-growing but metabolically active ('quiescent') E. coli cells. Growth arrest is achieved by the manipulation of endogenous cell cycle controls under culture conditions where nutrients are not limiting. This means that the entire metabolic resources of the cell can potentially be channeled towards the expression of product. The technology is simple to use and combines easily with existing E. coli cell culture techniques.

First Generation Technology

The original E. coli-based Quiescent Cell Expression System (Q-Cells) was geared towards the production of recombinant proteins (Rowe & Summers, 1999 (1.4Mb PDF)). The quiescent state was established by over-expressing a small regulatory RNA (Rcd) in a bacterial host expressing a specific mutant allele of hns. Quiescent cultures could be generated in shake-flasks and in fermenters at densities as high as OD₆₀₀=50. Q-Cells proved successful in producing a range of proteins including cytokines and an antibody fragment (Fab). The expression, folding and export of biologically-active Fab into the culture supernatant is more then ten times faster than in a conventional culture under the same conditions. ( Mukherjee et al 2004 (553k PDF) ).

Included here is a PDF version of David Summers' layman's guide to the original Q-Cell gene expression technology which was published by the SGM in Microbiology Today - 'A Quiet Revolution in the Bacterial Cell Factory' (358k PDF). To view this document you will need to have Adobe Acrobat Reader installed.

Second Generation Quiescent Cells

Limitations of the first generation Q-Cell system included its relative technical complexity and the need to induce quiescence by a temperature shift from 30°C to 42°C, which meant that product synthesis was invariably at the higher temperature. However basic research into the mechanism of action of the Rcd regulatory RNA (Chant and Summers, 2007) has enabled to the development of second generation Q-Cells technology with far greater flexibility and ease of use. Rcd was found to act by binding to the enzyme tryptophanase and stimulating its production of indole. In the second generation system quiescence is induced by adding indole directly to the growth medium of an E.coli culture carrying an appropriate hns mutant allele. This removes any constraint on the product expression temperature and simplifies the system for users because no Rcd expression plasmid is required.

With its greater simplicity and easy of use Cambridge Microbial is exploring the potential of the second generation Q-Cell system in a wide range of bio-factory applications. In particular interest is focused on its application to the production of chemical intermediates, biopolymers and biofuels, including microbial hydrogen.

Cambridge Microbial Technologies Ltd.

Email: info@cammicrobial.co.uk