Quiescent Microbial Cell Factories

It is widely recognised that the manufacture of commodity chemicals and fuels must switch from petrochemicals to a sustainable feedstock base. However it is also increasingly evident that development of processes for the bio-manufacture of fuels and chemicals will require technology improvements capable of increasing process productivity, robustness and economic competitiveness. A key component of improved bio-manufacture is the development of improved bacterial cell factories, which constitutes the focus of this application.

A universally accepted criterion for a bacterial cell factory development is that it must convert the maximum amount of raw material directly to the desired end product with minimum by-product accumulation. While much R&D effort is dedicated to eliminating the co-production of undesirable metabolites, the primary by-product, cell biomass accumulated due to microbial growth, is often dismissed as an inevitable consequence of the process. However, efficiency would be increased by uncoupling product formation from growth, keeping biomass constant while allowing the conversion of substrate to product for an extended period of time. This approach has been exploited in the production of certain amino acids by genetically modified strains but is not generally applicable for the majority of metabolic engineering strategies where growth is required to maintain the metabolic health of the bacterial cell.

The focus of this project is the use of quiescent cell (Q-Cell) technology developed in the Summers laboratory in the Department of Genetics at Cambridge University. Q-Cells are a non-growing but metabolically-active cell factory generated from the bacterium Escherichia coli. The bacterial host, possessing a specific genetic modification, can be induced into this state by the addition of indole as a chemical trigger of quiescence. In laboratory studies the productivity of Q-cells is up to 10-fold greater than conventional E. coli cultures, resources being channelled more efficiently into product in the absence of biomass generation. Moreover, pathway flux in central carbon metabolism remains high after the onset of quiescence, providing a constantly regenerated pool of metabolites that can be diverted into product.

The performance of Q-cells under conditions relevant to industrial application will be evaluated in this project. At Cambridge University Department of Genetics, work will be undertaken to improve the system on the laboratory scale, comparing the efficacy of a range of chemical triggers to induce quiescence. There will also be an assessment of the role of environmental factors (temperature) on the efficient operation of the system. It is hoped that these changes might avoid the need for genetic modification of the bacterial host strain, thus increasing the ease of use of the system as well as increasing its efficiency.

The investigations at Cambridge will be conducted in shake-flask culture and possibly in small-scale fermenters. However it is essential to determine whether good performance under these conditions will scale up to industrial conditions. This is where the role of the industrial partner CPI is crucial. CPI will initially conduct rigorous testing of the utility of the Q-Cell system in its present form. As work in Cambridge suggests potential improvements these will be incorporated into the CPI programme.

Bacterial cell factories offer a means to switch the manufacture of commodity chemicals and fuels from petrochemicals to a sustainable feedstock base. However their efficiency is reduced by the accumulation of unwanted biomass in addition to product. The Summers laboratory (Cambridge University) has developed an E. coli quiescent cell factory (Q-Cells). Metabolically active but non-growing Q-Cells are generated by treating a culture of hns mutant E. coli with indole to trigger the transition from active growth to quiescence. They show substantial improvements in efficiency of production of metabolites and recombinant proteins. The Cambridge contribution to this collaborative project is further improvement of the Q-Cell system through the identification of improved triggers of quiescence and the removal of the need for a specific mutant host strain.

Indole is a natural proton ionophore and its ability to arrest cell division and growth is due to the loss of the proton gradient across the cytoplasmic membrane. However, via a distinct mechanism, indole also inhibits DNA gyrase which is required for transcription. We will investigate whether non-biological ionophores (CCCP and DNP) that inhibit bacterial growth at substantially lower concentrations than indole also induce the Q-Cell state. These compounds are structurally distinct from indole and unlikely to inhibit DNA gyrase. We also speculate that using these alternative chemical triggers it might be possible to dispense with the requirement for the hns mutation in the host strain with benefits for ease-of-use. Finally we will conduct a study to determine the optimum temperature for the establishment and productivity of quiescent cells produced by indole or alternative chemical triggers. During the testing and optimisation process 3-hydroxybutyrate production and fluorescent protein expression will be used as indicators of Q-Cell productivity and metabolic activity.

As described in proposal submitted to Innovate UK