A Systems Biology Approach to Optimisation of (Fed-)Batch and Continuous Fermentation Processes for Recombinant Protein Production

This project will use a holistic approach that will focus on the complex interactions that give rise to the function and behaviour of a biological system. Specifically, this project will identify a large number of the enzymes and metabolites in the metabolic pathways of Escherichia coli cells, when the bacterium is used as a cell factory to produce large amounts of a recombinant enzyme, i.e. an enzyme produced from a gene that has been introduced into an E. coli cell using Molecular Biology techniques. The holistic approach, known as Systems Biology, will use a combination of two techniques, one to identify the enzymes in the cells, known as proteomics, and the other to identify the metabolites in the cells, known as metabolomics. These techniques require scientific equipment that allow the high resolution (good separation due to the large number of molecules being analysed together) and accurate mass (weight) determination of the enzymes and metabolites (and their components, as the enzymes and metabolites can be broken into fragments for analysis). It is hoped that this holistic approach will allow us to understand the relationships between enzymes and metabolites, external growth factors such as temperature of growth, change of acidity/alkalinity of microbiological growth media as the cells grow, constituents of the microbiological growth media and factors associated with the design of the introduced gene that produces the recombinant enzyme, so that commercial-scale recombinant enzyme production can be improved. Ultimately this would lead to, for example, cheaper more widely available medicines, as many medicines now have to be produced using recombinant enzymes, rather than environmentally unfriendly harsh chemicals.

The proposed research will be organised into 2 work packages over a 12 month period:
The first work package involves metabolome and proteome analysis method development. Objectives(s): establish appropriate diagnostic metabolomic and/or proteomic analyses for each of the fermentation samples supplied by the industrial partners. Output(s): 1) metabolomic and/or proteomic profiles of the 48 samples provided; in the case of the former analyses, profiles comprising numerous key abundance differences will be established, and in the case of the latter differentially expressed proteins will be unambiguously identified, 2) establish an untargeted approach to identify metabolomic abundance differences for the samples provided, and assign preliminary identities to the peaks, and perform proteomic analyses on the same samples to identify correlated differentially expressed E. coli proteins, towards confirmation of the metabolomic peak identities.
The second work package involves advanced metabolome / proteome analysis. Objective(s): further application of the systems biology methods developed, including a targeted metabolomics approach with quantification. In total it is envisaged approx. 300 samples will be analysed iteratively, generating metabolomic and proteomic data in each case.
The methods to be adopted are as follows: for metabolomic analyses, 5 microL of each culture will be extracted using a chloroform / methanol / water mixture, the samples analysed using LC(C18 and HILIC columns)-MS(high-resolution QExactive-MS) and the data analysed using Sieve2 / KEGG software; for proteomic analyses, 1 mL of each culture will be lysed, cleared and the recombinant protein removed (so that it does not mask native E. coli proteins during analysis) using Ni-chelating resin, the native E. coli proteins precipitated, analysed via GeLC-MS using LC(C18 column)-MS(high-resolution QExactive MS) and the data analysed using Progenesis QI / MASCOT software.

The beneficiaries of this research will be those producing and using recombinant enzymes. Examples of immediate beneficiaries include the Chemicals, Food Producers and Pharmaceuticals and Biotechnology industries, as well as academia. This research will significantly simplify and drive down the costs of recombinant enzyme research and development, increasing its uptake by industry and academia. In the longer term, the increasing number of the resulting non-petroleum-based (i.e. bio-based) products produced will contribute significantly to the alleviation of the consumption of resources that harm the environment in terms of biodegradability, toxicity, and pollution resulting in a beneficial effect on the health of the UK population. Importantly, the market for bio-based products is likely to increase significantly in the longer term, and has been identified as a lead market by the European Commission. Therefore it is imperative to wealth creation, and specifically the economic competitiveness of the UK, that the recombinant enzyme-using community has access to appropriate enzymes that can contribute to the manufacture of bio-based products.