Approach

Characterisation & ‘omics analysis

MeMBrane will perform multilayer phenotypic and genotypic characterisation of a large number of industrial and environmental strains of Saccharomyces and Propionibacteria to identify key resistant and non-resistant isolates. This will be combined with powerful ‘omics approaches to identify not only transcriptional changes under stress response but associated changes in membrane lipids and proteins. To aid analysis of large ‘omics datasets, we will develop MeMBrane-specific versions of DETOXbase integrating these into a database including a whole-genome metabolic model uniquely focussed on identification of stress responses during industrial fermentations.

Design of resistant membranes

cellThe ‘omics data will be used to inform the design of complementary in vitro and in silico membrane environments that will be tested for resistance to stress and used to delineate the molecular mechanisms and biophysical properties underpinning this. In vitro approaches will allow rapid screening of membrane compositions to determine which generate resistance to relevant stresses without time-consuming in vivo experiments. We will use model bilayers containing native lipids or bespoke formulations and incorporating membrane proteins that have been expressed in bacterial or yeast host strains and solubilised using styrene maleic acid polymers to retain their integrity before characterisation. In parallel, membranes of various compositions including proteins will be simulated and the effect of stresses studied at the molecular level; current Martini modelling framework parameters exist for most common bacterial membrane lipids and scripts are in place to include membrane proteins. Hundreds of compositions can be screened rapidly and optimised conditions retested in vitro to confirm maximal protective effects.

Membrane optimisation

Once desirable membrane compositions have been determined, these must be recreated in the relevant host system. Metabolic engineering of microbes is not always straightforward due to interconnecting or competing pathways within the cell. Therefore, we will combine synthetic biology and rational genetic improvement of strains to generate desirable membrane characteristics. Moreover, where these approaches are too complex or insufficient to create desirable membrane environments, we will integrate an additive-mediated approach of tailored lipid supplements for membrane co-manipulation and non-recombinant strategies, based on evolutionary engineering and intra- and inter-specific hybridisation.

High throughput screening and industrial scale tests

Once modified strains have been produced we will test their efficacy using high-throughput screening to characterise a large number of strains and growth conditions. Finally, the improved strains will be scaled up into relevant industrial environments to ensure increased performance is achieved in industrially-relevant process conditions.

Iterative improvement

MeMBrane will continue to improve strains by iterative cycling through these stages to maximise the potential benefits during the project.

flow

Environmental and social impact assessment

To evaluate the potential environmental impact of the new processes, a Life Cycle Assessment (LCA) will be performed, following ISO 14040 and 14044. The assessment will identify potential environmental hot spots, in order to iteratively guide the optimization of the research and compare the project’s products to existing counterparts and provide insight regarding the products’ overall environmental impact. This includes all processes from feedstock acquisition and conversion to the production of the final product (cradle-to-gate). The assessment will investigate a broad range of impacts, including global warming and fossil resource consumption.

cycle