DeTOX - Productive whole cell biocatalysis by engineering resistance to toxic products and substrates

Product toxicity is a major problem for many IBBE processes involving production of small molecules by living cells. Toxicity causes yield restrictions & cell lysis, & frequently affects the commercial viability of biomanufacturing. Likewise, small molecules in lignocellulosic feedstocks inhibit bacterial fermentations & ultimately depress product yields. In this CBMNet NIBB-led bid, a team of scientists from four Universities apply their fundamental expertise in systems & synthetic biology & membrane function, to engineer increased resistance to small molecules in the industrially relevant bacteria, E. coli & solventogenic Clostridia. Our innovation is to translate BBSRC-funded research in microbial stress responses, membrane structure & membrane transporters, into the development & commercialisation of innovative applications in IBBE by our 5 commercial partners. A key project output will be a commercial chassis strain, DeTox, with generally increased chemical resistance.

A major challenge in industrial biotechnology & bioenergy is to solve serious problems with yield restrictions due to product or substrate toxicity. Increasing product concentrations by >10-fold would deliver commensurate improvements in revenue from such processes, and is a viable target, given the millimolar product concentrations formed in many proposed bioprocesses at present. This is critical for commercially viable production of bulk & specialty chemicals by living cells, because many of these are toxic & need to be removed rapidly to avoid damage to the intracellular contents & cell membrane. It is also essential for the effective use of lignocellulosic substrates that contain fermentation inhibitors that exert their toxic effects by penetrating the cell. Our objective is to produce host strains with enhanced resistance to a broad range of chemical products & so provide highly-productive chassis for commercial synthetic biology. We will study the mechanisms of chemical toxicity and resistance in E. coli & solventogenic Clostridium spp., both by analyzing cellular responses during bioproduction and by experimental evolution of resistant strains. We will also apply world-leading membrane science (efflux pumps, proteomics, lipidomics & membrane biophysics) to execute novel, rational redesign of cell membranes to enhance resistance. We will combine knowledge of these systems to develop our DeTox strain platform by strain engineering, using synthetic biology standards. The chassis will be tested in small scale replicas of industrial bio-processes, & iteratively redesigned for maximum robustness under process conditions, using models describing cellular responses to toxin exposure. The outcome will be DeTox chassis, to be licensed to our partners, and DeTox gene cassettes, that can be ported to other hosts.

As described in proposal submitted to IUK