Rational Engineering of Advanced Clostridia for Transformational Improvements in Fermentation (REACTIF)

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CONTEXT n-Butanol is a valuable platform chemical with a large market (>3 M tonne/yr). It is used as a chemical precursor for a variety of polymers, plastics and solvents with a global market worth ~$8 billion). Average growth is about 3.2% with demand concentrated in N. America (28%), Western Europe (23%) and North East Asia (35%). Virtually all n-butanol today is synthetic and produced from propylene (derived from oil). The fermentation route to butanol is an old industrial process that was developed in the UK in 1912 and spread quickly to the N. America, S.Africa, Japan and Russia. It is reliant on a bacteria called Clostridia, which ferments sugar-rich liquors and converting them to butanol, in a similar way to which yeasts is employed in brewing to make ethanol. The commercial butanol fermentation process became uneconomic in the mid 1950's when it could no longer compete with petro-derived equivalents from low cost oil and gradually disappeared. More recently, the fermentation has been re-commercialized in China with at least 6 new plants and capacity for >300,000 T solvent/yr. However, these plants are struggling because the technology is not sufficiently advanced, the bacterial strains produce low amounts of butanol (due to butanol toxicity) and they rely on expensive starch-rich plant material as the source of sugar for the fermentation. Aside from the prohibitive cost of such feedstocks, their use for this purpose conflicts with a possible use as a food for human consumption. There is, therefore, a need to derive the sugar used from non-food plant derived material, the so-called lignocellulose component. Lignocellulose is the most abundant source of carbon on the plant. However, it is particularly recalcitrant to breakdown, and the treatments required (acid, alkali or steam explosion) to its conversion into sugar frequently leads to the formation of by-products that inhibit bacterial growth, so-called 'inhibitors'.AIMS & OBJECTIVESToday, advances in fermentation technology and the utilization of cheaper feedstocks, together with novel gene-based methods for strain improvement have the potential to transform fermentation process economics. We have assembled an exceptional group of academic and industrial partners, with specific expertise in the Clostridia fermentation. The industrial partners bridge the commercial supply chain (from feedstock to butanol production) whilst the academic groups focus on delivery of advanced microbes and superior fermentation performance using an array of advanced molecular biology techniques. Our objectives are to:-1. identify and characterise tolerance traits (to high butanol titres and lignocellulose-derived inhibitors), both in historical strains that were used commercially over four decades and in current production strains following the implementation of novel directed evolution strategies;2. transfer the identified alleles into current, robust production strains, together with rationale metabolic engineering to improve product titres, and;3. assessment of strain(s) performance on cellulosic feedstocks at lab and pilot scaleAPPLICATIONS AND BENEFITSThe project will deliver advanced Clostridia strains that offer a transformational change to fermentation performance and process economics of butanol production. Advanced strains not only improve fermentation performance but they will enable plant owners to switch to lower cost and more environmentally friendly cellulosic feedstocks. The production of butanol from renewable plant biomass as opposed to fossil fuels will provide a net reduction in greenhouse gas emission, contributing to a reduction in global warming and reducing the effects of environmental pollution on human health. Biobutanol has the potential to directly replace all synthetic butanol addressing a specific market need in the chemical industry for cost efficient renewable chemical replacements, particularly in Europe & N. America