One of the greatest challenges facing society is the future sustainable production of chemicals and fuels from non-petrochemical resources while at the same time reducing greenhouse gas emissions. The use of abundant and renewable lignocellulosic materials, that can be physically pretreated to yield both cellulosic and C5 fractions as feedstocks to make chemicals and fuels can be highly valuable if the process is efficient and does not lead to CO2 production.

In this project, SynConsor4Butanol, we will engineer synthetic consortia to convert, WITHOUT CO2 production, the cellulosic fraction from lignocellulosic materials to a value added product , n-butanol, that can be used both as a platform chemical or a biofuel.
Engineered synthetic consortia will be derived through a combination of interdisciplinary methodologies, synthetic biology (UNOTT, University of Nottingham; TUM, Technical University of Munich), metabolic engineering (INSA, University of Toulouse, UNOTT, University of Nottingham; TUM, Technical University of Munich), systems biology (UOG, University of Girona; INSA, University of Toulouse), and fermentation development (UOG, University of Girona).

Research Innovation (RRI) practices will be embedded within the programme of work through the participation of dedicated Social Scientists from the Synthetic Biology Research Centre (SBRC) at Nottingham. Life Cycle Analysis (LCA) and Techno-Economic Analysis (TEA) will be undertaken by Toulouse in partnership with BASF. SynConsor4Butanol will lead to the development of new "CO2 free" Sustainable production and conversion processes based on lignocellulosic feedstocks . This will lead to a value-added product, n-butanol, useful in the chemical industry and as a biofuel.

Ultimately, the developments made will lead to new sustainable industrial processes. Moreover, by combining resources and expertise, SynConsor4Butanol connects research partners in four different countries (Germany, UK, Spain, and France) with different but complementary scientific and technological expertise, thereby maximising resources and sharing the risks, costs and skills. The participants represent some of Europe's leading experts in Clostridial genetics, metabolism and engineering. The amalgamation of their expertise and resources provides the critical mass needed to compete with the rest of the world, in particular the US and China, where there is considerable activity in this field.

Technical Abstract:
1) Tool deployment will be reliant on gene transfer. C. carboxidivorans possesses a formidable restriction barrier comprising 10 R/M systems. We will optimise the current low level of transfer achieved (102-103CFU/ml per conjugation) through exclusion of target restriction enzyme recognition sites and the recombineering of appropriate methylase encoding genes into the R702 R-factor of the E. coli donor.

2) We will identify the most appropriate theophylline inducible riboswitches for Cas9 expression from our recently constructed riboswitch library (ACS Synth Biol. 2019; 8: 1379-90).

3) The most appropriate riboswitches will be used to in previously constructed CRISPR-Cas9 genome editing vectors (eg., ACS Synth Biol. 2016; 5: 1355-61) to build more effective editing tools tailored from C. carboxidiviorans. To exemplify, we will target genes with easily assayed phenotypes, such as pyrE and argH.

4) The same promoters will be used to implement CRISPR interference technology (CRISPRi) using dead Cas9 (dCas9). This may be used to knock-down gene expression in those instances where knock-out proves either impossible or not to result in the desired phenotype.

5) The tools developed will be used to implement the required metabolic engineering of C. carboxidivorans for the conversion of CO2, H2 and acetate to butyrate, a requirement of the proposed mixed consortium of clostridial strains.

6) Adaptive Laboratory Evolution will be used to evolve the final strain in chemostat cultures growing on CO2-H2-acetate mixtures to maximizethe rates of growth and butyrate formation. The evolved mutants will be characterized by DNA-seq and using a systems biology approach.

7) To ensure the adoption of RRI practices in the SynConsor4Butanol consortium, we will run a series of 3 workshops. A key output from the final workshop will be the drafting of a multi-disciplinary authored publication outlining insights from the RRI work in SynConsor4Butano

Potential Impact:
One of the greatest challenges facing society is the future sustainable production of chemicals and fuels from non-petrochemical resources while at the same time reducing greenhouse gas emissions. The use of abundant and renewable lignocellulosic materials, that can be physically pretreated to yield both cellulosic and C5 fractions as feedstocks to make chemicals and fuels can be highly valuable if the process is efficient and does not lead to CO2 production.

The focus of SynConsor4Butanol is to derive a process that utilizes artificial consortia, engineered to produce n-butanol at very high yield from cellulose and H2 without any loss of carbon in the form of CO2. N-butanol can be used both as a solvent, a building-block chemical and a jet fuel.Applicationsof n-butanol are diverse from paints, adhesives and inks to food ingredients, cosmetic and personal care, to pharmaceuticals, plastic and polymers. The global market for n-butanol is about 4M T/yr. and worth approx. $6B, rising to $9.2B by 2015. Supply is dominated by large chemical companies such as Dow, BASF, Eastman and Oxea. The average growth is around 3.2%/yr with demand concentrated in North America (28%), Western Europe (23%) and North East Asia (35%). Nearly all n-butanol produced today is synthetic and derived from a petrochemical route based on propylene oxo synthesis. Synthetic butanol production costs are linked to the propylene market and sensitive to the price of crude oil. Most butanol is converted into higher value esters i.e. butyl acetate, butyl acrylate and butyl methacrylate (BMA) or butyl glycol ethers for surface coatings, inks and cleaning. The European market for BMA > 100,000 T/yr, and worth >300 M euro at a cost of 3000 E/T. Renewable n-butanol also has huge potential as an advanced biofuel. The global biofuels market exceeds $50 B today and is forecasted to reach $80B by 2020. As a blend stock for gasoline alone, the biobutanol demand has been forecast at more than 120 million T/yr. Renewable n-butanol can also be converted through catalysis to jet fuel to provide renewable alternatives to the $270B global aviation fuels market.