Currently, the synthesis of the high explosive hexanitrohexaazaisowurtzitane (CL-20) is reliant on the availability of pre-cursors derived from non-sustainable fossil fuels and highly toxic metal catalysts. This process results in the generation of hazardous waste streams that pose significant hazards, which are also costly to safely dispose. As a result, the widespread application of CL-20 is limited, due to the prohibitive costs of current synthesis routes. This project aimed to develop an innovative, scalable, and environmentally sustainable biosynthetic route to benzylamine, a key CL-20 pre-cursor, using synthetic biology. 

Synthetic biology is a powerful tool that has been leveraged to produce an increasing number of high value compounds. Unlike other microbial hosts commonly used for synthetic biology applications, the use of the halophilic bacterium Halomonas facilitates the production of high value molecules under non-sterile aqueous conditions using renewable waste materials as feedstocks. This significantly reduces the costs associated with large scale fermentation required for production at an industrial scale. Here, metabolic engineering and synthetic biology workflows that utilize Design, Build, Test and Learn platforms developed by SYNBIOCHEM have been used to develop biosynthetic pathways that facilitate the biosynthesis of benzylamine from glucose & glycerol in both E. coli and Halomonas. 

Multi-enzyme biosynthetic pathways have been assembled for use in both E. coli and Halomonas, facilitating the production of benzylamine using glucose and glycerol as feedstocks. In addition, engineered E. coli strains have been evaluated in an effort to; increase pre-cursor availability, identify native pathways with unwanted activity and improve flux through the biosynthetic pathway. The insights gained using these strains will facilitate the engineering a Halomonas strain with an improved ability to produce benzylamine.

This work has demonstrated the feasibility of benzylamine production in both E. coli and the halophilic bacteria Halomonas from carbon sources present in waste feedstocks. The continued development of biological routes toward benzylamine synthesis in Halomonas will contribute enormously to reducing the overall costs and environmental challenges associated with the production of CL-20, whilst also further strengthening the role of bio-manufacturing as a commercially viable and sustainable option for chemicals manufacture.