Cell-free enzymatic production of glucaric acid

Cell-free biocatalysis with a number of enzymes is a fast growing and potentially high impact field in synthetic biology for the bio-manufacture of existing and novel fine or platform chemicals and biofuels. The assembly of multiple different enzymes to form synthetic pathways is a relatively new development in contrast to the single enzyme systems that have been used for decades. The cell-free approach employs enzymes outside of the cell, allowing for controllable reaction conditions and avoids metabolic repression. It prevents the diversion and loss of pathway intermediates or end-products into the cell's own metabolism. Multi-enzyme systems in a cell-free context are particularly attractive for the 'green' synthesis of high value compounds from inexpensive, simple and renewable substrates. These systems offer great versatility, allowing the investigator to 'pick, mix and test' without the need for genetic modification of the host organism and without interference from intracellular processes.Therefore, substrate conversion yields by cell-free biocatalysis can reach 100% of the theoretical value. However, major challenges for the industrial implementation of this approach includes costly enzyme preparation, enzyme stability and the dependency on expensive cofactors. Glucaric acid (GlucA) is one of the top 12 bio-based chemicals recognised worldwide for its potential impact and application in the synthesis of greener products. GlucA can be used in the production of (biodegradable) polymers, including (hydyroxylated) nylons and polyesters, offering a more sustainable and environmentally-friendly alternative to fossil fuel-derived products. The production of GlucA has been attempted mainly by chemical and microbial synthesis. This research describes a novel cell-free and multi-enzyme biocatalytic system developed for the synthesis of GlucA. The system is composed of a synthetic six enzyme pathway designed to facilitate the synthesis of GlucA from glucose-1-phosphate (G1P) which can be derived enzymatically from various natural polymers, such as cellulose or starch, and thus represents a promising approach to utilise crude biomass for GlucA production. An integrative framework was established to achieve an economical and efficient biocatalytic process which included; i)metabolic flux analysis for system optimisation, ii) the use of thermostable enzymes for improved stability and robustness of the system, iii) immobilisation and recycling of enzymes for reduced costs and iv) a cofactor regenerating tool to reduce cofactor requirements. To accomplish this framework, a novel analytical method was developed to engineer the GlucA production towards high titres and to monitor the pathway flux rapidly. It was based on ultrahigh performance liquid chromatography (UHPLC) and refractive index detection (RID) which enabled the simultaneous analysis of GlucA and its intermediates. All selected enzymes (mostly thermostable) were fused genetically to a synthetic peptide (referred to as the "linker") which displayed high affinity towards silica-based materials. Due to the non-invasive binding mechanism of the linker, the enzymes were co-immobilised successfully onto zeolite, a low costand commercially-available silica-based material. All the immobilised enzymes except the labile mouse myo-inositol oxygenase remained active, implying their suitability for application in a high temperature bioprocess, and allowed for their repeated use and recycling. Finally, an additional cofactor regenerating enzyme was integrated effectively into the cell-free process which maintained high cofactor levels while reducing the cofactor requirements of the pathway. In summary, this work presents the first cell-free production of GlucA and describes a powerful framework for viable cell-free biocatalysis based on an immobilised multi-enzyme synthetic pathway. Further work on the cell-free GlucA production is anticipated to extend the synthesis of GlucA from biomass such as cellulose and starch, and to allow increases in the overall efficiency of the system.