Optimisation of enzyme cascades for chiral amino alcohol synthesis in aid of host cell integration using a statistical experimental design approach.

Chiral amino alcohols are compounds of pharmaceutical interest as they are building blocks of sphingolipids, antibiotics, and antiviral glycosidase inhibitors. Due to the challenges of chemical synthesis we recently developed two TK-TAm reaction cascades using natural and low cost feedstocks as substrates: a recycling cascade comprising of 2 enzymes and a sequential 3-step enzyme cascade yielding 30% and 1% conversion, respectively. In order to improve the conversion yield and aid the future host strain engineering for whole cell biocatalysis, we used a combination of microscale experiments and statistical experimental design. For this we implemented a full factorial design to optimise pH, temperature and buffer type, followed by the application of Response Surface Methodology for the optimisation of substrates and enzymes concentrations. Using purified enzymes we achieved 60% conversion for the recycling cascade and 3-fold improvement using the sequential pathway. Based on the results, limiting steps and individual requirements for host cell metabolic integration were identified expanding the understanding of the cascades without implementing extensive optimisation modelling. Therefore, the approach described here is well suited for optimising reaction conditions as well as defining the relative enzyme expression levels required for construction of microbial cell factories.