Advancement of Cofactor F420 Accessibility for Biotechnology By Synthetic Biology and Metabolic Engineering

The deazaflavin cofactor ferredoxin 420 (F₄₂₀), synthesised by some Archaea and Eubacteria, is involved in energy metabolism, xenobiotic biodegradation, and biosynthesis of secondary metabolites. Due to its low redox potential, F₄₂₀ could potentially be used in novel industrial biocatalysis. However, one major barrier for such application is the production of cofactor F₄₂₀ at scale and at low cost. Here, through applying synthetic biology and metabolic engineering strategies, we present: (1) the development of an engineered E. coli production system for the biosynthesis of F₄₂₀, (2) improvement of F₄₂₀ space-time production yield by the recombinant E. coli up to ~40-fold higher than the yield from the wildly-used F₄₂₀ producer, Mycobacterium smegmatis, and (3) identification and construction of key gene knock-out E. coli mutants which further enhances F₄₂₀ productivity. The bacterial F₄₂₀ biosynthesis pathway was revised and chosen for heterologous expression in E. coli. The biosynthesis of main precursor for F₄₂₀, dehydro-F₄₂₀, was found to be dependent on the utilisation of phosphoenolpyruvate (PEP) in this revised pathway. In subsequent works, we showed that, in addition to the choice of carbon source as substrate, PEP is a key metabolite influencing intracellular F₄₂₀ concentrations. We further improved F₄₂₀ productivity by over-expressing PEP synthase. Additionally, we developed an updated genome-scale metabolic model of E. coli capable of predicting metabolic-level cellular response to the introduction of orthogonal F₄₂₀ biosynthesis pathway under given growth conditions. We identified gene knock-outs resulted in improved F₄₂₀ yields in silico. These results provide important insights on establishing E. coli as an industrial F₄₂₀-production system for applications in biocatalysis.