Advancement of Cofactor F420 Accessibility for Biotechnology By Synthetic Biology and Metabolic Engineering
Synthetic Biology Engineering Evolution Design SEED
2021
2021 Synthetic Biology: Engineering, Evolution & Design (SEED)
Poster Session
Poster Presenters - Accepted
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.