(664c) Design & Optimization of a Synthetic Entner-Doudoroff Pathway for Efficient NADPH Regeneration

NADPH is an essential cofactor for the biosynthesis of several high-value chemicals, including isoprenoids, fatty acid-based fuels, and biopolymers. Tunable control over all potentially rate-limiting steps, including the NADPH regeneration rate, will be crucial to maximizing production titers. We have engineered a synthetic pathway that increases the NADPH regeneration rate by up to 6-fold, compared to a wild-type E. coli MG1655 strain. To this end, we employed our newly developed Operon Calculator to design the 5-enzyme pathway, removing all cryptic genetic elements and optimizing sequences for maximum expression control. We integrated the synthetic bacterial operons into the E. coli MG1655 EcNR2 genome, and used MAGE genome mutagenesis approach to introduce ribosome binding site mutations. To direct genome mutagenesis, we employed the RBS Library Calculator to identify the smallest combinatorial mutation library that maximized the coverage of the 5-dimensional expression level space. We identified genome variants with high NADPH regeneration rate by screening with a NADPH-dependent blue fluorescent protein. Strains with optimized NADPH production were further confirmed through a furfural reductase assay. Finally, we showed that the synthetic ED pathway is able to improve Neurosporene production. The ability to rationally control the NADPH regeneration rate will substantially improve the biorenewable production of diverse chemical products.