Rational Design & Optimization of a Synthetic Entner-Doudoroff Pathway for Improved & Controllable 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 Entner-Doudoroff (ED) pathway that increases the NADPH regeneration rate by 10-fold, compared to a wild-type E. coli MG1655 strain, exceeding alternative approaches. To do this, we employed the 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 employed MAGE genome mutagenesis 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 coverage of the 5-dimensional expression level space. We quantitatively measured the NADPH regenerate rate of genome variants using a NADPH-dependent fluorescent protein, a reporter assay using a NADPH-sensitive promoter, and a furfural reductase absorption assay. Combining sequences and measurements, we are developing a sequence-expression-activity model to forward-design new ED pathways with proportional control over a wide range of NADPH regeneration rates. The ability to rationally control the NADPH regeneration rate will substantially improve the biorenewable production of diverse chemical products.