General approach to engineering ketol-acid reductoisomerase cofactor preference

Imbalances between cofactor consumption of an engineered biosynthetic pathway and the reducing equivalents provided by the cell can limit yield. This imbalance can be overcome, and yield increased, by adjusting the cofactor dependencies of the pathway enzymes to match the available cofactors. Engineering of the cofactor preference of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) has been typically made on a case-by-case basis with varying degrees of success. We have developed a straightforward, general recipe for altering the cofactor specificity of a class of biotechnologically-relevant NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). We identified key cofactor specificity determining residues and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies greater than those of the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair confirmed our design principles and revealed the molecular basis of the cofactor switch.