Engineering alanine dehydrogenase to favor nicotinamide cytosine dinucleotide for formate-driven alanine synthesis.
Abstract
To bypass the availability limitation and metabolic crosstalk associated with native reduced nicotinamide adenine dinucleotide (NADH) pools in L-alanine production, a non-natural cofactor system may offer a compelling strategy to secure independent reducing power. Here, we engineered an alanine dehydrogenase (AlaDH) from Geobacillus kaustophilus to shift its cofactor preference from NAD to the non-natural cofactor nicotinamide cytosine dinucleotide (NCD). Through three rounds of iterative mutagenesis and screening, an optimal triple mutant, A225P/V165A/S219E (designated as AlaDH*), was obtained. AlaDH* exhibited an 83-fold improvement in NCD preference, retaining 62% of the catalytic efficiency toward NCD relative to the wild-type enzyme toward NAD. Crystal structure analysis of the AlaDH*-NCD complex combined with site-directed mutagenesis revealed that cofactor binding cavity shrinkage and protein surface electrostatic map alterations contribute to NCD preference. Molecular dynamics simulations provided further insights into the mechanism of cofactor selectivity. Finally, we successfully constructed a formate-driven system by using NCD-preferring formate dehydrogenase (FDH*) and AlaDH*, demonstrating a dedicated reductive amination of pyruvate independent of NADH supply. Our results provide a new opportunity to engineer amino acid dehydrogenases for a more efficient production of amino acids, laying the foundation for future development of advanced cell factories by using NCD-linked enzymes.