Engineering of <i>Escherichia coli</i> Glyceraldehyde-3-Phosphate Dehydrogenase with Dual NAD⁺/NADP⁺ Cofactor Specificity for Improving Amino Acid Production

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the central metabolism of microbial cells. GAPDHs differ in cofactor specificity and use NAD+, NADP+, or both cofactors, reducing them to NADH and NADPH, respectively. Sufficient NADPH supply is one of the critical factors required for synthesis of the amino acids l-lysine, l-threonine, and l-proline in industrially important Escherichia coli-based producer strains. E. coli cells have NAD+-dependent glycolytic GAPDH. One reasonable approach to increase NADPH formation in cells is to change the specificity of the GAPDH from NAD+ to NADP+. In this study, we modified the cofactor specificity of E. coli GAPDH by amino acid substitutions at positions 34, 188 and 189. Several mutant enzymes with dual NAD+/NADP+ cofactor specificity were obtained, and their kinetic parameters were determined. Overexpression of the genes encoding the resulting mutant GAPDHs with dual cofactor specificity in cells of l-lysine-, l-threonine-, and l-proline-producing E. coli strains led to a marked increase in the accumulation of the corresponding amino acid in the culture medium. This effect was more pronounced when cultivating on xylose as a carbon source. Other possible applications of the mutant enzymes are discussed.

Keywords

• glyceraldehyde-3-phosphate dehydrogenase
• NAD⁺
• NADP⁺
• cofactor specificity
• l-threonine%22%2C%22default_operator%22%3A%22AND%22%7D%7D%7D"> <span style="font-variant: small-caps">l-</span>threonine
• l-lysine%22%2C%22default_operator%22%3A%22AND%22%7D%7D%7D"> <span style="font-variant: small-caps">l-</span>lysine