Developing a single strain for in vitro salvage synthesis of NAD⁺ at high temperatures and its potential for...

Thermostable enzymes have several advantages over their mesophilic counterparts for industrial applications. However, trade-offs such as thermal instability of enzyme substrates or co-factors exist. Nicotinamide adenine dinucleotide (NAD⁺) is an important co-factor in many enzyme-catalyzed oxidation-reduction reactions. This compound spontaneously decomposes at elevated temperatures and basic pH, a property that limits catalysis of NAD⁺/NADH-dependent bioconversions using thermostable enzymes to short timeframes. To address this issue, an "in vitro metabolic pathway" for salvage synthesis of NAD⁺ using six thermophilic enzymes was constructed to resynthesize NAD⁺ from its thermal decomposition products at high temperatures.

An integrated strain, E. coli DH5α (pBR-CI857, pGETS118-NAD⁺), that codes for six thermophilic enzymes in a single operon was constructed. Gene-expression levels of these enzymes in the strain were modulated by their sequential order in the operon. An enzyme solution containing these enzymes was prepared by the heat purification from the cell lysate of the integrated strain, and used as an enzyme cocktail for salvage synthesis of NAD⁺. The salvage activity for synthesis of NAD⁺ from its thermal decomposition products was found to be 0.137 ± 0.006 µmol min^-1 g^-1 wet cells. More than 50% of this initial activity remained after 24 h at 60 °C. The enzyme cocktail could maintain a NAD⁺ concentration of 1 mM for 12 h at 60 °C. Furthermore, this enzyme cocktail supported continuous NAD⁺/NADH-dependent redox reactions using only NAD⁺/NADH derived from host cells, without the need for addition of external NAD⁺.

The integrated strain allows preparation of an enzyme cocktail that can solve the problem of NAD⁺ instability at high temperatures. The strain simplifies preparation of the enzyme cocktail, and thus expands the applicability of the in vitro metabolic engineering method using thermostable enzymes. Further optimization of gene expressions in the integrated strain can be achieved by using various types of ribosome binding sites as well as promoters.