In Vitro Metabolic Pathway for the Salvage Synthesis of Nicotinamide Cofactor

Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. On the other hand, since thermophilic enzymes are optimally active at high temperatures, there is an inherent limitation in the application of these enzymes, namely their incompatibility with thermally labile substrates, products, and cofactors. Particularly, the relatively low thermal stabilities of nicotinamide cofactors (NAD⁺/NADH and NADP⁺/NADPH) tend to be a major obstacle in the application of thermophilic enzymes owing to their pivotal roles in a wide range of enzymatic redox reaction. Recently, we developed a novel approach to engineer an in vitro artificial pathway by assembling multiple thermophilic enzymes and demonstrated one-pot bioconversions of glucose to lactate, malate, and 1-butanol through the in vitro pathways. Although they could be accomplished at sufficient production rates and yields, the conversions stopped within several hours due to the thermal decomposition of nicotinamide cofactors ^1-3). To overcome this limitation, in the present study, we constructed an in vitro metabolic pathway consisting of eight thermophilic enzymes for the salvage synthesis of NAD⁺ from its thermal degradation products (nicotinamide and ADP-ribose). NAD⁺ concentration could be kept almost constant for 15 h at 60ºC in the presence of the salvage enzymes whereas that in the control experiments without the enzymes decreased to less than 25% of the initial level after the incubation for the same period. Thus, the in vitro salvage synthetic system can markedly improve the apparent stability of NAD⁺ at high temperatures and increase the feasibility of biocatalytic chemical manufacturing with thermophilic enzymes.

¹⁾ Ye X, et al. (2012) Microb Cell Fact, 11: 120

²⁾ Ye X, et al. (2013) J Biotechnol, 164: 34-40

³⁾ Krutsakorn B, et al. (2013) Metab Eng, 20: 84-91