In vitro metabolic engineering for the salvage synthesis of NAD(.).
Metabolic engineering
confidence
Key findings
In vitro salvage pathway for NAD+ synthesis from degradation products using eight thermophilic enzymes; NAD+ concentration kept constant for 15h at 60°C.
View source on PubMed (PMID 26912312) ↗
- Sample size
- Not reported
- Population
- In vitro enzyme system (recombinant E. coli heat-treated crude extracts)
- Dosing
- Optimized concentrations of eight thermophilic enzymes
- Duration
- 15h
- Route
- In vitro biocatalytic system
- Blinding
- not_reported
- Controls
- none
- Drug class
- coenzyme
Full abstract
Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h.