Polyphosphate

Compounds DHAP, DHA, l-GAL, AP from potato, isopropyl-β-d-thiogalactopyranoside (IPTG), polyphosphate, ATP, l-fructose, l-tagatose, glycerol, glucose, and antibiotics were purchased from Sigma-Aldrich. All restriction enzymes and DNA ligase were purchased from Novagen (Darmstadt, Germany). Ni–NTA affinity chromatography column was purchased from QIAGEN. The yeast extract and tryptone were purchased from OXOID LID, and brain heart infusion (BHI) was purchased from Becton, Dickinson and Company. All bacterial strains and plasmids are listed in Table 4.

Enzyme activities were determined as previously described [18 (link)]. Briefly, substrate was continuously converted to NADH through a coupled reaction at 60 °C, and NADH concentration was measured by absorbance changes at 340 nm using a UV-2450 spectrophotometer (Shimazu, Japan) (Additional file 1: Figure S2). The standard mixture was composed of 400 mM HEPES–NaOH (pH 8), 60 mM NH₄Cl, 10 mM MgCl₂, 1 mM polyphosphate (Cat: 28-2880-5; Sigma, Germany), 0.2 mM ATP, 1 mM glucose and an excess amount of glucose dehydrogenase (GDH). A moderate amount of the enzyme of interest and an excess of enzymes catalyzing downstream reactions of NAD⁺ salvage synthesis was added to the standard mixture, so that NADH production rate reflected the activity of the enzyme of interest. Substrates were used at the final concentration of 0.2 mM for determining enzyme activity as follows; deamino-NAD⁺ (NaAD) for NADS, nicotinate mononucleotide (NaMN) for NaMAT, nicotinate (NA) and phosphoribosyl pyrophosphate (PRPP) for NaPRT, NA and ribose-5-phosphate (R5P) for RPK, NA and ADP-ribose for ADPRP, and NAM and PRPP for NAMase, respectively. The overall performance of the integrated strain was assessed using NAM and ADP-ribose as substrates. Reaction mixtures were incubated at 60 °C for 1 min prior to substrate addition.