High-efficient preparation of β-nicotinamide mononucleotides by crude enzymes cascade catalytic reaction.

β-nicotinamide mononucleotide (β-NMN) is a key precursor of nicotinamide adenine dinucleotide, and becomes attractive in the nutrition and health care fields, but its enzymatic synthesis is expensive. In this study, a six-enzyme cascade catalytic system was constructed to produce β-NMN. Using D-ribose and nicotinamide as substrates, the β-NMN yield reached 97.5 % catalyzed by purified enzymes. Then, after knocking out the genes encoding proteins that consume β-NMN in E. coli BL21(DE3), the similar β-NMN yield, 97.2 %, using the crude enzymes could be also obtained. After that, β-NMN synthesis was performed under increased substrate concentration, and 'modular' crude enzymes cascade catalytic reaction system was proposed to reduce the inhibition of polyphosphate on ribose-phosphate diphosphokinase activity, and the β-NMN yield reached 78.4 % at 10 mM D-ribose, which is 1.82 times of that in 'one-pot' reaction and represents the highest β-NMN preparation level with phosphoribosylpyrophosphate as the core reported till now.