Microorganism and inoculum preparation
A gene-modified strain (B. subtilis168/pHT01-aprN1 ) was used to express NK. The medium for flask and fed-batch culture was as follows: yeast extract, 60 g L-1; glucose, 40 g L-1; glycerol, 20 mL L-1; NH4Cl, 3 g L-1; K2HPO4·3H2O, 1 g L-1; MgSO4·7H2O, 2 g L-1; MnSO4·H2O, 0.02 g L-1; FeSO4·7H2O, 0.05 g L-1; CoCl2·6H2O, 0.01 g L-1; ZnCl2, 0.01 g L-1; 2,6-pyridinedicarboxylic acid, 0.167 g L-1. Glucose and MgSO4·7H2O were autoclaved separately and aseptically added to the medium. Nutrient solution contained 100 g L-1 yeast extract and 250 mL L-1glycerol was used as feed medium. To avoid plasmid loss and prevent microbial contamination, chloramphenicol (5 μg mL-1) was added to all media after being dissolved in anhydrous ethanol and filtered through a 0.22 μm membrane.