Production of biochemical using a nar promoter based expression system in Escherichia coli

The nar promoter, one of dissolved oxygen (DO) dependent promoters, has been characterized for various reporter protein expression in E.coli. In this study, based on an advantage of cost effective expression and efficient protein expression system, the nar promoter expression system, in micro-aerobic conditions without nitrate. This promoter was evaluated by the expression of biosynthesis pathway enzymes for production of D-lactate, 2,3-butanediol (2,3-BDO), and 1,4-butanediol (1,4-BDO) and compared with constitutive lac promoter obtained by removing operator binding site. The D-lactate production was similar to constitutive lac promoter expression system. However, under control of nar promoter, the 2,3-BDO and 1,4-BDO production were yielded three-fold higher. In nar promoter expression system, the D-lactate, 2,3-BDO, and 1,4-BDO were produced 13.50 g/L (0.76 g lactate/g glucose), 10.67 g/L (0.53 g 2,3-BDO/g glucose), and 0.55 g/L (0.06 g 1,4-BDO/g glucose), respectively, in flask scale fermentation. Furthermore, to investigate the effect of DO level on the nar promoter, DO level was maintained at approximately 80% until the OD₆₀₀ reached 1.0 in aerobic conditions, and then it dropped to 1-2% for induction of each pathway enzyme using bioreactor. As a results, we could obtain similar titer for D-lactate and 2,3-BDO with flask scale fermentation. Interestingly, 1,4-BDO production was increased to 3.14 g/L (0.16 g 1,4-BDO/g glucose) which was about six fold higher than flask scale fermentation. In conclusion, the nar promoter can be introduced as a useful promoter for expression of biosynthesis pathway genes to optimize the pathway for enhanced production biochemical in microaerobic cultivation.