Part:BBa_K2569036

Nicotine oxidase NicA2

Expressing this BioBrick in E. coli enables the bacterium to degrade nicotine. Nicotine is one of the most harmful ingredients in cigarette and is the biggest resistance to smoking cessation. NicA2 is a monomer with a size of 53 kDa t, catalyzing the key first step in nicotine catalytic metabolism. Heterologously overexpressing NicA2 in E. coli can convert nicotine to pseudo-oxidation can be achieved by resting cells of E. coli as a catalyst in a whole-cell reaction.

Contribution: Botchan Lab Tokyo 2020

nicA2 expression in E. coli .

nicA2 gene from Pseudomonas putida S16 was cloned and expressed in Escherichia coli BL21(DE3). After IPTG induction, the lysate of E. coli containing pET28a-nicA2 and E. coli containing the vector, pET28a, were analyzed by SDS-PAGE (Figure1). Large amount of proteins approximately 50 kDa were found only in the lane of the lysate of E. coli containing pET28a-nicA2 .

Figure 1. SDS-PAGE analysis of E. coli expressing nicA2 gene. Lane M, protein molecular marker; lane 1, cell extracts of E. coli containing pET28a vector; lane 2, cell extracts of E. coli containing pET28a-nicA2.

E. coli cells containing pET28a-nicA2 were collected by centrifugation. After washing with PBS buffer, these cells were resuspended with ddH2O and 3 g l-1 nicotine. Thin layer chromatography analysis and gass chromatography mass spectrometry were conducted using them (figure2). The spots of nicotine and N-methylmyosmine were found in the TLC plate. GC-MS results show identifies N-mehtylmyosmine.

Figure 2. C. Mass spectra of N-methylmyosmine (P) as determined by GC-MS analysis. D. Thin layer chromatography analysis of products formed by incubation of nicotine and whole cells of transformant pET28a-nicA2. P stands for N- methylmyosmine.

Article Source: Systematic Unraveling of the Unsolved Pathway of Nicotine Degradation in Pseudomonas Tang H, Wang L, Wang W, Yu H, Zhang K, et al. (2013) Systematic Unraveling of the Unsolved Pathway of Nicotine Degradation in Pseudomonas. PLOS Genetics 9(10): e1003923. https://doi.org/10.1371/journal.pgen.1003923

Sequence and Features BBa_K2569036 SequenceAndFeatures