A study published in Nature on October 20, 2022, by a team of researchers from Peking University, the National Institutes of Health (NIH) led by Frank Gonzalez, the First Affiliated Hospital of Zhejiang University School of Medicine led by Chaohui Yu, Fudan University School of Basic Medical Sciences led by Yang Li, and the First Affiliated Hospital of Wenzhou Medical University led by Minghua Zheng, explored the process. They found that nicotine, ingested during smoking, accumulates in the gut and accelerates the progression of NAFLD. Notably, they discovered that nicotine can be efficiently degraded by the human gut commensal bacterium B. xylanisolvens.
To identify the key enzyme responsible for nicotine degradation, the research team utilized chromatographic and spectroscopic techniques and identified the nicotine metabolite as 4-hydroxy-1-(3-pyridinyl)-1-butanone (HPB), which significantly differs from host nicotine metabolism products, representing a novel nicotine metabolite. Subsequently, through whole-genome sequencing and functional gene analysis, the team identified the potential nicotine degradation enzyme NicX in B. xylanisolvens, and in vitro enzymatic assays confirmed NicX's role in nicotine degradation.
Cell surface display system involves fusing the target protein with an anchor protein to express the protein on the cell surface. In this project, we are utilizing E. coli surface display system, fusing NicX with four different anchor proteins. Tianjin University established the parts anchoring protein INPNC (BBa_K1921015) in 2016, and in 2023, we used INPNC to display NicX on the cell surface. This system allows our fusion protein to degrade nicotine in the environment without purification.
In conclusion, our research efforts are driven by the practical goal of contributing to the degradation of nicotine, thereby playing a key role in environmental protection and human health.
To identify the key enzyme responsible for nicotine degradation, the research team utilized chromatographic and spectroscopic techniques and identified the nicotine metabolite as 4-hydroxy-1-(3-pyridinyl)-1-butanone (HPB), which significantly differs from host nicotine metabolism products, representing a novel nicotine metabolite. Subsequently, through whole-genome sequencing and functional gene analysis, the team identified the potential nicotine degradation enzyme NicX in B. xylanisolvens, and in vitro enzymatic assays confirmed NicX's role in nicotine degradation.
Cell surface display system involves fusing the target protein with an anchor protein to express the protein on the cell surface. In this project, we are utilizing E. coli surface display system, fusing NicX with four different anchor proteins. Tianjin University established the parts anchoring protein INPNC (BBa_K1921015) in 2016, and in 2023, we used INPNC to display NicX on the cell surface. This system allows our fusion protein to degrade nicotine in the environment without purification.
In conclusion, our research efforts are driven by the practical goal of contributing to the degradation of nicotine, thereby playing a key role in environmental protection and human health.
Molecular Cloning
For molecular cloning, we selected pET28a as vector. We successfully amplified three gene segments of NicX (as control group), INPNC-NicX-Histag(Figure 1a). Then we digested and connected all the segments to pET28a vector through two restriction enzymes of NcoI and XhoI. At present, recombinant plasmids have been successfully constructed (Figure 1a).
Figure 1. (a) PCR results. M:marker 7,8,9:INPNC-NicX-Histag (1992bp) 19,20,21:NicX(1293bp)
Figure 1. (a) PCR results. M:marker 7,8,9:INPNC-NicX-Histag (1992bp) 19,20,21:NicX(1293bp)
Induced Expression
Figure 1. (a) SDS-PAGE of INPNC- NicX- histag(1989bp) & NicX(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 1:INPNC- NicX- histag(1989bp)Before induction 2, 3, 4, 5, 6:After induction; 2: 37℃ 0.3mM IPTG,3: 37℃ 0.5mM IPTG,4: 37℃ 0.7mM IPTG,5: 37℃ 1mM IPTG 6: NicX(1293bp) Before induction 7,8:After induction; 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG (b) 1: 37℃ INPNC- NicX- histag(1989bp)Before induction 2-6:After induction; 2: 37℃ 0.3mM IPTG,3: 37℃ 0.5mM IPTG,4: 37℃ 0.7mM IPTG,5: 37℃ 1mM IPTG；6: 37℃ NicX(1293bp) Before induction 7-8:After induction; 7: 37℃ 0.3mM IPTG,8: 37℃ 0.5mM IPTG
Figure 2. (a) SDS-PAGE of INPNC- NicX-histag(1989bp) & NicX(1293bp) transformed into BL21 expressing strains. Induction time: 15h M:GoldBand Plus 3-color Regular Range Protein Marker(8-180 kDa), 3: NicX (1293bp) Before induction 1,2: After induction; 1: 37℃ 0.5mM IPTG, 2: 37℃ 0.3mM IPTG 8:INPNC- NicX- histag(1989bp) Before induction 4,5,6,7:After induction; 4: 37℃ 1mM IPTG, 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG,7: 37℃ 0.3mM IPTG(b)3: 37℃ NicX (1293bp) Before induction 1-2: After induction; 1: 37℃ 0.5mM IPTG, 2: 37℃ 0.3mM IPTG 8: 37℃ INPNC- NicX- histag(1989bp) Before induction 4-7:After induction; 4: 37℃ 1mM IPTG, 5: 37℃ 0.7mM IPTG, 6: 37℃ 0.5mM IPTG,7: 37℃ 0.3mM IPTG
HPLC-MS Result
Due to budget constraints, we had to narrow down our testing to one concentration using LC-MS. We had roughly 320 cells in a 50μl reaction system, yielding about 0.5ng of protein at a reaction rate of 2μmol/min·μg. Here's the kicker: when we scaled up to 10ng of protein, the reaction rate soared to 4μmol/min·μg. And when we directly used 10ng of purified protein, the reaction rate matched, 3.9μmol/min·μg. Plus, surface display tech cuts out the need for cell disruption and purification, which means much lower time and cost compared to protein purification. It's exactly what we were aiming for. Our surface display experiment was a success!
References
1. Sun, F., Pang, X., Xie, T., Zhai, Y., Wang, G., & Sun, F. (2015). BrkAutoDisplay: functional display of multiple exogenous proteins on the surface of Escherichia coli by using BrkA autotransporter. Microbial Cell Factories, 14, 1-12. https://doi.org/10.1186/s12934-015-0316-3
2. Chen, B., Sun, L., Zeng, G., Shen, Z., Wang, K., Yin, L., ... & Jiang, C. (2022). Gut bacteria alleviate smoking-related NASH by degrading gut nicotine. Nature, 610(7932), 562-568. https://doi.org/10.1038/s41586-022-05299-4
2. Chen, B., Sun, L., Zeng, G., Shen, Z., Wang, K., Yin, L., ... & Jiang, C. (2022). Gut bacteria alleviate smoking-related NASH by degrading gut nicotine. Nature, 610(7932), 562-568. https://doi.org/10.1038/s41586-022-05299-4