Rational design of a flavoenzyme for aerobic nicotine catabolism.

Enzymatic therapy with nicotine-degrading enzyme is a new strategy in treating nicotine addiction, which can reduce nicotine concentrations and weaken withdrawal in the rat model. However, when O2 is used as the electron acceptor, no satisfactory performance has been achieved with one of the most commonly studied and efficient nicotine-catabolizing enzymes, NicA2. To obtain more efficient nicotine-degrading enzyme, we rationally designed and engineered a flavoenzyme Pnao, which shares high structural similarity with NicA2 (RMSD = 1.143 Å) and efficiently catalyze pseudooxynicotine into 3-succinoyl-semialdehyde pyridine using O2. Through amino acid alterations with NicA2, five Pnao mutants were generated, which can degrade nicotine in Tris-HCl buffer and retain catabolic activity on its natural substrate. Nicotine-1'-N-oxide was identified as one of the reaction products. Four of the derivative mutants showed activity in rat serum and Trp220 and Asn224 were found critical for enzyme specificity. Our findings offer a novel avenue for research into aerobic nicotine catabolism and provide a promising method of generating additional nicotine-catalytic enzymes. IMPORTANCE: Nicotine, the main active substance in tobacco, results in cigarette addiction and various diseases. There have been some attempts at using nicotine oxidoreductase, NicA2, as a therapeutic for nicotine cessation. However, it uses cytochrome c as it is electron acceptor, which is impractical for therapeutic use compared with using O2 as an oxidant. Thus, amino acid alteration was performed on Pnao using NicA2 as model. Five of the mutants generated degraded nicotine at a rate similar to NicA2, and one of the catabolic compounds was identified as nicotine-1'-N-oxide. Our research highlights a new direction in developing enzymes that efficiently catabolize nicotine without co-enzymes and suggests that structure-similar human original MAOA (or B) may assist with nicotine cessation after being engineered.

Rational Design of a Flavoenzyme for Aerobic Nicotine Catabolism.

Enzymatic therapy with nicotine-degrading enzyme is a new strategy in treating nicotine addiction, which can reduce nicotine concentrations and weaken withdrawal in the rat model. However, when O2 is used as the electron acceptor, no satisfactory performance has been achieved with one of the most commonly studied and efficient nicotine-catabolizing enzymes, NicA2. To obtain more efficient nicotine-degrading enzyme, we rationally designed and engineered a flavoenzyme Pnao, which shares high structural similarity with NicA2 (RMSD = 1.143 Å) and efficiently catalyze pseudooxynicotine into 3-succinoyl-semialdehyde pyridine using O2. Through amino acid alterations with NicA2, five Pnao mutants were generated, which can degrade nicotine in Tris-HCl buffer and retained catabolic activity on its natural substrate. Nicotine-1'-N-oxide was identified as one of the reaction products. Four of the derivative mutants showed activity in rat serum and Trp220 and Asn224 were found critical for enzyme specificity. Our findings offer a novel avenue for research into aerobic nicotine catabolism and provides a promising method of generating additional nicotine-catalytic enzymes.

Characterization on nicotine degradation and research on heavy metal resistance of a strain Pseudomonas sp. NBB.

The remediation of polluted sites containing multiple contaminants like nicotine and heavy metals poses significant challenges, due to detrimental effects like cell death. In this study, we isolated a new strain Pseudomonas sp. NBB capable of efficiently degrading nicotine even in high level of heavy metals. It degraded nicotine through pyrrolidine pathway and displayed minimum inhibitory concentrations of 2 mM for barium, copper, and lead, and 5 mM for manganese. In the presence of 2 mM Ba2+ or Pb2+, 3 g L-1 nicotine could be completely degraded within 24 h. Moreover, under 0.5 mM Cu2+ or 5 mM Mn2+ stress, 24.13% and 72.56% of nicotine degradation were achieved in 60 h, respectively. Strain NBB tolerances metal stress by various strategies, including morphological changes, up-regulation of macromolecule transporters, cellular response to DNA damage, and down-regulation of ABC transporters. Notably, among the 153 up-regulated genes, cds_821 was identified as manganese exporter (MneA) after gene disruption and recovery experiments. This study presents a novel strain capable of efficiently degrading nicotine and displaying remarkable resistance to heavy metals. The findings of this research provide valuable insights into the potential application of nicotine bioremediation in heavy metal-contaminated areas.

Guided by the principles of microbiome engineering: Accomplishments and perspectives for environmental use.

Although the accomplishments of microbiome engineering highlight its significance for the targeted manipulation of microbial communities, knowledge and technical gaps still limit the applications of microbiome engineering in biotechnology, especially for environmental use. Addressing the environmental challenges of refractory pollutants and fluctuating environmental conditions requires an adequate understanding of the theoretical achievements and practical applications of microbiome engineering. Here, we review recent cutting-edge studies on microbiome engineering strategies and their classical applications in bioremediation. Moreover, a framework is summarized for combining both top-down and bottom-up approaches in microbiome engineering toward improved applications. A strategy to engineer microbiomes for environmental use, which avoids the build-up of toxic intermediates that pose a risk to human health, is suggested. We anticipate that the highlighted framework and strategy will be beneficial for engineering microbiomes to address difficult environmental challenges such as degrading multiple refractory pollutants and sustain the performance of engineered microbiomes in situ with indigenous microorganisms under fluctuating conditions.