| Tobacco is an important cash crop,but it produces a large amount of nicotine-containing wastes in the production process.If these wastes are handled improperly,they will pollute the environment and affect human health.Therefore,tobacco and its wastes should be degraded or recycled by effective technology.Microorganisms can treat toxic chemicals more efficiently and economically than physical and chemical methods.Agrobacterium tumefaciens S33 isolated from tobacco rhizosphere degrades nicotine via a novel hybrid of the pyridine and the pyrrolidine pathways.Before the N-heterocycle can be broken down,at least six steps involved in oxidoreductive reactions are carried out in the hybrid pathway.In principle,the six steps should transfer electron from nicotine and its intermediates to the final acceptor O2 via the electron transport chain(ETC).In this thesis,the electron transfer of key enzyme in the nicotine degradation pathway of A.tumefaciens S33,6-hydroxypseudooxynicotine oxidase[Pno,which was renamed as 6-hydroxypseudooxynicotine dehydrogenase in this study(Pnd)],and the application of nicotine-converting engineered bacteria are mainly explored.The main work in the thesis are as follows:Pno has been characterized to catalyze the fourth step using the artificial electron acceptor 2.6-dichlorophenolindophenol(DCPIP).Here,biochemical,genetic,and LC-MS analysis were used to identify that Pno catalyzes the dehydrogenation of pseudooxynicotine,an analogue of the true substrate 6-hydroxypseudooxynicotine in vivo,using the electron transfer flavoprotein(EtfAB)as the physiological electron acceptor.NAD(P)+,O2,or ferredoxin could not act as electron acceptors in this reaction.It was proven that the oxygen atom in the aldehyde group of the product 3-succinoyl-semialdehyde-pyridine was derived from H2O.The growth rate of A tumefaciens S33 on nicotine decreased when etfAB genes in the nicotine-degrading gene cluster were knocked out.But the growth rate of A.tumefaciens S33 on 6-hydroxy-3-succinoylpyridine,an intermediate downstream of the hybrid pathway,did not change.It indicated that EtfAB plays an important role for efficient nicotine degradation.The electrons were found to be transferred from the reduced EtfAB to coenzyme Q by the catalysis of electron transfer flavoprotein:ubiquinone oxidoreductase(Euo).These results are helpful to understand the electron transfer process and energy metabolism involved in the oxidative degradation of nicotine,and provide novel insights into the bacterial catabolism of nicotine.In addition,nicotine was converted into a valuable compound 6-hydroxynicotine by using the engineered bacteria S33-?hno constructed earlier in the laboratory.Batch and fed-batch fermentation experiments were carried out under the optimum conditions(30?,pH 7.0,and initial nicotine concentration was 1.0 g/l).using cells cultured in glucose ammonium salt medium containing 0.1 g/1 HSP at 30? for 24 hours as biocatalysts.The molar conversion rate of each batch was 95%in batch fermentation,and the final molar conversion rate of fed-batch fermentation was up to 98.4%.The percent recovery of the product was 76.9%and the productivity was 83.9%.In summary,heterologous expression,purification,and determination of the reaction of EtfAB and Euo were performed in the thesis to confirm that EtfAB can be used as physiological electron acceptor of Pno and transfer electrons to CoQ by the catalysis of Euo,and then the electrons were further transferred to ETC.This conclusion puts the hybrid pathway of nicotine oxidative degradation by A.tumefaciens S33 to become more complete,and also provides a new insight into the electron transfer and energy metabolism of N-heterocyclic aromatic compounds in the process.The application of engineered bacteria for converting nicotine in the thesis not only provides an experimental basis for industrial production of high value compound 6-hydroxy nicotine,but also provides a new and green alternative way for the utilization of tobacco and its waste. |
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