Molecular Cloning,Expression And Functional Analysis Of Genes Involved In Nicotine-degradation Pathways In Ochrobactrum Intermedium SCUEC4 Strain

Nicotine,also known as nicotine,is a natural alkaloid found in Solanaceae plants.It is also a toxic nitrogen heterocyclic compound composed of a Pyridine ring and a Tetrahydropyrrole ring and is the most abundant in tobacco.Nicotine has a stable chemical structure,and is solvent-soluble in water,ethanol and etc.Meanwhile,it is difficult to be degraded in the natural environment.Nicotine produced during tobacco cultivation and tobacco production will infiltrate into the ground with rainwater and pollute the water.Among smokers,nicotine is the main substance causing addiction.Long-term intake of nicotine will cause cardiovascular and cerebrovascular diseases in human,and even causes cancer and teratogenesis and so on.Traditional nicotine degradation methods have a series of problems such as high cost,low efficiency,and potential secondary pollution.To date,the degradation of nicotine by microorganisms is a low cost,highly efficient,and environmentally friendly method.In the early stage of this study,a large number of strains with nicotine degradation ability were screened and isolated from the soil of the tobacco planting base in Xiangyang City,Hubei Province.One of the strains with high nicotine degradation ability was studied,which was identified as Ochrobactrum intermedia SCUEC4.The whole genome of O.intermedia was sequenced.Based on these sequences,we predicted the nicotine degradation metabolic pathway and analyzed the degradation gene clusters.Three genes ocnC,ocnE and ocnH related to the nicotine degradation and metabolism pathway of SCUEC4 strain were cloned and expressed.Then,the biological functions and enzymatic characteristics of ocnE and ocnH genes were analyzed.The main experimental results of this paper are shown as follows:(1)The whole genome of O.intermedia SCUEC4 strain obtained from Nanopore sequencing technology demonstrate that the strain genome contains two chromosomes and two plasmids.The results indicate that the total number of nucleotides of the genomic DNA is 4,696,857 bp with GC content 57.67%,a number of CDS 4666,a number of t RNA 72,and 8 gene islands.There are 3,217 protein genes annotated by COG functional annotation,and 1487 encoding genes by KEGG function.The genomic DNA contains two chromosomes and two plasmids,among which the tig00003 plasmid has related genes of the nicotine degradation gene clusters.After a comparative analysis,it was found that the gene clusters has a high similarity to the nicotine degradation gene cluster of Agrobacterium tumefaciens SCUEC1 strain.They are functional genes involved in the pyridine pathway and pyrrolidine pathways.They include the nicotine hydroxylase enconding gene ocnA,6-hydroxy nicotineoxidase enconding gene ocnB,aldehyde dehydrogenase enconding gene ocnC,6-hydroxy-3-succinylpyridine3-monooxygenase enconding gene ocnD,2,5-dihydroxy-pyridine dioxygenase enconding gene ocnE,N-formylmaleicacid deformase enconding gene ocnF,the maleamate amidase enconding gene ocnG,and maleate isomerase enconding gene ocnH and so on.These reulsts are helpful in predict the pathway of nicotine degradation of VPP in O.intermedia SCUEC4 strain.(2)The entire DNA of O.intermedia SCUEC4 strain was used as a template to obtain ocnC,ocnE,and ocnH gene fragments by PCR.The length of ocnC gene,ocnE gene and ocnH gene were 1,344 bp,1,029 bp and 585 bp,respectively.Bioinformatic analytic results demonstrate that the OcnC protein is a hydrophobic non-secreted protein with a molecular weitht 48.0 k Da.It has 91.7%homology with aldehyde dehydrogenase from O.intermedia sp.SJY1 strain and belongs to the aldehyde Catalase superfamily.The OcnE protein is a hydrophiclic non-secreted portein with a molecular weight 37.6k Da.It has 97.4%homology with 2,5-dihydropyridine dioxygenase in Ochrobactrum intermedia sp.SJY1 strain and leucine aminopeptidase in Rhizobiales strain,which belongs to the peptidase M29 superfamily.The OcnH protein is a hydrohobic non-secreted protein with a molecular weight 20.6 k Da.It has 100%homology with maleic cis-trans isomerase derived from O.intermedia sp.SJY1 strain and belongs to Asp/Glu racemase superfamily.In the secondary structures of OcnC,OcnE and OcnH protein,?-helix accounts for 46.98%,20.76%,and 44.85%respectively;while?-sheet accounts for 11.63%,23.98%,and 9.79%respectively.(3)The recombinant plasmids pET28a(+)-ocnC,pET28a(+)-ocnE and pET28a(+)-ocnH were transformed into E.coli BL21(DE3)strain for proteins expression.The effects of different concentrations of IPTG,induction temperature and induction time on the expression of the recombinant protein OcnC,OcnE and OcnH were studied.The results of SDS-PAGE showed that the sizes of OcnC,OcnE and OcnH proteins were the same as predicted.The optimal IPTG concentrations of OcnC,OcnE and OcnH proteins were 1.0 mmol/L,0.7 mmol/L,and 0.6 mmol/L,respectively.The optimal expression temperatures of OcnC,OcnE and OcnH proteins were 37??30?and25?).The best induced expression times of OcnC,OcnE and OcnH proteins were 20 h,30 h and 20 h,respectively.The solubility of the proteins OcnC,OcnE and OcnH were analyzed,while the OcnE and OcnH proteins were purified using nickel columns.The results showed that the proteins OcnC,OcnE and OcnH were mostly distributed in the precipitates,while were less detected in the supernatant.The OcnE and OcnH protein with high concentrations and purities could be collected by 200 mmol/L imidazole eluent.(4)The biological function of the OcnE protein was detected.Using2,5-dihydroxypyridine(2,5-DHP)as the reaction substrate,the purified OcnE protein was used for the enzymatic reaction.Within the time range from 0 to 35 min,the absorbance value OD₃₂₀ of the system decreased with the increase of reaction time.The absorbance value gradually decreased from 0.8017 to 0.1167,meaning the substrate2,5-DHP was decreasing.These results proved that the OcnE protein has2,5-dihydroxypyridine dioxygenase activity.To investigate the effects of different temperature,pH and metal ions on the biological activity of OcnE protein,as well as the thermal stability and pH stability of OcnE protein were analyzed.The results show that the optimal reaction temperature of OcnE protein is 25?,the optimal reaction pH is 7.0.Metal ions Fe²⁺and Mg²⁺have obvious promotion effects on the OcnE protease activity.Among them,Fe²⁺has the best effect on promoting enzyme activity.The OcnE protein was placed in 0?,37?,40?,50?,60?,and 70?water baths for 2 h,and sampled every 30 min.The OcnE protease activity in the 0?water bath for 120 min had less effect on enzyme activity,and the relative enzyme activity was 100%fell to 91.57%.After 120 min treatment in 37?,40?,50?,60?and 70?water baths,relative enzyme activities of the OcnE protein gradually decreased to 57.03%,37.36%,28.85%,7.47%and 1.70%,respectively.These data shown that the OcnE protein is relatively stable at 0?;Then,the OcnE protein was treated in Tris-HCl buffer at pH 5.0?8.0 for4 h at 0?and sampled every 1 h.The results demonstrated that the protease activity of the OcnE protein decreased with the increase of time.After 4 h treatment,the protease activity of the OcnE protein was at pH 5.0,6.0,7.0 and 8.0 buffers gradually decreased to 18.56%,36.62%,59.55%and 26.94%,respectively.(5)The functional of the OcnH protein was measured.The retention times of the maleic acid standard and the fumaric acid standard were 4.01 min and 5.47 min,respectively.Using maleic acid as the reaction substrate,the purified OcnH protein was used to perform the enzymatic reaction.The test results showed that the value of the maleic acid peak area was 9763.59 m AU·s and the fumaric acid peak area was 0 m AU·s when the enzymatic reaction time was 0 min.After incubation for 30 min,the peak area of maleic acid reduced to 5186.46 m AU·s,and the peak area of fumaric acid increased to 3856.63 m AU·s.Under the catalytic action of the OcnH protein,the peak area of maleic acid gradually decreases in a time-dependent manner,while the peak area of fumaric acid gradually increases.These data indicated that the OcnH protein has maleic acid cis-trans isomerase activity.Using different concentrations of maleic acid as a substrate,the Michaelis constant of the OcnH protein was measured using a double reciprocal method to obtain the equation y=0.02893x+0.06839.The Km of the OcnH protein was0.4230 m M,while the Vmax was 14.62 U/mg.