Ensifer Meliloti CGMCC 7333 Degrades The Nicotinic Insecticide Acetamiprid And Biotransforms 3-indole Acetonitrile

The author's laboratory isolated a N2-fixing bacterium Ensifer meliloti CGMCC 7333 with the ability of degrading neonicotinoid insecticide thiacloprid(THI)to the major metabolite THI amide derived from the cyano moiety by hydrolysis of THI.In present study,E.meliloti CGMCC 7333 was further studied on the metabolism of another neonicotinoid insecticide acetamiprid(ACE)and the transformation of plant hormones precursor 3-indole acetonitrile(IAN).Biotransformation of ACE by resting cells of E.meliloti CGMCC 7333 resulted in the formation of a polar metabolite with an HPLC retention time of 3.80 min,and no comparable peak was observed upon analysis of the controls containing the inoculated bacterium alone or the substrate alone.The metabolite was identified as IM-1-2 from the cyano moiety by hydrolysis of ACE,using liquid chromatography-mass spectrometry(LC/MS)and nuclear magnetic resonance(NMR)analysis.E.meliloti CGMCC 7333 degraded 13.5%of 2.12 mmol/L ACE in 12 h with a half-life of 63 h and 96%of the reduced ACE was converted to IM-1-2.ACE degradation by E.meliloti CGMCC 7333 was fited into the the exponential regression curve equation,y=2.1233e-0.0109t,and the correlation coefficient(R2)was 0.99.Escherichia coli Rosetta overexpressing the nitrile hydratase(NHase)from CGMCC 7333 converted 96.9%of 2.04mmol/L ACE in 100 min with a half-life of 19.7min and 93.3%of the reduced ACE was converted to IM-1-2.Its exponential regression curve equation was y=2.0206e-0.0352t and the correlation coefficient(R2)was 0.99.The bioefficacy examination of IM-1-2 against the horse bean aphid Aphis Craccivora Koch indicated that when the concentration of IM-1-2 was increased to 1 ppm,the mortality level was only 13.56%,which meaning that IM-1-2 showed significantly lower insecticidal activity than ACE against the aphid A.craccivora Koch.For ACE biodegradation,the molar conversion rates at 12,24,36,48,72,and 96 h were 96.0,94.6,90.5,73.7,57.2,and 43.8%,respectively.Interestingly,the molar conversion rate of ACE degradation declined significantly after transformation for 36 h.This result indicated that the IM-1-2 formed by hydration of ACE by E.meliloti CGMCC 7333 may have been further degraded.The experimental group with bacterial inoculation and the control without CGMCC 7333 inoculation showed apparent IM-1-2 degradation.After incubation for 8 d,the amount of IM-1-2 was reduced by 1.40 and 1.32 mmol/L with bacterial inoculation and with IM-1-2 alone,respectively,and the corresponding degradation rates were 67.5%and 62.9%,respectively.The half-lives for IM-1-2 degradation for the experimental and control groups were fitted into firstorder dissipation kinetics(R2=1.00 and 0.99,respectively)and were calculated to be 4.9 and 5.5 d,respectively.There was no significant difference(P>0.05)in IM-1-2 degradation between the experimental and control groups at any sampled time.These results indicated that IM-1-2 degradation did not result from bacterial transformation and instead resulted from spontaneous chemical degradation.Three new peaks with retention times of 4.26,4.53,and 6.84 min,respectively,were observed in the LC-MS analysis of the control group sample without bacterial inoculation after incubation for 8 days.These peaks represent IM-1-4,ACE-NH,ACE-NH2 respectively.The resting cells of E.meliloti CGMCC 7333 converted IAN to a polar metabolite with retention time of 4.18 min,while the control of substrate IAN alone or bacterium alone did not appeared this peak.The metabolite was identified as indole-3-acetoamide(IAM)using LC-MS and NMR analysis.After transformation for 48 h,the amount of IAN was reduced from 6.41 mmol/L to 0.06 mmol/L and the product IAM was increased to 5.99 mmol/L.The molar conversion rate was 94.4%.The NHase gene cluster of E.meliloti CGMCC 7333 is 1671-bp in length and contains an ?-subunit of 642-bp,an ?-subunit of 660-bp and a hypothetical accessory protein coding gene of 387-bp.In previous studies,a recombinant plasmid pET28a harboring the CGMCC 7333 NHase coding gene cluster involving the ?-subunit,?-subunit and the hypothetical accessory protein coding genes(pNABC)was constructed and overexpressed in E.coli Rosetta.It formed 4.14 mmol/L of IAM in 10 min.In present study,the pNAB plasmid involving the ?-subunit,?-subunit genes was further construted and overexpressed in E.coli Rosetta.The cells of E.coli-pNAB,formed 2.16 mmol/L of IAM in 10 min.which was only half of NHase activity of E.coli-pNABC.This result indicated that co-expression the accessory protein coding gene significantly improved the NHase activity.The plasmid pNA only containing ?-subunit gene was also constructed and overexpressed.As like as the control E.coli-pN with the empty pET28a,the cells of E.coli-pNA almost had no NHase activity.In SDS-PAGE,the recombinant plasmids pNABC,PNAB and pNA could be expressed the corresponding proteins in hosting E.coli Rosetta respectively.In soluble fraction,only the NHase overexpressed from pNABC could be clearly observed,while the NHase from pNAB and the ?-subunit from pNA could not.These results indicated that co-expression of the hypothetic accessory protein coding gene improved the protein solubility of the target NHase and which therefore increased the NHase activity.In conclusion,our present study first found that the nitrogenfixing bacterium E.meliloti CGMCC 7333 and its NHase specifically transform the neonicotinoid insecticide ACE to IM-1-2.IM-1-2 was not further degraded by CGMCC 7333,whereas it was spontaneously hydrolyzed to the derivate ACE-NH,ACE-NH2 and IM-1-4.CGMCC 7333 converted IAN to IAM,while no indole-acetic acid(IAA)was detected.Co-expression of the hypothetic accessory protein coding gene improved the protein solubility of the target NHase and which therefore increased the NHase activity.