Isolation&Characterization And Molecular Catabolic Mechanism Study Of A Picolinic Acid-Degrading Strain Alcaligenes Faecalis JQ135

2-picolinic acid is a 2-position substituted isomer of picolinic acid and is an important organic synthesis intermediate which is widely used in the pharmaceuticals,agrochemicals,food and feed industries.With the rapid development of pyridine pesticides which is of higher biological activity and higher selectivity,applications of picolinic acid in the field of pesticides will increase.At the same time,picolinic acid is a byproduct of the biodegradation of L-tryptophan in the organism and of microbial degradation of nitrobenzene and 2-aminophenol.The oxidation of 2-methyl-pyridine in coal tar and bone oil,photochemical degradation of herbicides such as picoram and diquat are also the sources of picolinic acid.Picolinic acid has biological toxicity,therefore the wide range of uses and sources of picolinic acid will make it accumulate in the environment and thus pose a threat to ecosystem and human health.Microbial degradation of picolinic acid is efficient and safe.However,study about it is few and limited to the strain level,and no gene has been reported.Therefore,it is necessary to conduct an in-depth study of the mechanism of picolinic acid conversion and degradation.This study aims to screen an efficient strain capable of degrading picolinic acid and to elucidate its degradation properties and degradation mechanism,which will provide theoretical and technical basis to evaluate the ecotoxity of picolinic acid after its extensive application,and provide biological resouces to control pollution.This study will also play reference significance to the microbial degradation study of pyridine and its derivatives.The main results are as follows:(1)A strain capable of efficiently degrading picolinic acid was isolated from Hangzhou municipal wastewater through continuous enrichment,separation and purification,and was named JQ135.It was identified as Alcaligenes faecalis based on the physico-biochemical characteristics and 16S rRNA gene(GenBank accession number KT988067)phylogenetic analysis.Strain JQ135 was deposited in the China Centre for Type Culture Collection under the accession number CCTCC M 2015812.(2)With the uses of UV scanning and High Performance Liquid Chromatogram(HPLC)to detect the residues of the substrate during the degradation process of picolinic acid by JQ135,a preliminary study was conducted on its degradation characteristics.Degradation by JQ135 was induced by picolinic acid and a 24 h lag phase was observed when JQ135 cultured in LB was collected and inoculated into MSM containing picolinic acid.With the 10%inoculation amount of the JQ135 cells pre-grown in the presence of picolinic acid,JQ135 could use 50-300 mg/L picolinic acid as the sole carbon source for growth in MSM liquid medium,and could degrade 50-300 mg/L picolinic acid completely within 30 h.The optimum pH and temperature for its degradation of picolinic acid was pH 7.0 and 30℃ respectively.The degradation efficiency was the fastest under the 100%inoculation amount.In addition,an intermediate metabolite 6HPA was identified by Liquid Chromatogram-Mass Spectrometry(LC-MS).Combined with other studies,a degradation pathway of picolinic acid by JQ135 was speculated,picolinic acid → 6HPA → 3,6-DPA→2,5-DHP→→→CO2+H2O+NH3.(3)A transposon mutant library of strain JQ135 was constructed through random transposon mutagenesis.A result of 12 picolinic acid degradation-deficient mutants were screened and classified as a,b,c three types.Type a includs 8 mutants that neither used picolinic acid as the sole carbon source for growth nor degraded it at all.Type b is Mut-G31,which could not use picolinic acid as the sole carbon source for growth but could convert it into equal molar ratio 6HPA with no further degradation,this mutant confirmed that 6HPA is an intermediate in picolinic acid degradation,and the mutant can be used for perparation of important organic synthesis intermediate 6HPA.Type c contains 3 mutants,which could not use picolinic acid as the sole carbon source for growth but could degrade it completely.(4)There was no significant difference of growth characteristics in LB between Mut-G31 and JQ135.The ability to accumulate 6HPA of Mut-G31 may be due to the inactivation of the gene for picolinic acid degradation.The transposon flanking sequences of Mut-G31 were cloned,sequenced and assembled,finally a 4103 bp DNA fragment(Genbank accession number KY195989)was obtained.Sequence analysis of this 4103 bp fragment identified three complete ORFs,designated as orf1,orf2,and orf3.The orfl encoded a hypothetical protein.The orf2 gene encoded a polypeptide which showed 32%identity to the 3-deoxy-D-manno-octulosonic acid kinase(KdkA)from Haemophilus influenzae,A protein encoded by orf3 was 48%identical to the putrescine-binding periplasmic protein from Pseudomonas aeruginosa PA14.Besides,the transposon insertion was found to be between 527 and 528 bp from the start codon of orf2.(5)Intact orf2 gene orf2-gtg and a truncated orf2 gene orf2-atg were cloned into the EcoR I-Pst I-digested pBBR1-MCS5,yielding the recombinant plasmids pBB-orf2gtg and pBB-orf2atg.Then the recombinant plasmids were transferred into the Mut-G31 and the ability of utilization of picolinic acid by complemented strain was detected.Mut-G31gtg grew well in MSM plate containing 500 mg/L picolinic acid and recovered the ability to completely degrade picolinic acid.This indicated that orf2 was started with GTG,and was essential for the biodegradation of picolinic acid in strain JQ135.However,orf2 is not the functional gene for picolinic acid degradation.This study provides the first evidence on the genetic mechanism of picolinic acid biodegradation.