The Hydroxylation Of Pyridine-related Compounds And The Relative Hydroxylase, Cloning And Functionial Expression

Recently,new insecticides containing the 3-chloropyridylmethyl group as a versatile building block have been developed,among which imidachloprid is one of the most promising insecticides.6-Hydroxynicotinic acid,the first intermediate of the bacterial degradation pathway of nicotinic acid,can be used as a starting material for the synthesis of imidachloprid.It is difficult to hydroxylate nicotinic acid at C6 position in a chemical synthesis way.However,biotransformation process has been applied to pruduce 6-hydroxynicotinic acid which shows a possible application in industry.In this paper,we used microbial transformation method to research the nicotinic acid hydroxylation by Comamonas testosteroni JA1,and construct a combined technology of growing culture hydroxylation of nicotinic acid and resting cells hydroxylation of 3-cyanopyridine.The complete gene sequence of the Nicotinate dehydrogenase(NaDH)from C.testosteroni JA1 and another pseudomonas strain named Pseudomonas putida KT2440,cloning,structural analysis and functional expression has been studied.In addition,we present evidence for analysis the NaDH substrate specificity difference of the two strains.Resting cells of JA1 with 3-cyanopyridine hydroxylation activity can also hydroxylate nicotinic acid to 6-hydroxynicotinic acid.Higher production of 6-hydroxynicotinic acid depended on the presence of 1%glucose,1%peptone and 1 %nicotinic acid which used as the inducer.In addition,the fermentation condition of JA1 strain was very ascendant.The nicotinic acid hydroxylation activity was not descend obviously but stayed highly with an increase from 25℃to 37℃,pH value from 6.5 to 7.0 and the medium volume from 10 mL to 40 mL.The ideal transformation condition of JA1 which transform nicotinic acid to 6-hydroxynicotinic acid was pH 7.0,temperature 30℃and nicotinic acid 3%.Under an appropriate condition,in a 1.5L and 30L liter fermenter,production yield of 6-hydroxynicotinic acid by JA1 was 87.35 g/L and 68.42 g/L.Moreover,electronic effects and space effects resulting from effects of substitute groups were discussed in this paper.It indicates some analysis of regioselective hydroxylation regulations of pyridine ring at position C6.Yasuda et al(1995)reported that hydroxylation of 3-cyanopyridine to 3-cyano-6-hydroxypyridine by C.testosteroni MCI2848 resting cells needed to be induced by 1～2%of nicotinic acid during cultivation.Here we reported that a strain of JA1 for hydroxylation of 3-cyanopyridine could hydroxylize nicotinic acid to 6-hydroxynicotinic acid with a very lower(in the absence of nicotinic acid in the medium,)or little(in the presence of nicotinic acid in the medium)capacity of degrading produced 6-hydroxynicotinic acid during the induced cultivation.Therefore the induced cultivation of JA1 for 3-cyanopyridine hydroxylation activity by nicotinic acid could be used to accumulate 6-hydroxynicotinic acid.In the combined procedure of growing culture transformation of nicotinic acid and resting cells transformation of 3-cyanopyridine,not only a extra 50.38 g of solid 6-hydrxoynicotinic acid was produced from 1 L cultivation broth with a 99.97%molar conversion yield,but also production yield of 3-cyano-6-hydrxoypyridine was raised to over 2 times more than that of the resting cells from induced cultivation broth at 8 h and 5.77 g of solid 3-cyano-6 -hydrxoypyridine was produced with 1 L cultivation broth of resting cells which is superior to 4.39 g/L cultivation broth of resting cells reported previously by Yasuda et al(1995).JA1 was found to resist to many antibiotics.Alkaline lysis extraction of the JA1 culture reveals a 19 kb plasmid.0.0025%SDS treatment of the strain eliminated the extrachromosal plasmid.The mutants lost the kanamycin resistance ability while keeping the nicotinic acid hydroxylation activity.It indicated that the gene encoding the kanamycin resistance resides in the plasmid,whereas gene responsible for the nicotinic acid hydroxylation does not.The same level of the nicotinic acid dehydrogenase activity between wild type and mutant further proves this.A method for large scale screening of 6-hydroxynicotinic acid-producing microbes requested urgently for shot-gun cloning.To this aim,a high through-put screening method for 6-hydroxynicotinic acid transformed by microbes was established by spectrum determination of 6-hydroxynicotinic acid based on 96-well microplate-Multiskan Spectrum.The determination wavelength and the reference wavelength of 6-hydroxynicotinic acid were 251 nm and 231 nm respectively.Beer's law is obeyed in the range of 0.5～11μg/mL(R²=0.9999)for determination of amount of 6-hydroxynicotinic acid.The average recovery rate was 99.11～100.81% with satisfactory results.The results showed that there was no apparent difference between current microplate method and previous HPLC method in the detection of the 6-hydroxynicotinic acid formation.The microplate method is simple,convenient and accurate therefore it has the potential for large scale(about 2000～5000 reactions/d) screening of 6-hydroxynicotinic acid-producing microbes.5000 transports gained for shot-gun screening were detected,but no positive clone that can transform nicotinic acid into 6-hydroxynicotinic acid was obtained.In 2006,Ashraf Alhaper firstly reported the complete primary sequence of the ndhF, ndhS,ndhL,and ndhM genes encode the 33-,23-,50-,and 37-kDa subunits of Eubcterium barkeri NaDH.Additionally,gene clusters associated with NaDH in the other nine bacteria were identified with database searches.The work on hydroxylation of nicotinic acid by P.putida NA-1 described earlier.P.putida NA-1 genome sequence had not been reported yet,therefore,the genomic DNA of a model strain P. putida KT2440 has been broadcasted.In the view of the same lineage,P.putida KT2440 was study on nicotinate hydroxylation.Similar result has been showed that KT2440 can aslo transform nicotinate into 6-hydroxynicotinate at a high level,and cannot hydroxylate 3-cyanopyridine at all.With database searches,only one presumable candidate ndhSL gene cluster was found due to the same sequential arrangement of the corresponding subunits or domains observed in Ashraf Alhaper paper.NaDH activity was detectable both in resting cells and crude extracts. 3-cyano-6-hydroxypyridine was not detected when 3-cyanopyridine was added as the substrate in resting cells and crude extracts.For the first time to our knowledge,the complete primary sequence of NaDH from the pseudomonas strain,cloning,structural analysis and functional expression has been reported.Based on the fact that highly conserved amino acid sequences are present in NaDH, we employed the PCR methods to clone the NaDH genes in JA1.An internal 1.5-kb fragment was amplified using the degenerate primers(JA1,JA2)corresponding to the conserved regions between two[2Fe2S]iron sulfur clusters and molybdenum(Mo) cofactor.The complete ndhSLM gene cluster and its flanking regions were obtained by three steps inverse PCR.NaDH activity was detectable both in resting cells and crude extracts.Quite remarkablely,same as wild-type C.testosteroni JA1, 3-cyano-6-hydroxypyridine was detected when 3-cyanopyridine was added as the substrate in resting cells and in crude extracts of P.entomophila L48 pNJASLM-P clones on the basis of HPLC analysis.Therefore,the property that NADH can also catalyze 3-cyanopyridine hydroxylation has been proved.Based on the NCBI alignment result,we proposed that the difference of the S subunit may relate to the substrate specificity difference.P.entomophila L48 pNKTS +NJALM-P clones harboring the ndhS genes from P.Putida KT2440 and ndhLM genes from C. testosteroni JA1 exhibit nicotinic acid hydroxylation activity.However,P. entomophila L48 pNJAS +NKTL-P clones harboring the ndhS genes from C. testosteroni JA1 and ndhL genes from P.Putida KT2440 exhibit the activity to hydroxylate nicotinic acid as well as 3-cyanopyridine.The results of subunits exchange within NaDH_KT2440and NaDH_JA1approved that relation,and the function that 2Fe-2S cluster-binding domains can influence the substrate specificity has been reported the first time within the study of the Mo-MCD-containing molybdenum hydroxylases in XO family protein.