Functional Characterization Of Key Genes For Catabolism Of Nicotinic Acid By Pusillimonas Sp.T2

Pyridine and its derivatives are ubiquitous in nature,6-hydroxynicotinic acid(6HNA)is an important chemical intermediate of pyridine derivative and it is difficult to hydroxylate nicotinic acid(NA)at C6 position in a chemical synthesis way.However,the conditions of microbial transformation are mild,the process is simple,and the production have high purity,so it has a wide prospect in application.In this study,the NA-degradation characteristics,metabolic pathway and molecular degradation mechanism were studied by using the nicotine-degrading strain T2.The encoding of three-component NA hydroxylase(NahAB1B2)genes were cloned from strain T2 and functionally characterized by genome sequencing and bioinformatics comparison,which provided theoretical methods and basis for green transformation of 6HNA production.The degradation characteristics of NA by strain T2 were studied and the metabolic pathway was speculated.The results showed that the optimum degradation conditions of NA was 30 ? and pH 7.0.In addition,300 mg/L NA could be degraded completely by strain T2 in 3.5 h when OD600= 0.60.And we sepeculated that the NA degradation pathway was NA?6HNA?2,5-DHP??TCA cycle.The whole genome sequences of strain T2 was determined.A total of 3.3 Mb filtered paired-end reads and with the percentage of 56.9%GC were assembled into scaffolds using the SOAPdenovo alignment tool.The draft genome excluding the gaps totally has 3054 predicted opening reading frames(ORFs).Protein-coding sequences were predicted and analyzed by the Glimmer v3.0 program and were then used for BLASTP searching againsr the RAST,nr,Swiss-Prot,COG,KEGG and GO databases.The key enzyme genes nahAB1B2 of NA degradation were cloned and heterologously expressed in the non-NA-degrading Shinella sp.strain HZN7.The results showed that resting cells of the recombinant strain HZN7-pBBR-nahAB1B2 could convert NA into equimolar 6HNA,while the recombinations HZN7-pBBR-nahAB1(lacking component B2)and HZN7-pBBR-nahAB2(lacking component B1)could not convert.Cell extract of HZN7-pBBR-nahAB1B2 exhibited NA hydroxylase activity.After addition of an artificial electron acceptor(such as phenazine methosulfate,PMS),the NA hydroxylase activity was significantly increased.The Km and Vmax values for NA were 65.94 ?mol/L and 260.80±5.69 mU/mg,respectively,using PMS as electron acceptor.In addition,the results about the function of metal ions showed that Fe2+ and Fe3+ had no effect on the enzyme activity,while Hg+ and Ag+ had strong inhibitory effects.Moreover,several structural analogs of NA were tested as substrates for NahAB1B2,however,none of these led to activities.In summary,these data indicate NahAB1B2 is specific for NA.