| Nicotinate dehydrogenases(EC 1.17.1.5)are a class of enzymes that can catalyze regioselective hydroxylation at C6 of pyridine derivatives,such as nicotinic acid,carboxypyridine,3-cyanopyridine(3-CP),etc.Using nicotinate dehydrogenase for the biocatalysis has the feature of high specificity,mild reaction conditions and friendly to environment,which make it possible to synthesize 6-hydroxypyridine derivatives in an efficient and eco-friendly way.Owing to the low activity of nicotinate dehydrogenase in wildtype bacteria,the study of recombinant expression and molecular modification of nicotinate dehydrogenase is of great value and significance.However,research of nicotinate dehydrogenase was limited due to the particularity of multi-subunit structure and catalytic function of nicotinate dehydrogenase.There are also few reports on the using of molecular modification strategy to improve specific enzyme activity of nicotinate dehydrogenase.Thus,a systematic research of efficient heterologous expression and molecular modification of nicotinate dehydrogenase from Comamonas testosteroni JA1(NDHase-JA)was carried out.(1)A recombinant expression system was constructed in non-model bacteria to achieve the heterologous function expression of NDHase-JA.NDHase-JA contained three subunits of NdhS,NdhL and NdhM.When expressed in Escherichia coli,the proteins were overexpressed but in the form of inclusion bodies.We selected several strains that were evolutionarily similar to Comamonas testosteroni and several broad-host plasmids that differed in copy number.An expression library containing twelve expression systems was constructed and a Comamonasoni testosteroni expression system was selected due to the better soluble expression and a higher catalytic activity towards 3-CP.The activity of NDHase-JA in this system reached to 40.6 U/L,far surpassing the previously reported level.(2)The expression level of NDHase-JA was significantly improved by using multi-level expression enhancement strategies to improve the efficiency of gene transcription and translation,protein folding and assembly.At first,the expression level of NDHaseJA improved obviously when MmP1 T7-like system was integrated in the expression system.Combined with molecular chaperone co-expression strategy to assist the correct folding and assembly of the three subunits of NDHase-JA,the activity of enzyme reached to 115.0 U/L.Secondly,promoter engineering strategy was used to regulate the expression level of MmP1 RNA polymerase.Further,we used N-terminal engineering strategy to improve the expression level of NDHase-JA.When using these strategies to improve the expression level of NdhL subunit,the enzyme activity got to 192.2 U/L(3)The key amino acid fragment of NDHase-JA which could catalyze 3-CP was identified through multiple sequences alignment strategy.Site-directed mutation was taken towards this fragment of amino acids and got a mutant of A86E.The specific enzyme activity improved by 10.7%to 5.6 U/mg,crude enzyme activity increased by 12.6%to 216.2 U/L.(4)Efficient preparation of 6-hydroxy-3-cyanopyridine was achieved through the optimization of fermentation process and catalytic process.After optimizing the medium composition and high-density fermentation,the enzyme activity reached 869.3 U/L.The catalytic process was optimized:The optimum temperature was 30℃;the optimum pH was 7.5;the optimal concentration of artificial electron acceptor phenazine methosulfate was 50 μM;the optimal substrate concentration was 100 mM.In 50 mL catalytic system,the conversion rate of 3-CP was more than 99%after 23 h.The 1 L reaction system was further evaluated,the substrate conversion rate was more than 99%after 20 h,the yield reached to 0.6 g/L/h under optimal conditions. |
