Directed Evolution Of E.Coli Aromatic Aminotransferase To Enhance Its Catalytic Activity Towards Dl-Homophenylalanine And D-Norleucine

Chemical synthesis of non-natural amino acids has many drawbacks, such as complicated procedures, difficulty in unfolding chiral compounds and unfriendly environment. Aminotransferases, able to produce chiral amino acids, are another choice for synthesis of non-natural amino acids. Compared to chemical methods, transaminases catalysis has some advantages, for instance, high catalytic efficiency, easy separation of products, and friend environments. However, non-natural amino acids are not natural substrates of aminotransferases, and enzymatic catalysis is very poor. To enhance catalytic efficiency of nature aminotransferases towards non-natural amino acids, we tried to use directed evolution of E. coli tyrB gene by error-prone PCR and using non-natural amino acids as a sole nitrogen source, and obtain mutated aminotransferases catalyzing DL-homophenylalanine and D-norleucine.The plasmid pET23a-tyrB DNA and the specific oligonucleotides for tyrB gene were used as a template and primers, and mutagenesis was carried out through4rounds of error-prone PCR. The amplified DNA fragments were inserted into pET23a expression vector at Ndel and Xhol restriction sites, and the recombinant plasmid DNAs were then transformed into E.coli BL21(DE3) to form a mutation library.The mutants obtained were inoculated into M9medium containing5mmol/L DL-homophenylalanine or10mmol/L D-norleucine as a sole nitrogen source. The wild type BL21(DE3)/pET23a-tyrB and BL21(DE3)/pET23a were separately used as control. To allow bacterial cells to utilize DL-homophenylalanine or D-norleucine as substrate more effectively, cell cultures were transferred into the selective medium supplemented with gradually decreasing of DL-Homophenylalanine or D-Norleucine concentrations for3rounds. After cultivation in the selective medium with1mmol/L DL-Homophenylalanine for70h, the OD600of the culture in2tubes showed3-fold higher than those of wild type. Similarly, the OD600of mutants increased markedly, but the OD6oo for two controls BL21(DE3)/pET23a-tyrB and BL21(DE3)/pET23a showed a little change. Single colony was isolated on an agar plate containing the selective medium and then incubated in the selective medium at37℃. Finally,7 colonies numbered as H-3, H-14, H-20, H-26, H-28, H-36, H-37growing rapidly in0.5mmol/L DL-homophenylalanine and other7colonies N-2, N-12, N-19, N-20, N-22, N-25, N-26growing better in lmmol/L D-norleucine were found.The color reaction produced by a couple reaction of transamination with glutamate dehydrogenation was employed to further determine catalytic efficiency of D,L-homophenylalanine or D-norleucine transamination.5colonies H-3, H-14, H-20, H-36, H-37towards D,L-homophenylalanine and4colonies N-2, N-12, N-19, N-20towards D-norleucine displayed high catalysis. The glutamate, the product of transamination, was separated by TLC and cut out from silica gel plate, and then interacted with ninhydrin to produce dark blue color, by which glutamate content was quantified. The result showed that transamination of H-14crude extract was5fold high as compared with that for the wild type, and of N-19crude extract was14.6fold higher than that of the wild type.Multiple sequence alignment showed that the mutated sites of tyrB gene in H-3and H-37are the same, and H-14and H-36are identical. The tyrB gene in all9colonies H-3, H-14, H-20, H-36, H-37, N-2, N-12, N-19, N-2shared3common mutated sites, two non-sense mutations A135G and T843C, and one mutated site T629G resulted in a replacement of Phe210with Cys. H-14shares the same mutated sites G433A, T488A with H-20, but has other additional sites T322G, T665C, C688T, C712A, A728G, T1076C. Its corresponding substitutions in amino acid sequence are Leu108Val, Met222Thr, Arg230Cys, Pro238Thr, Asn243Ser,Va1359Ala. The N-19shares the same mutated sites A439T with N-2, N-20, but has its own mutated sites A65T, A158G, A209G, A496T, T524A, A556G, T569C, A637G, T686C, A1022G, which result in amino acid changes at Glu22Val, Glu53Gly, Glu70Gly, Thr166Ser, Ile175Asn, Thr186Ala, Leu190Pro, Ile213Val, Ile229Thr, Phe339Val.