Isolation And Identification Of Halomonas Sp. ZL-4 And Its DAPDC Gene (lys A) Clone And Expression

DAPDC is a vitamin B6-dependent enzyme that stereospecifically converts meso-DAP to L-lysine. The enzyme is of interest because of its importance in bacterial growth and survival. Lysine is essentially required in protein biosynthesis and for their viability and development. So biosynthesis of L-lysine has come under increased scrutiny as a target for novel antibacterial agents.A strain of halophilic bacterium was isolated from a high NaCl concentration medium cultured with D.salina. Based on the analysis of bacterial 16S rDNA, cell morphology, physiological and biochemical characteristics, the strain was designated as Halomonas sp.ZL-4. Then total DNA of the strain was extracted and partially digested by Sau 3AI, the DNA fragments ranged from 1～6 kb were retrieved and linked with pUC19 to form reconstructed plasmids. Then they were transformed into E.coli DH5αto construct a partial gene library of Halomonas sp.ZL-4. Fifty clones growing on the screening medium were randomly selected, and then the DNA fragments within the reconstructed plasmids were sequenced and taken a homologous BLAST by dint of America GenBank to seek the gene we need until a gene (lys A) that coding for diaminopimelate decarboxylase (DAPDC) has been found. After that, the lys A gene was amplified by PCR and ligated with vector pET32a to reconstruct a new plasmid pET-lysA. The latter was then transformed into E.coli BL21. New transformant (with pET-lysA) was cultured and shaken at 37℃in LB medium supplemented with 1mmol/L isopropyl-β-d-thiogalactopyranoside (IPTG) over 8 h, then total- and extracellular- amount of L-lysine of the samples were detected. The results showed that the yield of L-lysine of transformant was markedly more than control, its L-lysine in the cell was excreted outside slowly. Furthermore, DAPDC activity has been tested between transformant and control by means of their crude extracts. Evidence proved that the activity of the former was 1.78 times than control. In sum, all the data mentioned above implied that the cloned foreign lys A gene has been transformed into E. coli BL21 and expressed high level catalyzing activity.