Study On L-Methionine Yield Of E.coli With DapA Gene Deletion

L-methionine(L-Met), an essential amino acid, involves in synthesis of adrenalin, bilineurine and creatine in vivo, and the transfer of active methyls, the metabolism of phospholipids in livers as well. With the development of feed industry in China, the demand for L-Met is increasing. However, domestic supply is insufficient and import of L-Met is indispensable. Microbial fermentation, which is an important method to produce other amino acids, is expected to be an efficient way to produce L-Met. As is known, the method has the remarkable features of lowering costs and pollution, and promoting the development of feed industry and animal husbandry nationwide.Dihydrodipicolinate synthase (DHDPS) is coded by dapA gene. And DHDPS is the enzyme that catalysts the first unique step of L-lysine biosynthesis in plants and microorganisms. It catalyzes the aldol condensation of L-aspartate-β-semialdehyde and pyruvate to dihydropicolinic acid via a Schiff base formation between pyruvate and a lysine residue. The functional enzyme is a homotetramer consisting of a dimer of dimers. DHDPS is member of dihydrodipicolinate synthase family that comprises several pyruvate-dependent class I aldolases that use the same catalytic step to catalyze different reactions in different pathways. The present study focuses on deleting dapA gene in E.coli to provide more precursors for high-yield of L-Met by means of gene targeting technique. The study might be a reference for producing other metabolites from microorganisms.A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defectiveλprophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42°C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defectiveλprophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries. pKD46 has an optimized ribosome-binding site for efficient translation of gam and expresses gam, bet, and exo from the arabinose-inducible ParaB promoter. It is also a temperaturesensitive replicon to allow for its easy elimination.L-lysine branch is one of Asp metabolic pathways in Escherichia coli. Its presence certainly affect the yield of another amino acid, L-methionine, which shares the same precursor as L-lysine. dapA codes the first key enzyme for L-lysine synthesis. Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis. It catalyzes the aldol condensation of L-aspartate-beta- semialdehyde and pyruvate to dihydropicolinic acid via a Schiff base formation between pyruvate and a lysine residue. The functional enzyme is a homotetramer consisting of a dimer of dimers. DHDPS is member of dihydrodipicolinate synthase family that comprises several pyruvate-dependent class I aldolases that use the same catalytic step to catalyze different reactions in different pathways.E.coli K12 with pKD46 plasmid (K12/pKD46)was involved in the experiment. Induced by arabinose, Red recombinases fromλbacteriophage were expressed, which provided the host with recombination ability. In overlapping-primer PCR, High-fidelity DNA polymerase and primers with EcoRⅠcleavage sites were used. DPSCAT fragments with 50bp of dapA gene at two ends and chloramphenicol-resistance gene in between were amplified. The linear fragment were cloned into pMD18-T vectors and then transformed into E.coli strain DH5α. By extracting plasmids, enzyme cutting identification, the results were as good as expected. By sequencing analysis, it indicated that the homogolous rate was 99% which confirmed the accuracy of the results.DPSCAT fragments cloned into vectors were amplified. The linear fragments were recombined and transformed into E.coli K12/pKD46 by electroporation. The positive strains of L-Lys deletion mutant were screened and identified.The L-Met in the samples of wild type E.coli K-12 and respectively were compared quantitatively by HPLC. The results indicate that the L-Met of recombinants was as 3.61 times as that of wild type E.coli K-12. The strains with dapA deletion were obtained and were characterized as L-Met high-yield.