Cloning Genes Having The Tolerace To L-Serine From Methylobacterium. Sp MB200 And Study On The Sera Gene

Methylotrophic bacteria are a class of microorganisms that can grow on the mediunm with the one-carbon compounds as well as multi-carbon compounds but not containg carbon-carbon bonds as the sole carbon source, which has the unique metabolic pathways and wide range of applications. It is siganificant both in theory and practical application for us to study the regulation and metabolic mechanisms of these organisms.Methylotrophic bacteria MB200,which used in this work can grow on the medium containing carbon compounds of non carbon-carbon bonds to synthesis L-Serine. But its tolerance to L-Serine concentration as low as 15mg/mL.We tried to screen and analyze the genes that tolerance to L-serine, for providing the basis to the study in metabolic and regulation of these organisms. Preniminary work has been successfully constructed the mutant library of methylotrophic bacteria MB200 using the plasmid transposon pTnMod-RKm'.177 mutants that tolerance to L-Serine were obtained with 20 mg/mL as the initial screening concentration.We extracted the mutants genomic DNA and digested them with Bam H I, then ligated into the pMD18-T which digested with the same enzyme, and later transferred into E.coli DH5a. The positive transformants were picked on the plates with antibiotics, correct plasmids were sequenced for further verified. Total of 18 genes were cloned as this method, then we analyzed them with bioinformatics. We found that the serA gene cloned from the mutant S046, shared 91% identity with the serA gene of M.extorquens DM4 at the nucleotide level reported in Genbank, shared 94% similarity with the reported serA of M.extorquens AM1 in the amino acid level. The gene size is 1,251bp, encoding the PGDH that catalzes the first committed step in the phosphorylated pathway of L-Serine biosynthesis, and there is a ACT domain in the presence of its C terminal.We futher confirmed that PGDH was activated in methylotrophic bacteria MB200, and its activity was sensitive to L-Serine, the PGDH activity was decreased by about 44% when added to a final concentration of L-Serine was 40μM,while little changes in its activity by continuing to increase the concectration of L-Serine. Besides,the two recombinant strains MB200/ pCM80serA and MB200/pCM80serA△77 were constructed with the expression vector pCM80. The results showed that the PGDH activity in the former strain was still sensitive to L-Serine, which was decreased by about 33% when added to a 40μM final concentration of L-Serine. While little changes on the PGDH activity in the latter strain, its PGDH activity was unsensitive to the L-Serine.In this study, the resting cell reaction systems were prepared of these two recombinant strains and the original strain, and found that the L-serine production of MB200/pCM80serA and MB200/pCM80serA△77 was 13.6 mg/mL and 16.8 mg/mL respectively, which higer than the MB200 of 6.4 mg/mL. The L-serine yield in the MB200/pCM80serA△77 represented a 2.6-fold increased from that of the wild strain.