A Preliminary Study On DNA Mismatch Repair Gene Mutl And Transcription Regulator Gene Trmf Of Methylobacterium Strain MB200

Methylobacterium sp.MB200 is a facultative methylotrophic bacterium isolated in this laboratory in the previous experimental research,which has the ability to synthesize L-serine.However,its low tolerance to methanol limits the development of bioprocesses.The high concentrations of methanol and its sudden changes in their concentrations have a toxic effect on these bacteria.2.5%(v/v)methanol completely inhibits the growth of bacteria,and there is no report on the improvement of methanol tolerance of methylotrophic bacteria.It mainly includes the following results:In the experiment,the suicide plasmid p K18 mob was used to construct the plasmid vector pk18mot-mutlps,and the inserted mutant strain MB200 m TB of the mutL repair gene of M.sp.MB200 was constructed by means of three-parent conjugation.With the content of 1.6% methanol as the initial concentration induced by methanol and a gradient of 0.2%,the concentration of methanol in the induced domestication environment was gradually increased.Under the continuous domestication and induction,10 strains were screened to grow in 7%methanol environment Strains.The recombinant expression plasmidpcm80-mutl was constructed by using the universal broad-host vector p CM80,which can carry out gene expression in methylotrophic bacteria.Mut L of mutation-repairing gene was replenished by the obtained strain resistant to high concentration of methanol,and the genetic stability was restored.A total of 4recombinant strains with stable inheritance were obtained.Through analysis of the growth of the strains,3 strains had a faster growth rate and increased bacterial volume than wild strains.Not only can they grow and reproduce quickly under 7% methanol,but also the amount of bacterial cells has been greatly improved compared to wild bacteria,and the tolerance of methanol has been increased by nearly 4 times.The analysis of HPIC shows that the production of L-serine is improved compared to wild bacteria M.sp.MB200.Among them,the L-serine yield of MB200 m TB1 was the highest,which was23.4 mg/m L,2.4 times higher than that of wild bacteria.Compared with the wild strain,the remaining strain only achieved high methanol tolerance,and the growth rate,growth amount and L-serine yield did not increase significantly.According to reports,precursor fermentation using glycine as a precursor is considered to be the most promising L-serine production method.In the previous work of this laboratory,a series of mutant strains were constructed by transposon frameshift mutations,and the mutant gene of one of the mutant strains encoded the transcriptional regulatory gene tmrf of the Mar R family,and its L-serine production was wild 1.8 times of bacteria.This gene may affect the amount of glycine as a precursor in and out of cells,and then lead to theproduction of L-serine.By constructing the inserted mutant MB200 t TB of trmf gene,constructing the restorer MB200 t HB of trmf gene and the overexpression MB200 trmf,and culturing them in the medium with different amount of glycine.It was found that the growth of MB200 t TB mutant in the medium with glycine addition was higher than that of wild bacteria,while the growth of MB200 t HB restorer was higher than that of wild bacteria.When the amount of glycine added in the medium was 5?M/m L,all the strains except MB200 t TB showed the phenomenon that the strain aggregated and deposited on the bottom of the medium,and stopped growing.By preparing a resting cell system,the fermentation broth was analyzed by high-performance liquid chromatography,and the HPIC results showed that the mutant strain MB200 t TB had an L-serine production of 16.3 mg/m L,which was 1.7 times that of wild bacteria.To sum up: In this study,by constructing a methylotrophic bacteria M.sp.MB200 mutation repair gene mutL deletion mutant MB200 m TB,through the methanol induction and domestication,4 strains were selected that could grow in a medium with a methanol content of 7%,among which 3 strains have higher growth rate and volume than the wild ones,and the other one strain has only achieved higher methanol tolerance than wild strains,and the growth rate and growth volume have not been significantly improved.At the same time,by constructing the insertion mutant MB200 t TB of M.sp.MB200 gene trmf,transcription regulator Mar R family,the replenishment strain MB200 t HB and the overexpressing strain MB200 trmf.Growth analysis showed that with theinactivation of gene trmf,the growth amount and growth rate of the strain in the medium increased to some extent,which indicated that the gene was related to glycine feedback regulation of M.sp.MB200.