| Methylobacterium sp.MB200 is a facultative methylotrophic bacteria that can grow on single-carbon or non-C-C bond low-carbon compounds as substrates and produce a variety of metabolites.It has shown strong adaptability and utilization ability to methanol in the previous experiments.In this study,transcriptome analysis was used to preliminarily investigate the intrinsic metabolism of methylotrophic bacteria MB200 under the conditions of formaldehyde and methanol,and to obtain mutant strains with higher tolerance and transformation ability of methyl compounds by mutating mismatch repair of genes muts and mutl and directional induction,and the ability of each strain to ferment L-serine with methanol as substrate was preliminarily analyzed.The main findings of the study are as follows:1.Transcriptome analysis of MB200 was carried out,and it was found that when formaldehyde was used as carbon source,the expression of genes related to methane metabolic pathway and intercellular signaling was significantly up-regulated,and the expression of genes related to glycolysis was down-regulated.The expression of genes related to the bacterial two-component system was significantly up-regulated,and related to carbon metabolism was significantly down-regulated under methanol condition.A number of genes belonging to different metabolic pathways with significant differential expression were found by analyzing the differential expression of MB200 strain in the two experimental groups(methanol and formaldehyde as substrate respectively),indicating that methylotrophic bacteria MB200 regulates multiple metabolic pathways when adapting to high concentrations of methanol and formaldehyde,respectively.This means that it may be necessary to pay attention to multiple pathways when targeted modification of methyl nutrient bacteria.2.Based on MB200,mutants with ultra-high tolerance to methanol and formaldehyde were obtained.In this study,muts and mutl were used to carry out insertion deletion mutation,and methanol,and directional induction was conducted with methanol and formaldehyde as the only carbon sources.High methanol tolerance mutant strains MB200 s HBc,MB200 l HBc,MB200 sl HBc and high formaldehyde tolerance mutant strains MB200 s HBq,MB200 l HBq,MB200 sl HBq were obtained.Among them,the methanol tolerance and growth of MB200 s HBc,MB200 l HBc and MB200 sl HBc were significantly improved compared with MB200,and the methanol tolerance of MB200 s HBc was the highest,from 8 g/L to 44 g/L,which was increased by more than 5 times and the growth rate was not affected.The mutant STRAINS MB200 s HBq,MB200 l HBq and MB200 sl HBq had no obvious improvement in tolerance to formaldehyde,and the highest tolerance concentration was 0.45 g/L.However,the growth of the three strains was significantly increased at this concentration(0.45 g/L),and the growth of the mutant MB200 l HBq was 1.69 times higher than that of MB200.3.The 7 genes with the most significant differences in expression of MB200 between the two experimental groups were further verified by q RT-PCR using six strains of back-compensating bacteria.The results were highly consistent with the transcriptome analysis,and the expression levels were correlated with the growth levels of the strains,indicating that the methyl nutrient bacteria adopted different coping strategies when adapting to and utilizing high concentrations of methanol and formaldehyde.4.In order to explore the biotransformation ability of mutant strains,L-serine fermentation experiment was carried out under the condition of 8 g/L methanol as carbon source and 40 g/L glycine as precursor.It was found that the L-serine production of five strains was significantly increased compared with MB200,and the yield of MB200 s HBc was up to 16.31 ± 0.8 mg/m L,which was2.26 times that of MB200. |
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