| Methylotrophic bacteria are ubiquitous in nature. They have particular one-carbon metabolism pathway, they can transforme a large of one-carbon compounds in nature like methanol and formaldehyde into the nutrients of the strains, producing a variety of by-products such as vitamin B12, poly-βhydroxybutyrate and so on. Therefore, it has important significance to do further study.L-serine is an important amino acid, which is widely used in pharmaceutical industry, food, feed, cosmetics, agriculture. L-serine is in great demand each year, but it's industrial production is difficult. At present, the production of L-serine mainly rely on microbial fermentation.In this study, three diffenent strains of methylotrophic bacterium WGP07, WGM16, WGP35 were isolated from different environments. Based on 16S rDNA, the morphological observation, physiological and biochemical identification, the strain WGP07 was identified as Paracoccus, the strain WGM16 was identified as Methylobacterium, the strain WGP35 was identified as Pseudomonas. All of them can produce L-serine with methanol. The production of L-serine in the strain WGP35 is the highest, which can accumulate 2.5 mg/ml L-serine.It is the key factor that the serine dehydratase encoded by sdaA gene catalyzed the degradation of L-serine to pyruvate, affecting the production of L-serine. In this experiment, the gene of sdaA in the strain WGP35 was mutated to investigate the production of L-serine. According to the gene sequence of Pseudomonas putida GB-1 in Genbank, the primers was designed to amplificate the sdaA's homologous fragment by PCR. The fragment was sequenced and indicated that it has 90% homology to the sdaA gene of Pseudomonas putida GB-1. The sdaA gene of the strain WGP35 was mutated to constructe the mutant strain WGP35TB with suicide plasmid PK18mob. The mutant strain WGP35TB showed auxotrophic L-serine. The production of L-serine in mutant strain is increased by 16%. |
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