The Study Of SERA Relative To Feedback Inhibition Of L-Serine In M.sp MB200

Methylotrophic bacteria can use single-carbon compounds as the sole carbon source for growth and energy metabolism. Some researches have been reported that these bacterias can convert single-carbon compounds to some valuable products, including L-serine. L-serine, a kind of amino acid, is extensively used, and it has been of great significance to study on producing L-serine by microbial fermentation and increasing its yield.To the fermentation of amino acid, relieving feedback inhibition has been an important method to increase yield.3-phosphoglycerate dehydrogenase (3-PGDH) catalyzes the first step in the biosynthesis of L-serine, and this step was also the rate-limiting reaction. Enzymatic activity of3-PGDH was affected by the regulation of feedback inhibition of end product (L-serine). Relieving feedback inhibition through molecular modification can facilitate the accumulation of L-serine.Combining references and bioinformatic prediction, this work speculated that three amino acid residues His360, Asn362, Asn380, which were the critical sites relating to feedback inhibition between3-PGDH with L-serine. Through site-directed mutagenesis, this work studied the mutant enzymes’activities and feedback inhibition by L-serine, and determined the exact sites in combination with feedback inhibition by L-serine.The study constructed mutant enzymes S1(H360A), S2(N362A), S3(N380A), S4(H360A/N362A), S5(N362A/N380A), S6(H360A/N380A), S7(H360A/N362A/N380A) by PCR, and expressed in BL21(DE3)-pET-30a(+). The inducing conditions were:temperature20℃, the final concentration of IPTG0.25mM, inducing time20h, speed120rpm. The purpose protein was purified by NTA-Ni-chelating affinity chromatography column under non-denaturing conditions. It was showed that protein S1, S2, S3were completely or partially relieved the feedback inhibition of L-serine, while their enzymatic activity reduced significantly. The M4had no obviously differences with that of the wild type in feedback inhibition of L-serine, but its enzymatic activity decreased (79%of the wild-type), While S5, S6, S7had no catalytic activity. The conclusion was as follows:In MB200, three amino acid residues(His360, Asn362, Asn380), which were in the regulatory region of3-PGDH, were the key sites relative to feedback inhibition of L-serine and had an effect on the catalytic activity.