Characterization, Directed Evolution And Application Of Serine Hydroxymethyi Transferase From Shewanella Algae And Arthrobacter Nicotianae

L-serine has been extensively used in synthesis of pharmaceuticals, chemicals, and the additives of food/feed and skin-care product because of its special chemical quality and physical function. There has been lots of reports about using glycine and methanol as substrates to produce serine with the help of the serine circulation route in vivo of the methylotrophic bacterium. Currently, L-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT, EC2.1.2.1) is the key enzyme, suggesting the importance of searching for a SHMT with high-yielding serine active aquatic bacteria are very important.In this study, methyl nutrition bacteria, with high SHMT activity, Shewanella algae (S. algae) and Arthrobacter nicotianae (A. nicotianae), were screened out from the deep mud and Southlake freshwater environment, respectively. The main enzyme involved in L-serine synthesis is SHMT which is encoded by the glyA gene. The SaglyA was obtained through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a417amino acid polypeptide. The full-length AnglyA was obtained through degenerate oligonucleotide-primed PCR, encoding a novel SHMT with54.3%similarity to the known SHMT from Escherichia coli. Recombinant glyA was expressed in Escherichia coli BL21(DE3). The SaSHMT showed the optimal activity at pH7.0and50℃, and a0.87-fold higher activity than Escherichia coli’s. Combination of the analysis of the catalytic mechanism of AnSHMT and the three dimensional structure of this protein predicated, site-ditrecetd mutagensis was conducted at249site. The catalytic efficiency of the mutant I249L was found to be2.78-fold higher than that of the wild-type, replacing isoleucine with leucine at the249position.In this work two methods have been tried to produce L-serine. First one was using resting cell system with original strain and this method is mainly used for screening high-yield strains of serine. The second one was using enzymatic reaction system by constructing pET-15b-SaglyA engineering bacteria. The substrates wree glycine (10g/L), and formaldehyde (13.3mmol/L, feeding), then the SaSHMT can produce77.76mM of L-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to L-serine being1.41-fold higher than that of E. coli. In summary, this study which based on site-directed mutagenesis rational design of the AnSHMT, lay the foundation of further exploration. The enzymatic reaction study of the SaSHMT may provide an efficient approach for L-serine sythesis in industry and the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate.