Characterization And Molecular Evolution Of Sugar Aminotransferase From E.coli

Valienamine is an important precursor of pharmaceuticals that treat type II diabetes including voglibose and acarbose.The common methods used to produce valienamine are chemical synthesis and semi-biosynthesis.However,both of them encounter problems like complex approaches and low yield.It is meaningful to develop one-step biosynthetic pathway of valienamine.Previously,researchers in our lab constructed artificial biosynthetic pathway of ?-valienamine by introducing heterologous sugar aminotransferase Btr R into Streptomyces hygroscopicus 5008.In this study,we characterize candidate sugar aminotransferases and carry out molecular evolution by establishing high throughput screening method.Through phylogenetic tree analysis,eight sugar aminotransferases with crystal structure from VIa and VIb subfamily and four from VIg subfamily are chosen as candidates.All the genes encoding twelve candidates are cloned and expressed in E.coli.After protein purification and catalytic activity measurement,the results show that Arn B_Sty and Ntd A_Bsu convert valienone into racemic valienamine;Wbp E_Pae,Sts C_Sgl and Spc S2_Ssp have catalytic activity towards ?-valienamine.Optically pure valienamine is achieved by Wec E,which is the starting scaffold for molecular evolution.An optical assay for sugar aminotransferase catalytic activity based on sugar aminotransferase-glutamate dehydrogenase coupling system is established by optimization of coupling enzyme loading,signal molecule NADH concentration and coupling time.The coupling enzyme loading is 0.5 U/m L,NADH initial concentration is 0.4 m M and the limitation of detection is 20 ?M.According to the results of 96 well screening,sugar aminotransferase-glutamate dehydrogenase coupling screening method is sensitive with weak background interference and efficient to distinguish positive mutants.We adopt semi-rational design and eighteen amino acid residues are chosen through crystal structure analysis,comparison of natural and unnatural substrate and multiple sequence alignment.Six mutants with higher activity in five amino acid residue sites(Lys223,Val318,Phe319,Tyr321,Ile322)are screened out.Y321 F shows highest catalytic activity which is 4.4 times that of wild type.The activity of V318 Q and V318 R are 3.6 times and 3.9 times that of wild type.The activity of F319 V and K223 I increases by 1 fold.Interestingly,I322 A converts valienone into racemic products.We combine the best mutation directly and catalytic activity of Y321F/V318 Q increases by 14.1 fold while Y321F/V318 R increases by 12.9 fold.All of the amino acid residues that improve catalytic activity are located on Loop 9,which reveals that Loop 9 plays a significant role in determining catalytic activity and stereoselectivity of Wec E towards valienone.The result of molecular dynamic simulation shows that the best mutant has more interaction with substrate,which stabilizes the transition state.This study establishes high throughput screening method for sugar aminotransferase Wec E from E.coli and adopts molecular evolution to increase catalytic activity towards valienone for the first time.It will lay foundation for one-step biosynthesis of valienamine and help us understand the relationship between structure and function of sugar aminotransferases.