Structure And Function Of Glycosyltransferases Involved In The Biosynthesis Of Steviol Glycosides

Excessive intake of high-calorie sugar in diets,such as glucose,fructose and sucrose,would result in various chronic diseases including obesity,diabetes,hypertension and cardiovascular diseases,which has attracted worldwide attentions.Thus,plant-derived sweeteners rebaudioside D（Reb D）and rebaudioside M（Reb M）from Stevia rebaudiana Bertoni have been considered as the desired substitutions of high-calorie sugars due to their pleasing features of low-or zero-calorie,high-intensity sweetness and less lingering bitter aftertaste.Nowadays,based on these advantages,they have been widely used as food additives in food industry.However,the low content of Reb D and Reb M in stevia plants leads to low production from traditional method such as phytoextraction,which restricts their commercial applications.Therefore,enzymatic preparation of Reb D and Reb M from stevioside or rebaudioside A（Reb A）,which were highly abundant in Stevia rebaudiana Bertoni,was developed as a promising solution.However,the problems of protein solubility and low enzymatic activity limiting the application of enzymatic preparation.In this study,we mined a bacterial glycosyltransferase Yoj K from Bacillus subtilis 168,which was not only able to catalyze Reb A to produce Reb D but also expressed with highly soluble expression in Escherichia coli（E.coli）BL21（DE3）.In order to improve its catalytic activity,protein structure of Yoj K was solved to perform structure-guided engineering and elucidate its catalytic mechanism.In addition,structure-guided engineering of the glycosyltransferase UGT76G1,which was responsible for the synthesis of Reb M from Reb D,was also performed.Meanwhile,molecular insights for the improvement of catalytic activity of UGT76G1 mutation was also investigated.The main results are as follows:1.Obtaining a novel bacterial glycosyltransferase Yoj K with the ability to glycosylate Reb A to produce Reb DIn order to screen a novel glycosyltransferase with the ability to synthesize Reb D from Reb A,six glycosyltransferases from different species including Yoj K,Ydh E,Os UGT,BCGT-1,UGT73AE1 and UGT72E2 were selected and expressed in E.coli BL21（DE3）.And the activity of them were determined whether they could glycosylate Reb A to produce Reb D.It was found that Yoj K could glycosylate Reb A to synthesize Reb D,which was verified by high performance liquid chromatography-mass spectrometry（LC-MS）and nuclear magnetic resonance（NMR）.Yoj K displays a Km value of 210.52±11.70μM and a kcat value of 1.08±0.05min^-1 toward Reb A.2.Highly efficient synthesis of Reb D enabled by structure-guided engineering of glycosyltransferase Yoj KTo improve the catalytic activity of Yoj K,the crystal structure of Yoj K was first solved and refined to 1.9（?）with a space group P 1 21 1（PDB:7VM0）.Next,structure-guided engineering of Yoj K was performed,and a variant Yoj K-I241T/G327N with a 7.35 folds increase of the catalytic activity toward Reb D was obtained through alanine scanning mutagenesis,site-directed saturation mutagenesis and combinatorial mutation.The Km and kcat values of the variant Yoj K-I241T/G327N were 193.07±7.30μM and 10.76±0.05 min^-1,respectively,indicating highly improved catalytic activity of Yoj K-I241T/G327N compared with wild type Yoj K.Moreover,based on the molecular dynamics simulations,the molecular mechanism of improved enzymatic activity of Yoj K-I241T/G327N was ascribed to the formation of new hydrogen bonding between the enzyme Yoj K-I241T/G327N and Reb A or uridine diphosphate glucose（UDPG）,which was beneficial to keep stable and favourable conformations for facilitating the efficient reactions.Finally,the variant Yoj K-I241T/G327N coupled with sucrose synthase was employed to produce 20.59 g·L^-1 Reb D from 19.32 g·L^-1Reb A with a yield of 91.29%in 15 h.3.Highly efficient synthesis of Reb M by rational directed-evolution of glycosyltransferase UGT76G1To improve the catalytic activity of UGT76G1,directed evolution of UGT76G1 was performed through alanine scanning mutagenesis,site-directed saturation mutagenesis and combinatorial mutation with the residues,which were predicted to interact with Reb D.A variant UGT76G1-T284S/M88L/L200A was obtained with a 2.38-fold increase in catalytic activity toward Reb D compared with UGT76G1-T284S.The variant UGT76G1-T284S/M88L/L200A displayed the Km of 324.25μM and kcat of 2.39 s^-1,which were higher than those of UGT76G1-T284S,indicating the improvement of affinity and catalytic efficiency.Moreover,molecular dynamics simulations illustrated that UGT76G1-T284S/M88L/L200A reduced distance between Reb D and catalytic residue H25 or UDPG by reshaping the hydrophobic pocket with better recognition of the diterpene skeleton of Reb D,which was favourable to formβ-1,3 linkage.Reb M was prepared in the cascade reaction of UGT76G1-T284S/M88L/L200A coupled with sucrose synthase At Su Sy.After optimizing the conditions,a fed-batch method was employed and 23.37 g·L^-1 Reb M was synthesized from 22.58 g·L^-1Reb D with a great yield of 90.50%after 7 h.In summary,structure-guided evolution was performed to improve the catalytic activity of glycosyltransferases Yoj K and UGT76G1,which allowed to highly efficiently produce Reb D and Reb M after the condition optimization of the cascade reaction.Thus,this study provides a potential industrial scale-production of Reb D and Reb M.