Molecular Modification Of The Sucrose Synthase And Its Application On High-Efficient UDP-Glucose Production

UDP-glucose（UDP-Glc）serves as a crucial precursor for the synthesis of UDP-glucuronic acid,UDP-galactose,and other UDP sugars,which are essential sugar donors for the precise synthesis of structurally complex oligosaccharides catalyzed by Leloir glycosyltransferases.However,the high cost of sugar donors has always been a significant challenge in the synthesis and application of oligosaccharides.Therefore,the large-scale and efficient production of UDP-Glc,as a key substrate for sugar donor synthesis,is necessary to address this cost issue.Sucrose synthase（Su Sy）has emerged as an important route for the one-step synthesis of UDP-Glc.However,the limited enzymatic activity and poor thermostability of Su Sy hindered the synthesis rate of UDP-Glc.Therefore,enhancing the catalytic activity and thermostability of sucrose synthase is crucial for achieving efficient and rapid synthesis of UDP-Glc.This study focused on the enzyme property improvement of sucrose synthase derived from Nitrosospira multiformis（Nm Su Sy）.A range of enzyme engineering strategies,including directed evolution and saturation mutagenesis were applied to enhance the property of Nm Su Sy.Remarkable improvements were successfully achieved in both the thermostability and catalytic activity of Nm Su Sy.Structure analysis revealed the pivotal role of subunit interactions reshaping in stability improvement,as well as the influence of hinge and hydrophobic patch-regulated gate anchoring on enzyme activity.Through the optimization of reaction conditions and the analysis of reaction kinetics,we were able to attain short-time and efficient production of UDP-Glc,yielding an impressive space-time yield of up to 136 g/L/h after 0.5 h.The main results of this study are summarized as follows:1.Computer-assisted prediction and beneficial mutations combined with the greedy accumulation strategy have successfully resulted in a significant enhancement of the thermostability of sucrose synthase,the reshaping of subunit interactions was found to play a pivotal role in improving the thermostability.Fireprot was utilized to predict the hotspots integrating energy-and evolution-based design methodologies with intelligent screening.Ten beneficial mutation sites were experimentally validated and effectively combined by applying the Greedy Accumulated Strategy for Protein（GRAPE）which utilized K-means clustering and a greedy algorithm.Finally,the optimal mutant Su Sy _M4（Q162W-G612P-A79F-S240Y）we identified.It exhibited a remarkable increase in the half-life of inactivation(T_1/2)to 33 min,representing a 27-fold improvement compared to the WT.Notably,at a UDP concentration of 200 m M,Su Sy _M4 achieved a conversion rate of 65%for UDP into UDP-Glc,resulting in a space-time yield of 37 g/L/h.The structural analysis at the monomeric level revealed that the A79F and S240Y mutations introduced newπ-πstacking and hydrogen bonding interactions,respectively.The G612P mutation enhanced the rigidity of the local structure.Furthermore,structural analysis of the tetrameric form unveiled a strengthened interaction at the subunit interface,leading to enhanced protein compactness.The Q162W mutation played a crucial role by facilitating the formation of new interaction interfaces,thereby bridging the four subunits and reshaping the subunit interactions into a stable triangular relationship.This newly formed modification significantly contributed to the improvement of Nm Su Sy’s thermostability.2.The semi-rational engineering of Nm Su Sy has significantly improved the enzymatic catalytic ability and the synthesis of UDP-Glc was efficiently improved by employing a combination of reaction condition optimization and an in situ conversion strategy for byproducts.The enzyme activity-enhanced mutant Su Sy _V148S-K769H was obtained through the utilization of directed evolution and site-saturation mutagenesis.The mutional enzyme exhibited enhanced relative enzyme activity and K_m/k_cat value which were 2.2 times and 1.7 times higher than the WT,respectively.Su Sy _V148S-K769H was applied for UDP-Glc production under optimal conditions,including 150 m M UDP,1.5 M sucrose,100 mg/L purified enzyme,and p H 6.5.The UDP-Glc concentration achieved to 110 m M after 2 h.The conversion rate of UDP reached73%with a product space-time yield of 31 g/L/h.In comparison,under the same condition,the WT only catalyzed 23%of the UDP to UDP-Glc.Furthermore,an in situ conversion strategy was employed to isomerize byproduct fructose to mannose by introducing MIase.Under the optimal conditions for UDP-Glc production,adding MIase to a concentration of 200 mg/L significantly accelerated the synthesis rate of UDP-Glc.Compared to single-enzyme catalysis by Su Sy _V148S-K769H,the production cycle was reduced from 2 h to 1 h.The resulting UDP conversion rate reached 83%by 10%increase,with an enhanced space-time yield of 70 g/L/h.3.The structural analysis of Su Sy _V148S-K769H elucidated that the enhanced stability of the hinge and the strengthened interactions within the hydrophobic patches contributed to the reduced latch distance,thereby improving the enzymatic catalytic activity.Molecular dynamics simulations were employed to investigate the conformation of ligand bound Su Sy _V148S-K769H.The mutations V148S and K769H near the hinge region resulted in a pre-closed state of the catalytic domains GT-B（A）and GT-B（D）,wherein the average latch distance between the"gate"residue E613 and the"anchoring residue"Y436 decreased from12.4（?）to 8.6（?）.This reduction facilitated rapid conformational transitions,enabling efficient exchange of substrates and products.Analysis of the local hydrogen bonding network revealed that the mutation V148S introduced new hydrogen bonds,thereby enhancing the stability of the hinge centre and its associated loop.Moreover,the reinforced hydrophobic interactions and increased hydrogen bonding within the hydrophobic patch resulted in a decreased latch distance,thereby promoting the conformational transition necessary for catalysis.Molecular dynamics simulations elucidated that the primary mechanism responsible for the improvement in catalytic activity was the modulation of latch distance by the hinge and hydrophobic patch,which served as crucial structural elements regulating the rate of conformational transition.4.The mutant Su Sy _M6 that omitted the activity-stability tradeoff was obtained and accomplished by developing a cell-free synthesis system,which enabled efficient synthesis of UDP-Glc and broad applicability in the manufacture of additional high-value sugars.By combining mutation sites from the thermostable Su Sy _M4 and the enzyme activity-enhancing enzyme Su Sy _V148S-K769H,the resulting Su Sy _M6 exhibited remarkable improvements in catalytic efficiency and thermostability,effectively overcoming the trade-off between stability and activity.Su Sy _M6 exhibited a 1.4-fold increase in enzyme activity compared to the WT,with its T_1/2 extending by 43-fold to 51 min.The k_cat/K_m value of Su Sy _M6 is 1.6-fold higher than that of the WT,indicating the enhanced catalytic efficiency.Under optimal reaction conditions,Su Sy _M6 facilitated the rapid synthesis of 129 m M UDP-Glc within 1 h,achieving an impressive UDP conversion rate of 86%.In the cascade catalysis of Su Sy _M6 and MIase,the synthesis rate of UDP-Glc is further enhanced.With the reaction time shortened to 0.5 h,120 m M UDP-Glc was produced,achieving a remarkable space-time yield of 136 g/L/h,currently the highest reported level.In the case of Su Sy _M6-Gal E multi-enzyme cascade catalysis,following the synthesis of 133 m M UDP-Glc by Su Sy _M6,the addition of Gal E enables the isomerization of UDP-Glc,yielding 10m M UDP-Gal.The cell-free catalytic system based on Su Sy _M6,which converts low-cost sucrose into high-value sugars,exhibits tremendous potential for practical applications.