摘要
Rebaudioside M8(Reb M8),a high-potency sweetener derived from natural Rebaudioside D found in Stevia rebaudiana,exhibits favorable organoleptic properties as an ideal sugar replacement candidate.However,the industrial biosynthesis of Reb M8 is hindered by the poor thermostability of its key biocatalyst,glycosyltransferase UGT94E13,limiting its application in long-term or high-temperature processes.In this study,a combinatorial strategy integrating computer-aided rational design as well as structural and evolutionary conservation analyses is utilized to enhance the thermostability of a catalytic-efficient UGT94E13-F169G/I185G(M0)to a superior variant M0-T102D/V110I/F208Y/N251E(M5)with a 5.4℃ increase in melting temperature(T_(m)),a 1.08-fold improvement in catalytic activity,and a 14.8-fold extension of half-life relative to M0.The simultaneous improvement of activity and stability is well explained by molecular dynamics simulations and structural analyses,revealing flexibility reduction,hydrophobic packing improvement,salt bridge formation,and electrostatics optimization as key factors for the improved thermal resilience.Furthermore,in a cascade biocatalytic system coupled with sucrose synthase AtSuSy,M5 enabled sustained biosynthesis and highproduction of Reb M8(35.30 g/L)under optimized fed-batch conditions.These findings establish M5 as a robust and scalable biocatalyst that overcomes the inherent activity-stability trade-off of glycosyltransferase UGT94E13,advancing the biomanufacturing potential of promising sweeteners in food and health industries.
基金
supported by the Natural Science Foundation of Jiangsu Province(BK20202002)
National Natural Science Foundation of China(22207044,22477047,32270082)
the Fundamental Research Funds for the Central Universities(JUSRP202404012,JUSRP124020).
