System Construction For Rare Sugars Synthesis By Escherichia Coli Whole Cells Based On Dihydroxyacetone Phosphate-dependent Aldolases

With the rapid development of society,rare sugar,because of its anti-cancer,neuroprotective,scavenging free radicals and other physiological activities,has a wide application prospect in medicine,diet and other fields.It is significant to establish a practical rare sugar resource pool and improve the production system.This research mainly focuses three different dihydroxyacetone phosphate?DHAP?-dependent aldolases coupled with glycerol phosphate oxidase,expressing in E.coli cells for one-pot processes catalysis DL-glycerol 3-phosphate?DL-3-GP?and D?L?-glyceraldehyde to produce value-added rare sugars.Whole-cell reaction system struggle the shackles of enzymatic synthesis of rare sugar,and effectively solve the serious problems in the production process of the rare sugar.Study uses three different aldolases included L-rhamnulose-1-phosphate aldolase?RhaD?from E.coli,D-fructose-1,6-biophosphate aldolase?Fru A?from Staphylococcus carnosus,L-fuculose-1-phosphate aldolase?FucA?from Thermus thermophilus HB8,respectively couple with glycerol phosphate oxidase?GPO?which come from Streptococcus pneumoniae,connected to the pET-28a plasmid.Three kinds of whole cell systems(RhaD_E.coli+GPO,FucA_T.HB8+GPO,FruA_S.car+GPO)successfully co-express in BL21?DE3?,and use DL-3-GP and D?L?-glyceraldehyde as substrates,to produce a variety of rare sugars included D-sorbose,D-psicose,L-sorbose and L-fructose and so on.The quantitative analysis of rare sugars was performed by thin-layer chromatography?TLC?and high performance liquid chromatography?HPLC?.Meanwhile,the reaction conditions of the whole cell system were optimized and the result was as follows:pH 7.0,37?,whole cell dry weight about 25 g·L^-1.Three kinds of whole cell system for rare sugar synthesis were successfully established.However,the aldolases may lose stereoselectivities when accepting glyceraldehyde as the acceptor.Wild type RhaD from E.coli lost its stereoselectivity when accepting D-glyceraldehyde as the acceptor.As a result,two rare sugars D-psicose and D-sorbose were generated with a ratio of 1:1.The molecular docking of RhaD with D-glyceraldehyde was performed by software discovery studio 4.0.Simultaneously,the key amino acids?29,115,188,etc.?interacted with the substrate was analyzed and mutated.The mutant RhaD^P188F was successfully obtained to exclusively synthesize high value-added D-psicose through the Iterative Saturation mutagenesis?ISM?.Finally,modified RhaD aldolase was applied to whole cell system and RhaD^P188F+GPO whole-cell reaction system was established using DL-3-GP and D-glyceraldehyde as substrates synthesized D-psicose.