(2S,　3R)-4-methylsulfonylphenylserine　[(2S,　3R)-MPS],　a　key　chiral　precursor　for　antibiotics　florfenicol　and　thiamphenicol,　could　be　asymmetrically　synthesized　by　L-threonine　transaldolase　(LTTA)　coupled　with　an　acetaldehyde　elimination　system.　The　low　efficiency　of　acetaldehyde　elimination　system　blocked　further　accumulation　of　(2S,　3R)-MPS.　To　address　this　issue,　strengthening　acetaldehyde　elimination　system　and　enzyme　selfassembly　strategy　were　combined　to　accelerate　biosynthesis　of　(2S,　3R)-MPS.　The　new　multi-enzyme　cascade　with　intensified　acetaldehyde　elimination　system　BL21　(PsLTTAD2/ScADH/BtGDH)　could　produce　(2S,　3R)-MPS　with　a　titer　of　157.6　mM,　1.7-folds　than　that　produced　by　the　original　system　BL21　(PsLTTAD2/ApADH/CbFDH).　Moreover,　self-assembly　of　PsLTTAD2　and　ScADH　by　respective　fusion　of　SpyTag　and　SpyCatcher　were　carried　out　to　develop　a　self-assembled　multi-enzyme　cascade　BL21　(ST-PsLTTAD2/SC-ScADH/BtGDH).　As　a　result,　the　yield　of　(2S,　3R)-MPS　was　up　to　248.1　mM　with　95%　de.　As　far　as　we　knew,　that　represented　the　highest　yield　of　(2S,　3R)-MPS　by　enzymatic　synthesis,　and　therefore　was　a　promising　and　green　route　for　industrial　production　of　this　valuable　compound.