L-threo-4-methylsulfonylphenylserine　ethyl　ester　[(2S,　3R)-1d],　a　key　building　block　of　florfenicol　which　synthesized　by　esterification　using　ethanol　and　L-threo-4-methylsulfonylphenylserine　[(2S,　3R)-1b]　as　substrates.　L-threonine　aldolase　(LTA)　is　a　promising　biocatalyst　for　producing　(2S,　3R)-1b　through　a　one-step　process　taking　4-Methylsulphonyl　benzaldehyde　(1a)　and　glycine　as　substrates　under　the　mild　condition.　However,　the　moderate　Cβ-stereoselective　blocked　the　industrial　application　of　LTA.　To　address　this　issue,　rational　design　by　combination　with　prereaction　state　molecular　dynamics　(MD)　simulation　and　per-residue　energy　decomposition　algorithm　are　employed　to　engineer　LTA　for　enhancing　Cβ-stereoselective.　As　a　result,　a　triple　mutant　N16A/E98S/Y314R　(Mu3)　is　screened　out　to　produce　(2S,　3R)-1b　with　93.7%　de　and　90.2%　conversion　under　the　300　mM　1a　substrate　loading.　Furthermore,　esterification　is　applied　to　synthesize　(2S,　3R)-1d　with　over　99%　chiral　purity　and　99%　product　purity.　The　success　of　this　study　provides　new　insights　for　the　rational　design　of　LTA　with　improved　Cβ-stereoselectivity　and　proves　a　chemo-enzymatic　route　for　green　synthesis　of　(2S,　3R)-1d.　©　2022