l-Threonine　aldolase　from　Actinocorallia　herbida　(AhLTA)　is　an　ideal　catalyst　for　producing　l-threo-4-methylsulfonylphenylserine　[(2S,3R)-1　b],　a　key　chiral　precursor　for　florfenicol　and　thiamphenicol.　The　moderate　Cβ　stereoselectivity　is　the　main　obstacle　to　the　industrial　application　of　AhLTA.　To　address　this　issue,　a　combinatorial　active-site　saturation　test　(CAST)　together　with　sequence　conservatism　analysis　was　applied　to　engineer　the　AhLTA　toward　improved　Cβ　stereoselectivity.　The　optical　mutant　Y314R　could　asymmetrically　synthesize　l-threo-4-methylsulfonylphenylserine　with　81　%　diastereomeric　excess　(de),　which　is　23　%　higher　than　wild-type　AhLTA.　Molecular　dynamic　(MD)　simulations　revealed　that　the　mechanism　for　the　improvement　in　Cβ　stereoselectivity　of　Y314R　is　due　to　the　acylamino　group　of　residues　Arg314　controlling　the　orientation　of　substrate　4-methylsulfonyl　benzaldehyde　(1　a)　in　the　active　pocket　by　directed　interaction　with　the　methylsulfonyl　group;　this　leads　to　asymmetric　synthesis　of　l-threo-4-methylsulfonylphenylserine.　The　success　in　this　study　demonstrates　that　direct　control　of　substrates　in　an　active　pocket　is　an　attract　strategy　to　address　the　Cβ　stereoselectivity　problem　of　LTA　and　contribute　to　the　industrial　application　of　LTA.　©　2021　Wiley-VCH　GmbH