The　synthesis　of　chloramphenicol,　a　kind　of　amphenicol　antibiotic　with　broad-spectrum　antibacterial　activity,　is　challenging　due　to　its　stereochemistry.　Here,　we　have　designed　a　four-step　chemoenzymatic　strategy,　including　a　biocatalytic　step　mediated　by　l-threonine　transaldolase　from　Pseudomonas　sp.　(PsLTTA)　to　convert　4-nitrobenzaldehyde　(1)　to　(2S,3R)-2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic　acid　(2)　followed　by　a　three-step　chemical　reaction　to　obtain　chloramphenicol.　A　rational　design　of　PsLTTA　was　devised　by　reshaping　the　substrate　binding　pocket　and　substrate　access　channel,　resulting　in　the　best　variant　PsLTTA-N35A/C57I/F59A/H69F　(PsLTTA-Mu9),　which　achieved　a　7.1-fold　higher　yield　of　2　than　wild-type　PsLTTA.　After　coupling　with　ScADH/CbFDH　to　remove　the　byproduct　acetaldehyde　and　optimizing　the　reaction　conditions,　the　whole-cell　catalyst　BL21(PsLTTA-Mu9/ScADH/CbFDH)　could　synthesize　200　mM　of　2　in　four　hours　with　99%　conversion　and　97.7%　de,　delivering　the　highest　time-space　yield　(11.3　g　L−1　h−1)　ever　reported.　Finally,　the　chemoenzymatic　approach　was　applied　for　the　gram-scale　synthesis　of　5　with　a　high　overall　yield　(54%).　The　success　of　this　strategy　demonstrates　the　great　advantage　of　the　chemoenzymatic　approach　in　the　asymmetric　synthesis　of　chloramphenicol　and　may　contribute　to　its　industrial　synthesis.　©　2023　The　Royal　Society　of　Chemistry.