Norepinephrine,　a　kind　of　β-adrenergic　receptor　agonist,　is　commonly　used　for　treating　shocks　and　hypotension　caused　by　a　variety　of　symptoms.　The　development　of　a　straightforward,　efficient　and　environmentally　friendly　biocatalytic　route　for　manufacturing　norepinephrine　remains　a　challenge.　Here,　we　designed　and　realized　an　artificial　biocatalytic　cascade　to　access　norepinephrine　starting　from　3,　4-dihydroxybenzaldehyde　and　L-threonine　mediated　by　a　tailored-made　L-threonine　transaldolase　PsLTTA-Mu1　and　a　newly　screened　tyrosine　decarboxylase　ErTDC.　To　overcome　the　imbalance　of　multi-enzymes　in　a　single　cell,　engineering　of　PsLTTA　for　improved　activity　and　fine-tuning　expression　mode　of　multi-enzymes　in　single　E.coli　cells　were　combined,　leading　to　a　robust　whole　cell　biocatalyst　ES07　that　could　produce　100　mM　norepinephrine　with　99%　conversion,　delivering　a　highest　time-space　yield　(3.38　g/L/h)　ever　reported.　To　summarized,　the　current　study　proposed　an　effective　biocatalytic　approach　for　the　synthesis　of　norepinephrine　from　low-cost　substrates,　paving　the　way　for　industrial　applications　of　enzymatic　norepinephrine　production.　©　2024　Elsevier　B.V.