Hydroxytyrosol,　a　naturally　occurring　chemical　with　antioxidant　and　antiviral　properties,　is　widely　used　in　the　nutrition,　pharmaceutical,　and　cosmetic　industries.　In　the　present　study,　a　modularized　cascade　composed　of　Modules　1　and　2　was　designed　and　implemented　to　convert　L-tyrosine　to　hydroxytyrosol.　Module　1　was　a　four-enzymatic　cascade　for　converting　L-tyrosine　to　tyrosol.　Engineering　Module　1　by　fine-tuning　the　expression　of　the　desired　enzymes　resulted　in　a　robust　whole-cell　catalyst,　BL21　(M1-13),　which　converted　L-tyrosine　to　tyrosol　at　high　substrate　loading.　Module　2　involved　a　4-hydroxyphenylacetate　3-monooxygenase　(HpaBC)-catalyzed　reaction　to　hydroxylate　tyrosol　to　form　hydroxytyrosol.　The　rational　design　of　the　HpaB　subunit　led　to　a　positive　variant,　HpaB-Mu　(T292S/R474A),　which　was　subsequently　applied　to　Module　2　for　tyrosol　hydroxylation,　yielding　a　robust　whole-cell　catalyst,　BL21　(M2-05).　The　two　designed　modules　were　merged　for　one-pot　conversion　of　L-tyrosine　to　hydroxytyrosol　by　adjusting　the　ratio　and　total　amount　of　whole-cell　catalyst　loading,　capable　of　converting　40　mM　of　L-tyrosine　to　35.8　mM　of　hydroxytyrosol　with　a　high　space–time　yield　(1.38　g/L/h).　The　current　study　proved　that　engineering　HpaB　at　the　substrate　tunnel　was　a　feasible　way　to　boost　its　activity　and　proposed　an　effective　method　for　synthesizing　hydroxytyrosol　from　low-cost　substrates,　which　has　great　economic　potential.　©　2025　Elsevier　Inc.