Esomeprazole　is　the　most　popular　proton　pump　inhibitor　for　treating　gastroesophageal　reflux　disease.　Previously,　a　phenylacetone　monooxygenase　mutant　LnPAMOmu15　(LM15)　was　obtained　by　protein　engineering　for　asymmetric　synthesis　of　esomeprazole　using　pyrmetazole　as　substrate.　To　scale　up　the　whole　cell　asymmetric　synthesis　of　esomeprazole　and　reduce　the　cost,　in　this　work,　an　Escherichia　coli　whole-cell　catalyst　harboring　LM15　and　formate　dehydrogenase　from　Burkholderia　stabilis　15516　(BstFDH)　were　constructed　through　optimized　gene　assembly　patterns.　CRISPR/Cas9　mediated　insertion　of　Ptrc　promoter　in　genome　was　done　to　enhance　the　expression　of　key　genes　to　increase　the　cellular　NADP　supply　in　the　whole　cell　catalyst,　by　which　the　amount　of　externally　added　NADP+　for　the　asymmetric　synthesis　of　esomeprazole　decreased　to　0.05　mM　from　0.3　mM　for　reducing　the　cost.　After　the　optimization　of　reaction　conditions　in　the　reactor,　the　scalable　synthesis　of　esomeprazole　was　performed　using　the　efficient　LM15-BstFDH　whole-cell　as　catalyst,　which　showed　the　highest　reported　space-time　yield　of　3.28　g/L/h　with　50　mM　of　pyrmetazole　loading.　Isolation　procedure　was　conducted　to　obtain　esomeprazole　sodium　of　99.55　%　purity　and　＞　99.9　%　ee　with　90.1　%　isolation　yield.　This　work　provides　the　basis　for　production　of　enantio-pure　esomeprazole　via　cost-effective　whole　cell　biocatalysis.　©　2024