The　synthesis　of　steroids　is　challenging　through　multistep　steroidal　core　modifications　with　high　site-selectivity　and　productivity.　In　this　work,　a　novel　enzymatic　cascade　system　was　constructed　for　synthesis　of　testolactone　by　specific　C17　lactonization/Δ1-dehydrogenation　from　inexpensive　androstenedione　using　an　engineered　polycyclic　ketone　monooxygenase　(PockeMO)　and　an　appropriate　3-ketosteroid-Δ1-dehydrogenase　(ReKstD).　The　focused　saturation　mutagenesis　in　the　substrate　binding　pocket　was　implemented　for　evolution　of　PockeMO　to　eliminate　the　bottleneck　effect.　A　best　mutant　MU3　(I225L/L226V/L532Y)　was　obtained　with　20-fold　higher　specific　activity　compared　to　PockeMO.　The　catalytic　efficiency　(kcat/Km)　of　MU3　was　171-fold　higher　and　the　substrate　scope　shifted　to　polycyclic　ketones.　Molecular　dynamic　simulations　suggested　that　the　activity　was　improved　by　stabilization　of　the　pre-lactonization　state　and　generation　of　productive　orientation　of　4-AD　mediated　by　distal　L532Y　mutation.　Based　on　that,　the　three　genes,　MU3,　ReKstD　and　a　ketoreductase　for　NADPH　regeneration,　were　rationally　integrated　in　one　cell　via　expression　fine-tuning　to　form　the　efficient　single　cell　catalyst　E.　coli　S9.　The　single　whole-cell　biocatalytic　process　was　scaled　up　and　could　generate　9.0　g/L　testolactone　with　the　high　space　time　yield　of　1　g/L/h　without　steroidal　by-product,　indicating　the　potential　for　site-specific　and　one-pot　synthesis　of　steroid.　©　2024