Abstract:　Monascus　sp.　is　an　important　food　microbial　resource　with　the　production　of　cholesterol-lowering　agent　lovastatin　and　other　healthy　metabolites.　However,　the　mycotoxin　citrinin　naturally　produced　by　Monascus　sp.　and　the　insufficient　productivity　of　lovastatin　limit　its　large-scale　use　in　food　industry.　The　aim　of　this　paper　is　to　modify　a　lovastatin-producing　strain　Monascus　pilosus　GN-01　through　metabolic　engineering　to　obtain　a　citrinin-free　M.　pilosus　strain　with　higher　yield　of　lovastatin.　The　citrinin　synthesis　regulator　gene　ctnR　was　firstly　disrupted　to　obtain　GN-02　without　citrinin　production.　Based　on　that,　the　lovastatin　biosynthesis　genes　(mokC,　mokD,　mokE,　mokF,　mokH,　mokI,　and　LaeA)　were,　respectively,　overexpressed,　and　pigment-regulatory　gene　(pigR)　was　knocked　out　to　improve　lovastatin　production.　The　results　indicated　ctnR　inactivation　effectively　disrupted　the　citrinin　release　by　M.　pilosus　GN-01.　The　overexpression　of　lovastatin　biosynthesis　genes　and　pigR　knockout　could　lead　higher　contents　of　lovastatin,　of　which　pigR　knockout　strain　achieved　76.60%　increase　in　the　yield　of　lovastatin　compared　to　GN-02.　These　studies　suggest　that　such　multiplex　metabolic　pathway　engineering　in　M.　pilosus　GN-01　is　promising　for　high　lovastatin　production　by　a　safe　strain　for　application　in　Monascus-related　food.　Key　points:　•　Disruption　of　the　regulator　gene　ctnR　inhibited　citrinin　production　of　M.　pilosus.　•　Synchronous　overexpression　of　biosynthesis　gene　enhanced　lovastatin　production.　•　pigR　knockout　enhanced　lovastatin　of　ΔctnR　strain　of　M.　pilosus.　©　2023,　The　Author(s),　under　exclusive　licence　to　Springer-Verlag　GmbH　Germany,　part　of　Springer　Nature.