Streptomyces clavuligerus(S.clavuligerus) produces a number of β-lactam compounds,including clavulanic acid.An efficient procedure for creating clavulanic acid high producing S.clavuligerus mutant by using the polymerase chain reaction(PCR)-targeting system was described.PCR-targeting system was applied to mutate the lat gene of S.clavuligerus to obtain lat gene knocked-out,unmarked mutant S.clavuligerus lat:scar.Through PCR-targeting system to knock out the lat gene of S.clavuligerus,clavulanic acid of the mutant S.clavuligerus lat:scar was 2.8-fold to the original strain.This procedure allows repeated use of the same resistance marker for making multiple knock-outs in the same strain.This provides another efficient procedure for creating Streptomyces mutants.