The standard gene disruption and replacement to delete the actinorhodin biosynthetic gene cluster (Act) in Streptomyces coelicolor was inefficient, and the polymerase chain reaction-targeting of the cosmid could efficiently delete the Act, but still was a time-consuming procedure for markerless gene replacement. By using optimal Streptomyces codons, we synthesized a sceS gene encoding identical amino acid sequence as the chromosome rare-cutting meganuclease I-sce I of the Saccharomyces cerevisiae mitochondria. Expression of sceS gene in S. coelicolor resulted in promotion of homologous recombination and subsequently, successful achieved markerless deletion of the Act. The sceS system may be useful for the sequential markerless deletions of chromosomal segments in Streptomyces.