Abstract: To increase rifamycin yield，the Amycolatopsis mediterranei mutant of the inactivated S-malonyltransferase gene（fabD）was constructed. S-malonyltransferase gene homologous recombinant vector was constructed using fusion PCR，and then transformed into A. mediterranei by electroporation，making the homologous recombination occurr. Then fabD-inactivated strain was screened by apramycin as a marker. Further，the rifamycin production between mutant strains was wmpared and the parental strain by fermentation. The results showed that the homologous recombinant vector of fabD was successfully constructed，and the A. mediterranei △fabD was obtained；the production of rifamycin SV in the mutant strain was 168.08 mg/L，with 9.94% increase in contrast to the parent strain. The inactivation of S-malonyltransferase gene weakened the fatty acid synthesis of mutant strain，subsequently strengthened the synthesis of rifamycin.

Key words: Amycolatopsis mediterranei, rifamycin, S-malonyltransferase, fusion PCR, gene inactivation