Abstract: Calcimycin is an essential ionophoric antibiotics exhibiting multiple biological effects.The gene cluster for calcimycinbiosynthesis has been cloned from the genomic DNA of Streptomyces chartreusis NRRL3882,but the calcimycin biosynthetic pathwayremains unclear.This study aims to analyze the function of the cal U3 gene in calcimycin biosynthesis gene cluster.The cal U3 genedisruption plasmid was constructed by PCR-targeting.The Δcal U3 mutant of Streptomyces chartreusis NRRL3882 was generatedthrough introducing the cal U3 disruption vector into the S.chartreusis strains by conjugation transfer and homologous recombination.For complementary strain,the cal U3 complemented plasmid was introduced into the Δcal U3 mutant via conjugation.The metabolites ofΔcal U3 mutant and complementary strain were analyzed by high performance liquid chromatography（HPLC） and liquid chromatogra-phy-mass spectrometry（LC-MS）.The cal U3 over-expression vector was constructed,and recombinant N-terminal His6-tagged Cal U3protein was over-expressed in E.coli and purified,and its UV/Vis absorbance spectra were analyzed.The result showed that the Δcal U3mutant was unable to produce calcimycin and N-demethyl-calcimycin,whereas cezomycin was still produced,and the complementationstrain restored the antibiotic production ability of the wild-type.The purified His6-tagged CalU3 has a distinctive yellow color,and the major absorption wavelengths of Cal U3 were at 377 nm and 455 nm.The above results indicated that cal U3 was related to the formation ofthe substituent group at the 3-position of the calcimycin benzoxazole ring.Cal U3 possesses a binding site for FAD-NAD（P）,and may bea FAD-NAD（P）-dependent oxidoreductase,which is responsible for catalyzing the 3-hydroxylation of the benzoxazole ring in activatedcezomycin to generate 3-Hydroxycezomycin.This study preliminarily elucidated the modification function of cal U3 gene on the 3-posi-tion of the benzoxazole ring of calcimycin,which laid a solid foundation for a comprehensive analysis of the biosynthetic pathway of cal-cimycin.