Lincomycin, a clinically important antibiotic for the treatment of diseases caused by Gram-positive bacteria, is formed by a bifurcated biosynthetic pathway. The latest research reported that two low-molecular-weight thiols, mycothiol and ergothioneine were discovered in the sulfur of lincomycin. The coupling of two bacterial thiols could be biosynthesized by histidine, cysteine and methionine, which indicates the relationship between the three amino acids and the enhancement of the two thiols in the lincomycin biosynthesis. In 15-l stirred bioreactor, methionine and the combination of the three amino acids were added, and the lincomycin productions were increased by 24.6 and 47.5% compared with control (6051 μ/ml). Along with last S-methylation gene lmbG, the key biosynthesis genes of mycothiol and ergothioneine and the cyclic regeneration genes in lincomycin biosynthesis, mshA, egtD, lmbT, lmbV and lmbE, were analyzed by real-time-quantitative PCR, and the results shown the expression levels of these genes were higher than the control.