Abstract: Lomofungin, generally biosynthesized by Streptomyces lomondensis S015, is a kind of phenazine compound which has broad-spectrum antifungal, antibacterial, and antitumor activities. The low-level production of lomofungin in S015 wild-type strain has been a limitation for its widely application. In order to improve the production of lomofungin and explore the function of related genes and fermentation conditions of S015 strain, this study first established a phylogenetic tree to analyze the endogenous S-adenosylmethionine synthetase metk, then overexpressed metK gene in wild-type S015, and finally optimized the carbon source through dynamic fermentation ex periment. The results showed that the production of lomofungin reached（27.4±2.8） mg/L by overexpressing metK gene, about 2.3-fold higher than that in wild-type S015. Carbon source optimization showed that the highest lomofungin production（130.6±8.0） mg/L was obtained while using 20 g/L xylose, increasing about 4.8-fold compared with the unoptimized condition. This study took advantage of up-stream gene manipulation and down-stream fermentation optimization strategy, significantly enhanced the production of lomofungin.