Abstract
Clavulanic acid is a product of the fermentation of Streptomyces clavuligerus. Clavulanic acid is a specific and irreversible inhibitor of a wide range of bacterial β-lactamases. Clavulanic acid has been used in human therapy for several years in combination with amoxicillin. In this study, the effects of different precursors on the biosynthesis of clavulanic acid using S. clavuligerus in batch and fed-batch fermentations were investigated. An intermittent feeding strategy was employed and the amount of feed medium was optimized to improve the production of clavulanic acid.The intermittent feeding of medium containing glycerol was the feeding strategy in shake flask cultures, which resulted in the maximum production of clavulanic acid was 2.3-fold higher than without glycerol feeding culture. Clavulanic acid biosynthesis was doubled by adding ornithine. The optimal initial ornithine concentration for clavulanic acid biosynthesis was 5~10 mM. Simultaneous intermittent feeding glycerol and ornithine in shake flask cultures, which resulted in the maximum production of clavulanic acid was 3.1-fold higher than without glycerol feeding culture. The optimal amount of glycerol and ornithine feeding twice daily with 12 hours interval for clavulanic acid production were 1.0 g-glycerol/100 ml-broth．day and 1.0 mmol-ornithine/100 ml-broth．day. The optimal mode of intermittent feeding strategy was feeding six times daily with 4-hour intervals.Feeding studies demonstrate that glycerol is precursor of the β-lactam moiety(C-5,C-6, and C-7), whereas ornithine is precursor of C5-moiety. Glycerol and ornithine exert a stimulation of the biosynthesis of clavulanic acid and both precursors are most efficiently incorporated into clavulanic acid.