Effect of urea on clavulanic acid production in Streptomyces clavuligerus

Abstract:

[Objective] Clavulanic acid (CA) is a β-lactamase inhibitor produced by Streptomyces clavuligerus. Since urea is a byproduct of CA biosynthesis, we investigated the potential inhibitory effect of urea on CA biosynthesis. [Methods] We designed urea addition, ammonium addition, urease inactivation and pH gradient experiments to research effect of urea on CA production. [Results] Addition of urea inhibited CA production in the wild type, and the production was completely abolished when the concentration of urea reached 20 mmol/L. To prevent urea hydrolysis, the genes encoding urease were disrupted and the corresponding mutant was analyzed: which accumulated higher concentration of urea, reaching 10mmol/L, but this did not inhibit CA production. Therefore, urea itself was excluded as the factor inhibiting CA biosynthesis. So the inhibitory effect of urea on CA production in the wild type may be due to the elevated ammonium concentration or pH resulting from urea hydrolysis by urease. To test these possibilities, ammonium salt were added in the fermentation medium, which showed no inhibitory effect on CA production. Furthermore, the gradient experiment of pH confirmed that different pH had a large influence for CA production. [Conclusion] pH increase due to urea hydrolysis was identified as the real reason to inhibit CA production in the wild type strain. This study laid the foundation for further elucidation of the regulatory mechanisms controlling CA biosynthesis in S. clavuligerus.