脲对棒状链霉菌棒酸合成的影响

Effect of urea on clavulanic acid production in Streptomyces clavuligerus

摘要:

【目的】棒酸(Clavulanic acid)是棒状链霉菌(Streptomyces clavuligerus)产生的β-内酰胺酶抑制剂, 其合成过程中产生副产物脲, 旨在探讨脲对棒酸合成的影响。【方法】通过发酵过程中脲和铵盐添加实验、阻断脲酶活性以及pH梯度实验研究脲对棒酸合成影响。【结果】脲添加实验结果表明: 低浓度脲降低棒酸产量, 当添加脲浓度达到20 mmol/L时, 完全抑制棒酸合成。由于脲酶可以把脲水解为铵离子, 导致铵离子浓度及pH提高, 因此, 通过阻断棒状链霉菌脲酶活性, 可以更准确地反映脲对棒酸合成的影响。结果发现, 脲酶敲除株发酵液中脲大量积累, 浓度高达10 mmol/L, 但棒酸产量没有明显降低, 说明在该浓度下脲自身并不能抑制棒酸合成。添加脲降低野生菌棒酸产量, 可能是脲被水解为铵离子或其引起的pH变化所致。而棒酸发酵液添加铵盐的结果显示铵离子对棒酸产量没有抑制作用; 另外, pH梯度实验证实不同pH对棒酸产量影响较大。【结论】排除了脲和铵离子对棒酸合成的抑制作用, 证实了脲酶水解脲导致pH提高是脲添加导致野生菌棒酸产量降低的真正原因, 为进一步阐明棒酸合成调控机制提供了根据。

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.