极端耐盐放线菌白色普氏菌YIM 90005T四氢嘧啶及5-羟基四氢嘧啶合成相关基因的克隆

Cloning and characterization of gene cluster for biosynthesis of ectoine and 5-hydroxyectoine from extreme halotolerant actinomycete strain Prauserella alba YIM 90005

摘要:

摘要：【目的】 为了研究耐盐放线菌对高盐环境的适应机理。【方法】 用HPLC定量检测了极端耐盐、丝状产孢放线菌——白色普氏菌（Prauserella alba） YIM 90005T在不同盐浓度下胞内相容性溶质的种类和含量。【结果】 结果发现，四氢嘧啶和5-羟基四氢嘧啶是其主要的相容性溶质。在培养基NaCl浓度为10%时，四氢嘧啶在胞内累积浓度最大，为18.77 μg/mg干菌体重。之后随NaCl浓度的升高，胞内的四氢嘧啶含量逐渐减少，而5-羟基四氢嘧啶的含量逐渐增加，在该菌耐受的最高NaCl浓度下（24% w/v），胞内5-羟基四氢嘧啶含量达到最大值，为22.98 μg/mg干菌体重。设计兼并引物，利用染色体步移，克隆得到四氢嘧啶及5-羟基四氢嘧啶合成相关基因ectABCD。序列分析表明，ectABCD位于一个操纵子中。进一步对不同NaCl浓度培养条件下ectB,D的表达量进行定量分析，结果表明该基因簇表达量随着培养基中NaCl浓度的增加而增大。【结论】 研究结果证实5-羟基四氢嘧啶是P. alba YIM 90005T在极高盐浓度条件下起渗透调节及保护的相容性溶质。

Abstract:

Abstract: [Objective] To study adaptive mechanism in hypersaline environments of extreme halotolerant filamentous actinomycetes. [Methods] Using HPLC we analyzed compatible solutes from extreme halotolerant filamentous actinomycete strain Prauserella alba YIM 90005T that was cultivated at different NaCl concentrations. [Results] Ectoine and 5-hydroxyectoine were two major compatible solutes for strain Prauserella alba YIM 90005T. Ectoine accumulated to the maximum content of 18.77 μg/mg dry cell weight after being inoculated in 10% NaCl (w/v). And 5-hydroxyectoine reached 22.98 μg/mg dry cell weight after being inoculated in 24% NaCl (w/v). The ectA (acyltransferase), ectB (aminotransferase), ectC (ectoine synthase) and ectD (ectoine hydroxylase) genes cluster encoding genes on ectoine and hydroxyectoine synthesis were further cloned by designing the degenerate primer and genome walking methods. The sequence analysis indicated that ectABCD was an operon. Furthermore, the expression of ectB and ectD inoculated at different salt concentrations was quantified by real-time PCR, and the results indicated that the expression of the gene cluster would be increasing as the salt concentration increased. [Conclusion] 5-hydroxyectoine was the major compatible solute for osmotic regulation of strain Prauserella alba YIM 90005T to adapt high salt concentration.