Difficidins生物合成基因簇中蛋白激酶基因difB功能研究

Characterization of difB in the difficidins biosynthetic gene cluster

目的对海洋芽孢杆菌Bacillus methylotrophicus B-9987 difficidins化合物生物合成基因簇中difB基因功能进行探究。方法通过生物信息学手段对difB的功能进行预测;构建difB双交换阻断突变株及高表达株;通过高效液相色谱法(HPLC)分析野生株和突变株发酵产物的差异;在大肠杆菌中表达DifB蛋白,以脱磷酸difficidin(3)作为底物,探究DifB的体外功能。结果得到了ΔdifB双交换阻断突变株及difB高表达株;HPLC分析结果表明,相比较野生株,ΔdifB突变株中difficidins化合物峰不再产生,difB高表达株中difficidins的产量与野生株无显著差别;DifB蛋白不能催化脱磷酸化difficidin(3)发生磷酸化反应。结论difB是difficidins生物合成所必需的,可能与磷酸基团的组装相关,但磷酸化机制有待进一步研究。

Objective To investigate the function of difB in the difficidins biosynthesis gene cluster dif in marine-derived Bacillus methylotrophicus B-9987.Methods difB was analyzed by bioinformatic analysis.The mutant strain and high-expression strain of difB were constructed.The mutant strain,high-expression strain and wild-type strain were fermented under the same conditions,and the fermentation broths were analyzed by HPLC.difB was expressed in E.coli,and the dephosphorylated difficidin 3 was used as the substrate to study its function in vitro.Results Bioinformatic analysis showed difB encoded a putative protein kinase.ΔdifB mutant and B-9987/pADIFB high-expression strain were constructed successfully.HPLC analysis showed that difficidins were no longer produced in ΔdifB mutant strain compared to that of the wild-type strain;the yield of difficidins in difB high-expression strain B-9987/pADIFB did not change obviously.DifB protein was purified,but it could not catalyze the phosphorylation of dephosphorylated difficidin 3.Conclusion difB is necessary for the biosynthesis of difficidins and may be related to the assembly of C9 phosphate group in difficidins.The phosphorylation mechanism of difficidins needs further study.