StyS激酶自磷酸化对Pseudomonas putida B3中靛蓝生物合成的调节作用

Regulatory Effect of StyS Kinase Autophosphorylation on Indigo Biosynthesis in Pseudomonas putida B3

在恶臭假单胞菌Pseudomonas putida B3中,靛蓝生物合成关键酶基因styAB上游存在一个二元调控系统StyS-StyR。StyS蛋白属于激酶家族,是磷酸化信号转导的重要媒介,但激酶StyS的自磷酸化传导机制对靛蓝生物合成的调节作用尚未探明,因此,研究以Pseudomonas putida B3中激酶蛋白StyS作为研究对象,发现了两个磷酸化结合位点,构建了磷酸化位点突变株。通过分析野生菌株P.putida B3、styS基因缺失菌株P.putida B3-ΔstyS、styS基因回补菌株P.putida B3-ΔstyS-8和磷酸化位点突变株P.putida B3-ΔstyS-1之间的靛蓝产量及酶活发现,P.putida B3产量为10.14 mg/L,P.putida B3-ΔstyS-8产量为3.63 mg/L,磷酸化位点突变菌株无激酶活性且失去了产生靛蓝的能力。结果表明,StyS自磷酸化作用在靛蓝发酵体系中起重要作用。研究结果将为靛蓝生物合成途径的进一步研究提供参考。

The two-component StyS-StyR regulatory system locates in the upstream of the styAB gene,which encodes a key enzyme for indigo biosynthesis in Pseudomonas putida B3.As a kinase,StyS is an important mediator of phosphorylation-dependent signal transduction.However,the effect of StyS trans-autophosphorylation on indigo biosynthesis has not been clearly studied.Therefore,the StyS kinase protein in Pseudomonas putida B3 was used as the research object,and two different phosphorylation binding sites were discovered.Then a phosphorylation site-mutant strain was constructed.Indigo production and enzyme activity in the wild-type P.putida B3,a strain lacking the styS gene(P.putida B3-ΔstyS),a styS gene-backup strain(P.putida B3-ΔstyS-8),and the phosphorylation site-mutant strain(P.putida B3-ΔstyS-1)were compared.The results showed that the indigo yields of P.putida B3 and P.putida B3-ΔstyS-8 were 10.14 mg/L and 3.63 mg/L,respectively.It suggested that the phosphorylation site-mutant strain had no kinase activity and lost ability to produce indigo.The results indicated that StyS autophosphorylation played an important role in indigo production,which might provide references for further research on indigo biosynthetic pathway.