地中海富盐菌PhaE蛋白乙酰化修饰对其功能的影响

摘要

[Objective] The purpose of this study is to study the acetylation of PhaE,one subunit of polyhydroxyalkanoate (PHA) synthase (PhaEC),and its function in PHA biosynthesis in Haloferax mediterranei.[Methods] PHA granules were collected by sucrose density gradient centrifugation.The acetylated sites of PhaE were identified by LC-MS/MS.The acetylated lysine (K) sites were mutated to arginine (R,mimics deacetylation) or glutamine (Q,mimics acetylation),and the mutated gene was knocked into the chromosome on its original location through pop-in/pop-out method.Taking wild-type strain as control,the cell growth,ability of glucose consumption,and the PHA accumulation were detected in different mutated strains.The relative abundance of PhaE on the PHA granule was also analyzed by Western blot in wild-type and mutated strains.[Results] Two acetylated sites (K105 and K170) were identified on PhaE.Six different mutant (K105R,K170R,K105Q,K170Q,K105R/K170R and K105Q/K170Q) strains were constructed.The fermentation experiments showed that none of the single-site mutation could affect the cell growth and PHA synthesis,and no obvious effect was observed when the two sites were simultaneously mutated to Q.However,the PHA accumulation significantly decreased when these two acetylated sites were mutated to R at the same time.Interestingly,Western blotting showed that abundance of PhaE on the PHA granules almost decreased to 58％ of the wild type.[Conclusion] The deacetylation of PhaE decreased the PHA synthesis from glucose metabolism,which was likely resulted from the inefficient interaction between the deacetylated PhaE and the PHA granules (or PhaC on the PHA granules),thus led to reduction of the activity of PhaEC synthase for PHA biosynthesis.%[目的]研究地中海富盐菌PHA合酶(PhaEC)中PhaE亚基乙酰化修饰对其功能的影响,探讨乙酰化修饰对菌体生理代谢的调控作用.[方法]蔗糖密度梯度离心收集PHA颗粒,质谱鉴定颗粒结合蛋白PhaE的乙酰化位点.将乙酰化位点(赖氨酸,K)分别突变为精氨酸(R)(模拟去乙酰化)或谷氨酰胺(Q)(模拟乙酰化),利用同源双交换原理,将突变后的基因原位敲入基因组.以野生型为对照,检测突变对菌体生长、葡萄糖消耗和PHA合成能力的影响.利用Western blot检测PHA颗粒上PhaE的含量,进一步分析乙酰化修饰对蛋白功能的影响.[结果]在PhaE蛋白105位和170位赖氨酸(K)2个位点检测到乙酰化修饰.利用遗传操作系统将突变的基因原位敲入,共得到6种突变株.发酵结果表明,任何一种单突变对菌体生长及PHA合成的影响均不明显.但当2个位点同时突变成精氨酸(K105R/K170R)时,突变株生长及合成PHA的能力均受到明显抑制,2个位点同时突变成谷氨酰胺(K105Q/K170Q)则无明显影响.进一步的Western blot结果表明,突变成精氨酸的双突变株的PHA颗粒上,PhaE蛋白的含量相较于野生型约降低了一半.[结论]PhaE蛋白的去乙酰化能够导致菌株利用葡萄糖合成PHA的能力显著降低,其可能原因是降低了PhaE与PHA颗粒或PHA颗粒上PhaC的结合能力,从而降低了PhaEC合酶的活性.