Acetylation regulates the function of PhaE in Haloferax mediterranei

Abstract:

[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.