The Function Research Of FabG From Synechocysis Sp.PCC 6803

Polyhydroxyalkanoates(PHA)is a kind of polyester synthesized by microbe and algae grown in condition that are sufficient with carbon source and lack of nitrogen or phosphorus source in vivo,meanwhile it act as an important energy storage material and exist in cell as granules.PHA has great quantities of advantages except for biodegradable and it was considered as potential alternative to raw petroleum materials.?-Ketoacyl-ACP reductase(FabG)is the first NAD PH-dependent reductase in the fatty acid synthesis pathway.Acetoacetyl-ACP which is the substrate of FabG is similar to acetoacetyl-CoA that is the substrate of PhaB in the PHB synthesis pathway.The only difference between them is the thioester bond connects to ACP and CoA,respectively,it can be supposed that FabG might participate in PHA synthesis regulation from previous work.In this paper,photo-autotrophic cyanobacterium Synechocystis sp.PCC 6803 was as experimental material,and SpFabG(FabG from Synechocystis sp.PCC 6803)was cloned,heterologous expressed and purified in vitro to investigate its kinetic characteristics as reductase,and the optimum temperature and pH that SpFabG catalyze acetoacetyl-CoA reaction were researched,furthermore the Km,Vmax and kcat values were evaluated by double reciprocal plots.Meanwhile,fabG overexpressed on ?phaB strain(OvfabG+?phaB),fabG overexpressed strain(OvfabG)and fabG knocked down strain(fabG::C.K2)were constructed,then wild-type(WT)and four mutant strains were cultivated in N-repletion and N-deprivation with 0.4%sodium acetate conditions and physiology features were measured to research the reglution function of SpFabG,the results are as followed:1.The results in vitro showed that,the optimum temperature of SpFabG was 30 ? and optimum pH was 7,the Km,Vmax,and kcat values were 2.3 mM,92.59 ?mol/L/min and 19.85 s-1,respectively,compared with FabG from other organisms SpFabG has a low affinity with acetoacetyl-CoA and its catalytic efficiency lies in middle level.2.OvfabG and fabG::C.K2 showed no obvious difference with WT on fatty acid(FA)content,and the expression level of fabG didn't affect FA synthesis pathway,which indicated SpFabG was essential but not rate-limiting for fatty acid synthesis.3.When phaB was knocked out,?phaB strain didn't accumulate PHB under N deprived condition,and overexpression of fabG on ?phaB recovered the ability of PHB accumulation reached 31%level of WT,the results above demonstrated when Synechocystis sp.PCC 6803 lose the ability of PHB accumulation,SpFabG could function as SpPhaB,however it still can't reach the level of SpPhaB yet.The FA content of OvfabG+AphaB decreased 27%compared with WT,this might result from FabG participated in the PHB synthesis pathway which led to part of carbon flux flowed to PHB synthesis pathway and the decrease of FA content eventually.Furthuremore,OvfabG and fabG::C.K2 showed no obvious difference with WT on PHB content,which indicate SpFabG doesn't regulate PHB synthesis when SpPhaB exists.These results above lay a theoretical foundation for utilizing cyanobacterias to fix CO2 to produce mcl-PHA photoautotrophicly and promote output of PHA.