The Physiological Roles Of Cg1360 And Cg1361 In ATP Synthase From Corynebacterium Glutamicum

ATP synthase is a membrane-bound multi-subunit complex,which is essential for living organism to synthesize energy.ATP synthases synthesize ATP by a rotary mechanism driven by a transmembrane proton motive force,and play a vital role on physiology and cell metabolism.Generally,the intact ATP synthase is composed of two major subcomplexes,the membrane-embedded Fo sector and the water-soluble FI sector.The Fo sector consists of three subunits(a1,b2 and C10o-15)and can function as a proton channel.The F1 sector consists of five subunits(?2?3?1?1?1)and contains three catalytic sites at the interface of a and ? subunits.In most bacteria,the atp operon contains nine open reading frames(ORFs),including eight structural genes(atpBEFHAGDC)and one preceded atpl gene.AtpI,also known as UncI,is a small membrane protein and is shown to play a role in the c-subunit ring formation during the assembly of some bacterial ATP synthases.Corynebacterium glutamicum is used as an industrial workhorse for amino acid production,and its atp operon contains eight structural genes and two additional genes of cg1360 and cg 1361 located in the upstream of atp operon.Some studies have focused on the roles of the entire atpBEFHAGDC cluster,but the physiological roles of cg1360 and cg1361 have not been explored so far.In this study,we analyzed Cg1360,Cg1361 and other bacterial AtpI proteins by bioinformatics tools,and found that Cg1360 is a hydrophobic membrane protein with four transmembrane helices(TMHs),which is very similar to AtpI.Cg1361 consists of 80 amino acids with no apparent TMH.Deletion of cg1360 resulted in a 30%or 69%reduction in the growth of cells in a medium with glucose or acetic acid as a carbon source,respectively,while the absence of cg1361 did not significantly affect the growth of the strain.The analyses of transcription and protein levels of ATP synthase in Acg1360 and Acg1361 mutants revealed that only the deletion of cg1360 down-regulated the expression of ATP synthase,reduced the F1 sector in the membrane,and decreased the ATP-driven proton pumping activity and ATPase activity,suggesting that Cg1360 plays an important role in ATP synthase function.We further explored the energy metabolism levels of the Acg1360 and Acg1361 mutants,and found that deletion of cg1360 resulted in a 42%increase in cellular glucose consumption rate,a decrease in ATP concentration to 72%of the wild type,and an increase in NADH and NADPH levels by 29%and 26%,respectively,while the deletion of cg1361 did not appear to affect the energy metabolism of the strain.This may be attributed to the fact that the lack of cg1360 resulted in functional defects of ATP synthase and impaired oxidative phosphorylation pathway.Cells can compensate for ATP deficiency by substrate phosphorylation by enhancing glycolysis,and the up-regulation expression of key glycolytic enzyme supports this speculation.The regulation of ATP supply is considered as a promising strategy to improve cellular metabolism for enhanced bioproduction yields.The productions of pyruvic acid,acetic acid and alanine have been successfully improved in some ATP synthase-deficient bacteria.L-valine is produced from pyruvate without the requirement of ATP via the four-step enzymatic steps,and NADPH acts as a limiting cofactor in the second step.In view of the above findings that deletion of cg1360 led to a significant increase in the NAD(P)H levels,the effect of deletion of cg1360 on the production of L-valine was examined in L-valine producing V-10 strain.The final titer of L-valine in the V-10-?cg1360 mutant reached 9.6±0.3 g/l in shake flasks,which was 17%higher than that of the V-10 strain.Therefore,Cg1360 can be used as an effective engineering target to enhance L-valine production in C.glutamicum by altering cellular energy metabolism.