Directional Modification Of Rhodobacter Sphaeroides To Improve The Production Of Coenzyme Q10

Coenzyme Q10 is an essential cofactor in the electron transport chain of aerobic respiration,consisting of a benzoquinone ring and a hydrophobic isoprenoid chain conjugate.Remarkable effect of CoQ10 on many diseases have been reported,and the production of coenzyme Q10 by fermentation has been favored by many researchers.R sphaeroides is one of the natural coenzyme Q10 producing strains.As a result of the complex genetic operation system,the research of this strain is mainly focused on the optimization of fermentation technology and the traditional mutation breeding.In this experiment,the genetic manipulation system of R.sphaeroides GY-2,an industrial producing strain of coenzyme Q10,was studied and a markerless gene replacement method was developed.This study not only benefits to improve the yield of coenzyme Q10 by regulating the metabolic pathway,but also provides a new idea for improving the production of high value-added products.It has important theoretical significance and practical application value.(1)Knocking out the upp gene of R.sphaeroides GY-2.The upp gene of R sphaeroides GY-2 was disrupted by the modified suicide plasmid and a new chassis cell R.sphaeroides GY-2?upp which was resistant to 5-FU was obtained.Compared with R.sphaeroides GY-2,the R.sphaeroides GY-2?upp showed no significant change in the growth,biomass,coenzyme Q10 production.The sensitivity to 5-FU was restored by upp gene complementation.(2)A genomic markerless editing tool based on the upp reverse screening marker mediated by the suicide plasmid pSUP202 was established.The double gene knockout strain R.sphaeroidesAuppACm whose chorismate mutase gene was disrupted was constructed using this method.Compared with starting strain,the coenzyme Q10 productivity and yield of the resulting strain was increased by 20.4%and 15.3%,while the biomass did not change significantly.Meanwhile,0.5 mM pHBA was detected in the supernatant of R.sphaeroides GY-2AuppAcm.(3)Using established genetic editing methods,mutant strains of R.Sphaeroides GY-2?upp-crtA::ubiG and R.SphaeroidesGY-2?upp-crtD::ubiE were successfully obtained.Compared with starting strain,the coenzyme Q10 productivity and yield of the R.Sphaeroides GY-2?upp-crtA::ubiG strain was increased by 13.39%and 13.1%while the biomass did not change significantly;The biomass and the coenzyme Q10 productivity and yield of the R.Sphaeroides GY-2Aupp-crtD::ubiE strain was no significant change.