Metabolic Engineering Of Rhodobacter Sphaeroides For Enhanced Production Of Coenzyme Q₁₀

Coenzyme Q10(CoQ10)is a kind of membrane bound compounds,which transports the electron in the electron transport chain of aerobic respiration,with ubiquitous occurrence in living organisms.CoQ10 is composed of a quinone ring and a side chain of ten monosaturated isoprenoid units.Because CoQ10 could strengthen respiratory chain and enhance energy supplement,it has been shown to be beneficial to the treatment and prevention of many diseases,such as cardiac diseases,hypertension and Parkinson’s diseases.In addition,due to its well-recognized function as an antioxidant,there is a growing demand for CoQ10 from the pharmaceutical and cosmetic industries.In this study,we analyzed the influential factors on CoQ10 synthesis in Rhodobacter sphaeroides,and comprehensively regulated the CoQio biosynthesis pathway and other pathways that affect CoQ10 biosynthesis.Current efforts towards construction of engineered CoQ10 producers are mostly focused on rational engineering of the CoQ10 synthesis pathway.Although these strategies have achieved good results,we cannot ignore the many other metabolic pathways that influence the synthesis of CoQio.For example,some pathways compete for the CoQ10precursors,like quinones and terpenes,and other pathways have connections to the respiratory chain.In this research,we regulated these non-CoQ10 synthesis pathways,and then combined the regulation of these pathways with that of the CoQio synthesis pathway to enhance the CoQ10 production.In R.sphaeroides,synthesis of coenzyme Q10 shares the same precursor geranylgeranyl diphosphate(GGPP)with carotenoids.Therefore,suppression of carotenoids synthesis is supposed to pose positive effects on accumulation of CoQio.In this study,ppsR was over-expressed to properly repress the transcriptional level of crt genes,which led to obvious decrease of carotenoids and increase of CoQio production by 28%.Although overexpression of ppsR enhanced CoQio production,the transcription level of crtE,the key gene for GGPP synthesis,was also repressed to half of the wild type.To further enhance the production of CoQio,crtE was constitutively co-overexpressed with ppsR to improve the supply of GGPP as a key precursor for the isoprenoid side chain of CoQ10.At last,the CoQ10 production reached 73.2 mg/L,which was enhanced by 47%compared to the wild type.As a hydrogen carrier,the main function of CoQ10 is delivering electron from NADH to the respiratory chain.So,there is a strong connection between redox potential and CoQ10 function.In this research,overexpression of glyceraldehyde-3-phosphate dehydrogenase type I(gapA-1)in R.sphaeroides elevated the NADH/NAD+ ratio by 2 times and meanwhile enhanced the CoQ10 production by 29%.This phenomenon demonstrated the positive correlation between CoQio production and NADH/NAD+ratio.But in this engineered strain,the biomass was decreased,which affects the final CoQio titer.To this end,Vitreoscilla hemoglobin was heterologously expressed to enhance the oxygen uptake ability of the cells,leading to a significant improvement of biomass.To further enhance the CoQio production,gapA-1 and vgb were co-expressed in the form of a single cistron.The CoQio titer was enhanced to 83.24 mg/L,representing 71%improvement as compared to the wild type.Overexpression of gapB2 and gapB3 in R.sphaeroides also elevated the NADH/NAD+ ratio and the CoQio production.This phenomenon confirmed the positive correlation between NADH/NAD+ ratio and the production of CoQio.RspPEGV strain was constructed by combining the regulation of the carotenoid synthesis pathway,the redox potential and oxygen utilization rate.The pathway of carotenoid synthesis was significantly repressed as expected.But because the biomass was declined,the production of CoQ10 was decreased to 62.71 mg/L,lower than those of the strains regulated separately.Considering that the CoQio production did not increase after combining the regulation of the two non-CoQ10 synthesis pathways,we turned to the combinatorial engineering of the two non-CoQ10 synthesis pathways with CoQio synthesis pathway respectively.First,we constructed RspMQPE strain to simultaneously regulate the carotenoid synthesis pathway and CoQio synthesis pathway.The production of carotenoids was repressed to a very low level.Although the CoQio content was increased compared to RspPE,the biomass was decreased.At last,the production of CoQio did not increase as compared to RspPE.Then RspMQGV strain was constructed to regulate the redox potential,oxygen utilization rate and CoQ10 synthesis pathway at the same time.The biomass and NADH/NAD+ ratio of RspMQGV were both enhanced as compared to RspMQd(regulation of MEP and quinone modification pathway,constructed by our lab).The CoQ10 production of RspMQGV was enhanced by 19%compared with RspMQd,and 2.36 times compared to RspMCS.Furthermore,the influence of air supplement condition on CoQ10 production was investigated.Sufficient air supplement at the beginning of fermentation and limited air supplement at the middle and late phases of fermentation was found to be the best condition for CoQ10 synthesis.Finally,600 mg/L of CoQio production was achieved in fed-batch fermentation,which is,to the best of our knowledge,the highest CoQio production among R.sphaeroides strains constructed by metabolic engineering.