Metabolic Engineering Of Rhodobacter Sphaeroides 2.4.1 For Pinene Production

Pinene is an important chemical raw material,and is widely used in industrial and medical fields.The pinene dimers have density and heating value similar to that of JP-10,and have the potential to be the advanced fuel.Pinene dimers can be generated via pinene dimerization using chemical catalysis.Growing concerns over fossiol-energy shortage have stimulated efforts to develop pinene production in microorganisms.In this study,we engineered and optimized the Rhodobacter sphaeroides 2.4.1 terpenoid pathway for the production of pinene.Given that researchers improved the yield of pinene in microorganisms through the fusion of Geranyl Pyrophosphate Synathase(GPPS)and Pinene Synthase(PS).So in this study,we optimized and synthesized gppsps and psgpps gene.Then,these two genes were applied to express in Rhodobacter sphaeroides 2.4.1.By comparing the final yield,we chose the gppsps gene for pinene production.Then we optimized the expression condition including incubation temperature,expression hours,the concentration of IPTG and the induction time.We have successfully constructed a inducible system of pinene synthesis in Rhodobacter sphaeroides 2.4.1.In order to improve the final yield of pinene,we firstly constructed the MVA pathway in Rhodobacter sphaeroides 2.4.1 for pinene production and analysed the reason why it didn't work as that in E.coli.Afterwards we overexpressed the eight enzyme genes from 1-Deoxy-D-xylulose 5-phosphate pathway(DXP pathway)and found that isopentenyl pyrophosphate isomerase(IDI)and 1-Deoxy-D-xylulose 5-phosphate synthase(DXS)may contribute to the final yield.So we chose to express the DXP pathway gene one by one with IDI and DXS.The engineered bacteria with IDI,DXS,1-Deoxy-D-xylulose 5-phosphate reductase(DXR)and 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase(ispD)achieved the highest yield.Finally,we changed the RBS sequence before gppsps gene to regulate the flux of the lower pathway.The balance of the upper pathway and the lower pathway achieved 65.8 ?g/L ?-Pinene and 138.32 ?g/L ?-Pinene,which were increased 100% and 110% respectively.We have successfully achieved heterologous expression of pinene in Rhodobacter sphaeroides 2.4.1,and improved the final yield through the overexpression of DXP pathway.Furthermore,the substitution of the gppsps gene in plasmid may make this engineered bacteria a terpenoid production platform.