| Lycopene and ?-carotene are the most popular and widely used carotenoids.Lycopene is the most efficient biological carotenoid singlet oxygen quencher,?-carotene is an important precursor of vitamin A in human body.They not only have a bright red or orange color,but also play a critical role in anti-oxidative and anti-cancer.Due to the distinctive biological properties,lycopene and ?-carotene have been used in a variety of fields including the functional food,nutraceutical,pharmaceutical and cosmetic industries,and has garnered increasing demand in the global market.Rhodobacter sphaeroides,a carotenogenic and phototrophic bacteria,was usually used as a model organism to study photosynthesis and membrane proteins.With the development of genetic manipulation and metabolic engineering,R.sphaeroides has been proved to be an efficient and practical host for high value-added natural products synthesis.It harbors a photosynthetic gene cluster containing the seven native crt genes involved in carotenoid synthesis,thus it can accumulate large amounts of carotenoids naturally.It equipped with rich membrane system that favors lycopene and ?-carotene aggregation in membranes.Furthermore,the fermentation of R.sphaeroides is also cost-effective as it can naturally synthesize carotenoids under anoxygenic and photosynthetic conditions.In this study,we explored the potential of metabolically engineered R.sphaeroides as a novel platform to produce lycopene and ?-carotene.The main contents and results are described below:(1)Construction of a lycopene biosynthetic pathway in R.sphaeroides.The basal lycopene-producing strain was generated by introducing an exogenous 4-step dehydrogenase gene crtl4 from Rhodospirillum rubrum firstly to replace the native crtI3 and deleting crtC to block lycopene-consuming pathway in R.sphaeroides.The basal lycopene-producing strain termed RL1 was able to produce lycopene exclusively as the end-product,and the lycopene content was 4.17 mg/g DCW after fermentation for 96 h.(2)Enhancing lycopene production by engineering the central carbon metabolic pathway and the MEP pathway.Deleting the 6-phosphoglucose dehydrogenase gene zwf blocked the pentose phosphate pathway and allowed for lycopene content improvement by 88%(7.82 mg/g DCW).Then,the MEP pathway was reinforced by amplifying dxs gene combining with zwf deletion,which further increased the lycopene content and the final engineered strain produced lycopene up to 9.26 mg/g DCW.(3)Clarifying the regulatory mechanism of carotenogenesis in engineered strains and the identification of fermentation products.The expression levels of crtE,crtI3,crtl4 and dxs were separately studied in wild type,RL1,RL2 and RL3 strain by fluorescence quantitative PCR.Results implied that the expression level of crtI4 was positively correlated with the synthesis of lycopene,which indicated the carotenogenic genes are co-regulated.The fermentation process of engineered RL3 was analyzed 10.32 mg/g DCW and found that there is still room for the yield improvement.The fermentation product of RL3 was extracted,purified,crystallized and quantitatively analyzed by HPLC,NMR,and IR,which further confirmed the product is lycopene.(4)Construction of the P-carotene biosynthetic pathway in R.sphaeroides.On the basis of engineered strain RL1 and RL3,an exogenous 4-step dehydrogenase gene crtIPa and a lycopene cyclase gene crtYPa from Pantoea agglomerans were introduced and resulted two ?-carotene-producing strain RC1 and RC3.Both strains finally achieved5.76and7.29mg/gDCW ?-carotene accumulation separately.This study described a novel lycopene and ?-carotene producer and provided a good insight into the photosynthetic bacteria as a cell factory for lycopene and ?-carotene biosynthesis. |
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