Optimizing The Synthesis Of NADPH And Glycerol Metabolic Pathway To Improve The Production Of ?-carotene

The oil hydrolysis industry is playing an increasingly important role under the background of increasingly tense oil resources and global warming. Glycerol, an inevitable by-product of fatty acids and biodiesel industry,has a large output and has become a cheap carbon resource as abundant sources gradually. By using glycerol as a raw material for fermentation to produce ?-carotene, lycopene and other natural products, the efficient utilization and transformation of glycerol can be realized.Multiple genes of the glycerol metabolic pathway were modulated in a previously engineered high ?-carotene producing Escherichia coli strain CAR015 to enhance glycerol utilization capability for improving isoprenoids production. The glpR gene, encoding glycerol 3-phosphate repressor, was deleted firstly. The glpFK, glpD and tpiA genes were then modulated by three artificial regulatory parts M1-37, M1-46 and M1-93 respectively.?-carotene titer reached to 72.45 mg/L after modulating glpD with M1-46 and obtaining the best strain Gly005, which was 5.44 times higher than CAR015, and glycerol consumption rate also increased 98%. This result demonstrated that glpD is a rate-limiting step in glycerol metabolic pathway. Modulating glpFK, glpD and tpiA genes in combination resulted in the best strains Gly016, which produced 69.69 mg/L ?-carotene. The titer was 5.23 times of the parent strain CAR015 and glycerol consumption rate also increased 85%.NADPH is an important cofactor for the synthesis of terpenoids compounds. The pntAB and yfjB genes, which are related to the synthesis of NADPH, were modulated by three artificial regulatory parts in the strain CAR015. This study demonstrated that modulating the yfjB of CAR015 by M1-46 resulted in the best strain HYL004, which produced 19.62 mg/L ?-carotene with a yield of 6.9 mg/g DCW and the titer increased 47.19 % compared with the parent strain CARO 15.Finally,this study combined the best modulation effects of NADPH synthesis and glycerol metabolic pathway. Modulating the yfjB gene in strain Gly005 by M1-46 led to a little increase of ?-carotene titer and obtained the strain YD001, which produced 78.71 mg/L ?-carotene with a yield of 20.75 mg/g DCW. Modulating the yfjB gene in strain Gly016 by M1-46 led to a little increase of ?-carotene titer and obtained the strain YD001,which produced 66.06 mg/L ?-carotene with a yield of 17.17 mg/g DCW.The titer of this YD001 increased 3.23% compared with the parent strain Gly016. This study showed that it is effective to improve the production of ?-carotene with the combined modulation of NADPH synthesis and glycerol metabolic pathway.In addition, modulating glpFK led to a little decrease of ?-carotene titer. In this study,the production of ?-carotene recovered after the promoter of glpFK in the strain Gly016 reverting to the original type, and this result demonstrated that the three kinds of promoters are not suitable for the regulation of glpFK. Modulating glpFK of the strain Gly016 by mRS library obtain the best strain Gly01814, which produced 95.26 mg/L P-carotene and consumed 11.52g/L. This result demonstrated that glpFK is also a rate-limiting step in glycerol metabolic pathway.