Study On High Cell Density Fermentation For Production Of Mevalonate By Methylobacterium Extorquens

Microbial cell factories are important biosynthesis platform for production of bulk chemicals,biofuels,and natural products.Using methylotrophic cell factories to produce high value-added compounds from methanol is a promising way of solving the bio-production problem of"competing with food"and technological economy caused by sugar feedstock fermentation.Therefore,it is of great significance to develop methanol-based microbial cell factories and their fermentation process.Methylobacterium extorquens AM1 is a typical model organism of methylotrophs,which can use methanol as the sole carbon and energy source for growth and metabolism.In this thesis,M.extorquens AM1 was used as the host and mevalonate synthesized frommethanol via reconstructed pathway as the target product.In order to solve the problem of dissolved oxygen supply limitation during the fermentation of M.extorquens AM1,which is a major problem for high cell density fermentation aiming at high mevalonate productivity,oxygen carriers were added in the fermentation process and the Vitreoscilla Hemoglobin was introduced through genetic engineering.Further integration of the mutated QscR transcriptional regulator and mvt operon into the pCM110plasmid yielded an engineered strain with improved productivity of mevalonate.Futhermore,the M.extorquens AM1 mutant strains capable of tolerating high methanol concentration and maintaining high genetic stability were screened and evaluated by utilizing the novel atmospheric pressure room temperature plasma（ARTP）mutagenesis combined with laboratory adaptive evolution.The growth of M.extorquens AM1 was enhanced by the addition of different oxygen carriers during shake flask fermentation.When 3%hexadecane was added after fermentation of M.extorquens AM1 for 24 h,the OD₆₀₀00 of M.extorquens AM1 was 12.56,which was 19.9%higher than that of the non-addition group.After 120 hours of fermentation,the final yield of mevalonate in the control group without the addition of the oxygen carrier was 143.7 mg/L,and the final yield of mevalonate in the culture system with the hexadecane group was 163.7 mg/L.The addition of hexadecane increased the yield of mevalonate.Increased by about10%.The integration of the vgb gene in M.extorquens AM1 by genetic engineering has also correspondingly increased the growth ability of M.extorquensAM1.Expressionofthevgbgenestrain AM1-Δcel/pCM110-P_maxF-vgbwiththemethanoldehydrogenase promoter on the pCM110 plasmid resulted in a OD₆₀₀00 of up to 19.84,whereas strain AM1-Δcel/pCM110-P_vhb-vgb expressing the vgb native promoter was obtained.The highest growth OD₆₀₀00 reached 18.73.The highest growth OD₆₀₀00 of the control group AM1-dcel::mvt was 10.06.The two strains AM1-Δcel::(P_maxF-vgb)and AM1-Δcel::(P_vhb-vgb)which express the vgb gene on the genome of M.extorquens AM1 showed no significant difference from the control strains.At the same time,the QscR transcriptional regulator and the mvt operon were integrated into the pCM110 plasmid,and expression in the M.extorquens AM1 also increased mevalonate production.Its mevalonate production of 387.65mg/L in shaking flasks for 144 hours is the highest production of mevalonate in shake flasks.In the 5L tank methanol feed,AM1-Δcel/pCM110-P_maxF-vgb-mvt-qscr had the highest OD600 growth of38.56,which is the highest value at present,and the mevalonate production was 1.03g/L.