Optimization Of Fermentation Process Of Methylobacterium Extorquens AM1 For Efficient Mevalonate Production

The methanol-based microbial cell factory can produce high value-added products from methanol,Which can solve the problem of 'competing with food'to a certain extent.Therefore,it is of great practicability tostrengthen the methanol-based biological process.In order to improve the production of mevalonate(MEV)produced by M.extorquens,the fermentation process was optimized at the shake-flask level.The strategy of adding methanol in batch was used to reduce the inhibition of methanol growth and production of MEV by methanol,and sodium formate was added to increase the intracellular reducing power.The optimal fermentation conditions were as follows:1%initial methanol concentration,0.33%methanol was added every 24 h after the start of culture,and 20 mmol/L sodium formate was added after 120 h of fermentation.The yield of MEV was 450.74 mg/L,which was higher than that before optimization.The cell density OD600 value reached 23.96,which was 86.46%higher than that before optimization.Methanol usage was reduced by 33.3%compared withbefore optimization.After adding sodium formate for 24 h,the intracellular reducing power was increased by 61.85%,of the sodium formate group the increase of MEV.On the basis of the optimization of shake flask,the fermentation process of 1L and 5 L fermenter was further optimized.The determination of 1%methanol concentration as the optimal starting concentration was carried out,and the influence of nitrogen source was quantitatively studied.The 3 times nitrogen source of starting nitrogen source is the best for the growth of the bacteria.Under this condition,the highest OD600 value reaches 37.15;while the 5 times nitrogen source relative to the starting nitrogen source is the best for MEV production,the highest yield reaches 948.42 mg/L.On this basis,the nitrogen source was added during the fermentation process.The total nitrogen source was 5 times that of the original medium nitrogen source.The folw was added by constant flow addition.The addition time was 4-day,and the final OD600 value was reached 41.5.MEV production reached 1130 mg/L.Add 20 mmol/L sodium formate at 120 h,on the basis of simultaneous addition of carbon source and nitrogen source,the highest OD600 value is 42.43 and the mevalonate is 1450 mg/L.In this study,the optimal methanol addition strategy was obtained at the shake flask level.The addition of sodium formate solved the problem of insufficient intracellular reducing power;the optimum flow conditions of carbon and nitrogen sources were obtained at the fermenter level,and the bacterial cells were improved for the development of high value-added products methanol-based cell plants.