Study On 3-hydroxypropionic Acid Production From Glycerol By Recombinant Escherichia Coli W3110 Strain

3-Hydroxypropionic acid(3-HP)can be used for synthesis of many important chemicals due to its lively chemical properties.In resent years,glycerol was over produced and the price was dropped.Therefore,3-HP production by recombinant strains from glycerol is intensively investigated.In this study,the strain.E.coli W31110_?glPR(pCDFDuet-tac-gpd1-TUkgsadh/pACYCDuet-tac-dhaB)was constructured,and the basic culture conditions in shake flask was optimized,in addition to the 3-HP biotransformation by resting cell.The fermentation process of 3-HP from glycerol by the strain in a 5L bioreactor was also investigated.The main results were as follows:(1)The shake flask fermentation for 3-hydroxypropionic acid production was optimized by single factor experiments.The results showed that the optimized culture media were composed of(g/L):glycerol 30,glucose 20,NaCl 1,MgSO4·7H2O 0.25,Na2HPO4·12H2O 22.7,KH2PO4 3,yeast extract 4,(NH4)2SO4 4,VB12 0.005,initial pH 7.0.The seed culture time at 10 hour;inoculums size of 4%(V/V);the amount of added IPTG was 0.05 mM,and the inducing temperature was 30?.Under these optimized conditions,the 3-HP production reached 5.19 g/L which was 2.08 times higher than that of the original condition.(2)The reaction system for 3-HP production with the resting cell was optimized by single factor experiments.The concentration of cell and glycerol were all 20 g/L;the concentration of VB12 and NAD+were 10 mg/L and 0.15 mM,respectively;and the temperature was 35?,the buffer solution was 0.05 mol/L Tris-HCl(pH 7.0).Under the condition of the above reaction after 6 h,the production of 3-HP reached 4.12 g/L,and the yield was 0.51 g/g glycerol.(3)The fermentative process was investigated in a 5L bioreactor.The 3-HP production was 7.31 g/L when pH was controlled at 7.0,0.9 times higher than the pH was not controlled.Then the effect of VB12 added on fermentation was investigated,and the optimal strategy was adding of 5 mg/L every 12 h,and the production of 3-HP was improved to 8.56 g/L,which was 0.17 times higher than the control.In the end,glucose was added for redox balance,and 20 g/L glucose made 3-HP produced improved to 10.23 g/L.In the fed-batch process,glycerol was feeding to maintained at 15-20 g/L,and 3-HP production was 12.23 g/L,and when glucose was added,the 3-HP production could reach 15.4 g/L.